A novel near-infrared xanthene-based fluorescent probe for detection of thiophenol: In vitro and in vivo

Article abstract of DOI:10.1039/d0nj03370g

Thiophenol (or PhSH) is widely utilized for producing pesticides, dyes, and pharmaceuticals. However, high-concentration thiophenol not only has adverse effects on living organisms, but also causes environment pollution. Herein, we designed and synthesize

Full text of DOI:10.1039/d0nj03370g

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A novel near-infrared xanthene-based fluorescent probe for
detection of thiophenol in vitro and in vivo
Received 00th January 20xx,
Accepted 00th January 20xx
Yongquan Wu,a,b Aiping Shi, Huiying Liu, Yuanyan Li, Weican Lun, Hong Zeng* and Xiaolin Fan*
a
a
a
a
a
a
DOI: 10.1039/x0xx00000x
Thiophenol (or PhSH) is widely utilized for producing pesticides, dyes, and pharmaceuticals. However, high-concentration
thiophenol not only has adverse effects on living organisms, but also causes environment pollution. Herein, we designed
and synthesized a novel near-infrared (NIR) xanthene-based fluorescent probe (NOF) for real-time detection of thiophenol
in vitro and in vivo. The probe NOF had good sensitivity and selectivity, and fast response towards thiophenol. The
thiophenol-sensing mechanism was verified by high resolution mass spectra (HR-MS) and density functional theory (DFT)
calculation. The probe NOF was also successfully applied to detect thiophenol in real water samples. Finally, owing to its
NIR-emitting properties, it was successfully used for NIR fluorescent imaging of thiophenol in living cells and mice.
have a shallow penetration depth, which is one of the causes
that have limited their applications in imaging of deep tissues
and the body. In addition, the body is a complex environment
that can cause short-lived auto-fluorescence and tissue
scattering, which can inevitably interfere with the imaging.
1
. Introduction
Thiophenol (PhSH), also known as benzenethiol, belongs to a
class of highly reactive and toxic aromatic thiols widely used
3
5-38
1
,2
for preparing pesticides, pharmaceuticals, and dyes. Despite
its usefulness, thiophenol is highly toxic, therefore is
categorized as a class of pollutant compounds; it also has been
Therefore, the development of an NIR-emitting fluorescent
probe that can sensitively and selectively detect and image
thiophenol is highly desirable; however, such probes have
3
listed as one of the prioritized pollutants. Animal model
3
9-41
rarely been reported in the literature.
studies have revealed that the median lethal dose in fish of
Xanthene fluorescent dyes have excellent photophysical
properties, such as high fluorescence quantum yields, high
tolerance to photobleaching, and high molar extinction
coefficients, thus are good candidates for use as skeletons of
4
thiophenol is 0.01–0.4 mM and that in mouse is 46.2 mg/kg.
Exposure to thiophenol can cause many symptoms, including
vomit, nausea, muscular weakness, coughing, increased
breathing, and even death, as it can cause damage to the
central nervous system. As a consequence, it is necessary to
develop an easy, rapid, efficient method that can
quantitatively detect thiophenol in the environment and in
living organisms.
4
2-46
fluorescent probes.
Herein, we present a novel NIR-
5
-7
emitting xanthene fluorescent probe (NOF) with a ‘off-on’
response mechanism that can sensitively and selectively
detect thiophenol. The probe NOF was synthesized by
attaching a strong electron-withdrawing 2,4-dinitrophenyl
ether group to a xanthene skeleton, which served as an
electron acceptor; the attachment caused the probe to emit
weak fluorescence. But upon binding to thiophenol, the probe
NOF exhibited a remarkably enhanced fluorescence signal. The
probe NOF could effectively discriminate between thiophenol
from aliphatic thiols and that from nucleophilic sulfides. It was
also successfully applied to detect thiophenol in real water
samples and to image thiophenol in living cells and in mice.
The developed probe NOF can be applied to detect thiophenol
in the environment or in other biological samples.
Fluorescence probing is one of the most prominent methods
for detection of thiophenol due to its intrinsic advantages such
as high sensitivity, good selectivity, technical simplicity, non-
8
-12
invasion, and in situ and real-time detection capability.
A
number of fluorescent probes for the detection of thiophenol
1
3-34
have been designed and developed to date.
most of these fluorescent probes have slow response rates
longer than 10 min), relatively short emission wavelengths (in
Nevertheless,
(
the visible region), and small Stokes shifts. The short emission
wavelengths (λmax < 650 nm) cause these fluorescent probes to
a
School of Chemistry and Chemical Engineering, Gannan Normal University,
Shiyuan South Road, Ganzhou 341000, P. R. China, E-mail: fanxl@gnnu.edu.cn;
zenghong@gnnu.edu.cn
Jiangsu Key Laboratory for Biosensors, Institute of Advanced Materials (IAM),
Nanjing University of Posts and Telecommunications, 9 Wenyuan Road, Nanjing
b
2
10023, P. R. China
Electronic Supplementary
DOI: 10.1039/x0xx00000x
Information
(ESI)
available.
See
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COOH
DOI: 10.1039/D0NJ03370G
O
COOH
O
F
N
O
2
N
O
O2N
NO2
O
N
DMF/K2CO3/KI/50 0C
CH3COOH/CH3COOK/90
oC/ N2
O2N
OH
O2
NO2
O
NO2
NOF
1
COOH
O
COOH
OH
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N
O
AcOH/KAc, 90 o
N
O
C
2
NOF-OH
OH
Scheme 2. Synthetic route of NOF and NOF-OH.
Scheme 1. Sensing mechanism of NOF in response to thiophenol (PhSH).
2
.3.1 Synthesis of compound 1
p-Hydroxybenzaldehyde (305 mg, 2.5 mmol) was mixed with
potassium carbonate (414 mg, mmol) and N,N-
dimethylformamide (3 mL) in a 50-mL three-necked flask. After
that, 1-fluoro-2,4-dinitrobenzene (930 mg, 5 mmol) and
3
2
. Experimental section
2.1 Materials
All commercial chemicals were purchased from commercial potassium iodide (830 mg, 5 mmol) dissolved in N,N-
suppliers and used without further purification. 2-(4- dimethylformamide (3 mL) were added to the flask. The
diethylamino-2-hydroxybenzoyl) beozoic acid, acetic anhydride, mixture was heated to 50 °C under N
cyclohexanone, and HClO
(70%) were purchased from Engery magnetically stirred for 5 h. After the reaction was completed,
Chemical Technology Co., Ltd (China). Thiophenol, 3- the reaction solution was suction filtered to remove the solid.
aminothiophenol, 2,4-dinitrofluorobenzene,
p- The filtrate was transferred to a separatory funnel, into which
hydroxybenzaldehyde, and potassium acetate were purchased an appropriate amount of deionized water and
from Adamas Regent Co., Ltd (China). Potassium carbonate, dichloromethane were added for extraction. The extraction
potassium iodide, 4-hydroxybenzaldehyde was purchased was repeated three times, and the products in the organic
2
atmosphere while being
4
from Aladdin Technology Co., Ltd (China). Concentrated H
2
SO
4
layer were collected. Anhydrous sodium sulfate was added to
was purchased from Jiangsu Tong Sheng Chemical Reagent Co., the products to remove water for 2 h. The residue was
Ltd (China). Anhydrous DMF was purchased from J&K Scientific subsequently evaporated to dryness under reduced pressure.
Ltd (China). Cysteine (Cys), and glutathione (GSH) were The crude product was purified by silica gel column
purchased from Aladdin Technology Co., Ltd (China). chromatography using CH
Homocysteine (Hcy) was purchased from Sigma-Aldrich Co., eluent, from which a white solid was obtained. H NMR (400
2
Cl
2
: petroleum ether (3:1) as an
1
Ltd (China). MTT and PBS were purchased from Beyotime MHz, CDCl
Biotechnology Co., Ltd (China). RPMI 1640 was purchased from = 9.2, 2.8 Hz, 1H), 8.02 – 7.95 (m, 2H), 7.28 (d, J = 2.1 Hz, 1H),
3
) δ 10.00 (s, 1 H), 8.86 (d, J = 2.7 Hz, 1 H), 8.42 (dd, J
Thermo Fisher Scientific Co., Ltd (USA).
.2 Instruments
7.26 (d, J = 2.0 Hz, 1H), 7.23 (d, J = 9.1 Hz, 1H).
2.3.2 Synthesis of probe NOF
2
Compound 2 was synthesized according to a previously
NMR spectra were obtained on a Bruker DRX-400 NMR
spectrometer (Germany) with tetramethylsilane (TMS) as
internal standard. High resolution mass spectra (HRMS) were
obtained with an ESI ionization sources on Agilent 6540 Mass
Spectrometer (USA). UV-Vis absorption spectra were recorded
on a Shimadzu UV-2700 spectrophotometer (Japan). Emission
spectra were recorded on an Edinburgh FS5 fluorescence
spectrophotometer (UK). Cell imaging experiments were
performed on an Olympus FV1000 laser scanning confocal
microscopy (Japan). The absorbance values in MTT assay were
measured using a Varioskan LUX microplate reader (Thermo
Fisher Scientific Inc, USA). Mouse imaging experiments were
carried out using an IVScpoe 7550 in vivo imaging system
4
7
reported method and was used without further purification.
Compound 1 (288 mg, 1 mmol), compound 2 (376 mg, 1
mmol), potassium acetate (98 mg, 1 mmol), and acetic
anhydride (6 mL) were mixed in a 50-mL three-necked flask.
2
The mixture was stirred at 90 °C for 5 h under N atmosphere.
After the reaction, an appropriate amount of water was added
into the reaction mixture to remove excess acetic anhydride,
and an appropriate amount of sodium hydrogen carbonate
was slowly added thereafter. The precipitate was filtered
under reduced pressure to obtain a solid crude product. The
crude product was purified by silica gel column
: ethyl acetate (3:5) as an eluent,
from which a purple solid was obtained. H NMR (400 MHz,
3
OD) δ 8.88 (d, J = 2.8 Hz, 1H), 8.47 (dd, J = 9.2, 2.8 Hz, 1H),
.11 (dd, J = 7.1, 2.0 Hz, 1H), 8.05 (s, 1H), 7.74 (d, J = 8.7 Hz,
chromatography using CH
2
Cl
2
1
(Shanghai CLINX Science Instruments Ltd., China).
CD
8
2.3 Synthesis section
The synthetic routes of compound 1, the probe NOF, and NOF- 2H), 7.68 – 7.59 (m, 2H), 7.30 (dd, J = 9.0, 4.0 Hz, 3H), 7.22 –
OH are shown in Scheme 2. Their structural characterization 7.17 (m, 1H), 7.11 (d, J = 3.7 Hz, 3H), 3.67 (d, J = 7.1 Hz, 4H),
can be found in ESI.
2.95 (d, J = 7.6 Hz, 2H), 2.53 – 2.30 (m, 2H), 1.81 (d, J = 8.5 Hz,
H), 1.29 (d, J = 7.1 Hz, 6H). HR-MS (ESI), chemical formula:
2
C
+
37
H
32
3
N O
8
, calcd, 646.2184; found: 646.2186.
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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2
.3.3 Synthesis of NOF-OH
were used as the model cells and were cultured in RPMI 1640
View Article Online
DOI: 10.1039/D0NJ03370G
medium supplemented with 10% (v/v) fetal bovine serum. The
p-Hydroxybenzaldehyde (0.10 g, 0.82 mmol) was reacted with
compound 2 (0.34 g, 0.90 mmol) in 15 mL acetic acid overnight.
The crude product was then purified by silica gel column
cells used for cell imaging were cultured on a confocal imaging
dish overnight at 37 °C under 5% CO2 atmosphere for 24 h.
In vitro cytotoxicity of the probe was determined using MTT
assay and MCF-7 cells grown to the logarithmic phase in a 96-
chromatography to obtain a dark purple solid (0.10 g, yield =
1
2
1
8
3
2
C
2
4%). H NMR (400 MHz, Methanol-d
4
) δ 8.14 (q, J = 4.2, 3.3 Hz,
4
well flat-bottomed microplate (cell density = 1 × 10 cells per
H), 8.08 (d, J = 16.4 Hz, 1H), 7.71 – 7.60 (m, 2H), 7.56 (q, J =
.2 Hz, 2H), 7.22 – 7.09 (m, 4H), 6.91 (q, J = 7.6, 6.6 Hz, 2H),
.76 – 3.58 (m, 4H), 2.94 (s, 2H), 2.59 – 2.35 (m, 2H), 1.83 (s,
H), 1.34 – 1.29 (m, 6H). HR-MS (ESI), chemical formula:
well). Cell viability was determined as the amount of the
formazan product formed in each well measured by OD490 in
relative to OD690. Cell viability was calculated using the
following formula: cell viability (%) = (average of (OD490 -
OD690) of sample/average of (OD490 - OD690) of blank) ×
0
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+
+
31
H
30NO
4
[M+H] : calcd, 480.2169; found: 480.2346..
.4 Measurement of UV–vis absorption and fluorescence
1
00%. The data were expressed as mean of 4 parallel
spectra
experiments ± standard deviation.
The UV–vis absorption and fluorescence spectra of thiophenol
was carried out in DMSO/PBS solution (v/v, 1:1, pH=7.4) by
which a certain amount of thiophenol standard solution (2 mM)
was added dropwise to the solution of the probe NOF. The
mixture was stirred for 10 min before being subjected to the
test. The UV-vis absorption spectra were collected at a
wavelength range of 400 to 800 nm. The fluorescence spectra
were collected at an excitation wavelength of 670 nm and an
emission wavelength range of 690 to 850 nm.
2
.9 Cell imaging
In the control group, the cells were incubated with the
probe NOF (10 μM) in a medium containing 0.5%(v/v) dimethyl
sulfoxide at 37 °C for 30 min. In the experimental group, the
cells were pretreated with various concentrations of
thiophenol in DMSO for 2 h. After that, they were washed
three times with PBS to remove excess thiophenol before
being treated with the probe NOF (10 μM) for 30 min. All
treated cells were subjected to fluorescence imaging using a
confocal fluorescence microscope (Olympus FV1000, Japan).
2
.5 Determination of selectivity towards thiophenol
The UV-visible absorption and fluorescence spectra of the
probe NOF (10 μM) were measured in DMSO/PBS (v/v, 1:1,
pH=7.4) in the presence of various analytes, including
2
.10 Luminescence imaging in mice
All animal experiments were carried out in accordance with
the guidelines of the Animal Protection and Use Committee of
the Gannan Normal University and approved by the Animal
Ethics Committee of the Department of Science and
Technology of Jiangxi Province. Prior to the injection, Kunming
mice were anesthetized with a certain amount of chloral
hydrate (10% wt) and were then carefully placed in the animal
imaging system. One side of the groin of the mice was injected
with the probe NOF (20 μM, 50 μL), whereas the other side
was first injected with thiophenol (50 μM, 50 μL) before being
injected with the probe NOF (20 μM, 50 μL). The change of the
fluorescence intensity in the mice was observed after a certain
period of time. The fluorescence imaging was performed on an
IVScope 7550 imaging system (Shanghai CLINX Science
Instruments Ltd., China) using a long pass filter to collect
fluorescence signals greater than 650 nm under excitation at
-
dithiothreitol (DTT), GSH, Hcy, Cys, Phe, Trp, Leu, ONOO ,
2
-
3-
2- 2-
-
-
+
+
2+
2+
2+
2+
2 8
S O , NO , NO , S , ClO , SCN , Na , K , Ca , Cd , Cu , Ni ,
2
+
2+
2+
2+
3+
Pb , CO , Zn , Mg , and Cr , as well as of thiophenol.
Before the measurement, the probe solution was incubated at
3
7 °C for 10 min.
2
.6 Detection of thiophenol in real water samples by the probe
NOF
The detection of thiophenol in real water samples (including
tap water and mineral water from NongFu Spring) by the
probe NOF was carried out in PBS buffer (10 mM, pH = 7.4)
containing 1 mM CTAB at 37 °C. The water samples were
spiked with thiophenol at various final concentrations (0, 5, 10,
and 15 μM) prior to being added with the probe NOF (final
concentration = 10 μM). The fluorescence spectra of the
samples were then measured at 740 nm. The experiments
were carried out in triplicate, and the data were reported as
mean ± standard deviation.
6
35 nm. The fluorescence imaging images were analyzed using
ImageJ software.
2
.7 Computational details
3
. Results and discussion
To further understand the optical properties of the NOF and
NOF-OH, we optimized the molecular structure of the ground
and excited states of the NOF and NOF-OH at the B3LYP/6-31G
3
.1 Design and synthesis of probe NOF
According to the literature, nitro-substituted phenyl ether can
serve as a potential reaction site for thiophenols. Therefore, in
this work, 2,4-dinitrophenyl moiety was used as an effective
thiophenol-sensing group to modify xanthene skeleton to
synthesize the probe NOF. Due to the fluorescence quenching
effect of the nitro group, the fluorescence of this molecular
system should be quenched by the 2,4-dinitrophenyl moiety.
However, upon interaction with thiophenol, the 2,4-
4
8,49
(d) level using density functional theory (DFT).
Time-
dependent DFT (TD-DFT) calculations were performed to get
the nature of the excited states based on the optimized
structures.50 All calculations were performed using Gaussian
0
2
9 Rev. D.01.51
.8 Cell culture and cytotoxicity assay
To MCF-7 cells, which were provided by the Institute of
Biochemistry and Cell Biology (Chinese Academy of Sciences),
This journal is © The Royal Society of Chemistry 20xx
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dinitrophenyl moiety can be uncaged, in turn causing the period of 60 min under excitation at 670 nm (Fig S3). This
View Article Online
DOI: 10.1039/D0NJ03370G
observation indicated that NOF is responsible for sensing with
1-fluoro-2,4- no obvious photobleaching.
Compared with other reported probes (Table S1), this probe
fluorescent to be turned on (turn-on signal).
Firstly, 4-hydroxybenzaldehyde and
dinitrobenzene were used as the raw materials to synthesize
compound 1 under basic conditions. The compound 1 was has advantages for thiophenol detection including relatively
then used to react with xanthene precursor 2 through low detection limit and more red-shift excitation and emission
Knoevenagel method to form the target probe NOF (Scheme 2). wavelengths. As a result, this probe can be used for in vivo
NOF-OH, which was used as a control fluorescent molecule, imaging by excited at longer wavelength.
was also synthesized from 4-hydroxybenzaldehyde and
xanthene precursor 2 via a one-step synthesis method. The
structures of the probe NOF and NOF-OH were characterized
by nuclear magnetic resonance spectroscopy (NMR) and high-
resolution mass spectrometry (HR-MS) (Details can be found in
0
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the synthesis section and SI).
3
.2 Photophysical properties of the probe NOF in the presence of
thiophenol
The UV-vis absorption and fluorescence spectra of NOF in the
presence of thiophenol at different concentrations (0 – 30 μM)
were first determined in PBS: DMSO (1:1, v/v) at 37 °C for 10
min. Initially, we examined the absorption spectra of NOF
upon continuous addition of thiophenols, from which we
found that continuous addition of thiophenols (0–3.0 equiv.)
into buffer caused the absorption peak to become gradually
red-shifted to 675 nm. This is likely due to that the 2,4-
dinitrophenyl moiety in NOF was transformed into a phenol
group to generate NOF-OH (Scheme 1). The fluorescence
response of NOF to thiophenol was subsequently examined. As
can be seen in Fig. 1b, free NOF exhibited a relatively weak
fluorescence signal after being excited at 670 nm. Upon the
Fig. 1 Changes in emission spectra of NOF (10 μM) upon addition of increasing
addition of thiophenol, the fluorescence signal at 740 nm was concentrations of PhSH (0 − 30 μM), PBS:DMSO=1:1 (v/v, pH=7.4). λex = 670 nm.
increased by up to 10 folds. Furthermore, the fluorescence
titration assay revealed that the increase of the fluorescence
intensity was in a dose-dependent manner; the fluorescence
reached saturation when 30 μM thiophenol was added (Fig.
2
1
b). A good linear relationship (R = 0.996) between the
fluorescence intensity at 740 nm and the concentration of
thiophenol (2−18 μM) was also observed (Fig. S1). The
thiophenol-detection limit of NOF (calculated by 3σ/k) was
found to be as low as 0.120 μM, indicating that the probe NOF
can detect trace amounts of thiophenol.
The fluorescence intensity of NOF (10 μM) as a function of
time in the presence of thiophenol (1.5 and 2.5 equivalents) is
displayed in Fig. 2. The fluorescence intensity was drastically
enhanced within a few minutes after thiophenol was added,
and reached a plateau after 10 min. Such fluorescence
response suggests that the probe is suitable for real-time
detection of thiophenol.
In order to extend further applicability of probe NOF under
biological conditions, fluorescence intensity changes of NOF in
aqueous solution were investigated at different pH values in
presence or absence of thiophenol. As shown in Fig. S2, this
probe can detect PhSH effectively under physiological pH
condition (PH=7.4). Secondly, to determine whether NOF was
photostable under excitation for a long time, its photostability
in solution was assessed. The fluorescence intensity of was
maintained over 95% of its initial value when monitored over a
Fig. 2 Changes of fluorescence intensity at 740 nm of NOF (10 μM) + PhSH (15 and 25
μM) with different time. λex = 670 nm, λem = 740 nm.
3
.3 Selectivity towards thiophenol
To examine the selectivity of the probe NOF towards
thiophenol, we measured the fluorescence response of the
probe NOF (10 μM) in the presence of various analytes (5.0
equivalents each), including small molecules (Cys, Hcy, GSH,
-
-
-
2-
2-
3 2 2 8
Trp, Leu, Phe, DTT) and anions (NO , NO , ONOO , S O , S ,
-
-
ClO , SCN ) and compared them with the response in the
presence of thiophenol. As shown in Fig. 3 and Fig. S4, only the
presence of thiophenol caused significant fluorescence
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enhancement, whereas the presence of other analytes only
ARTICLE
To gain insights into the electronic properties aVniedw Atrhticele‘Oonflfin−e
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caused slight change of fluorescence. The significant increase on’ fluorescence response of the probe NDOOFI: 1in0.1t0h3e9/pDr0eNsJe0n3c3e70oGf
of fluorescence of the probe NOF was not observed in the thiophenol, we employed calculated DFT/TD-DFT data for both
presence of active sulfur-containing compounds (Cys, HCy, and NOF and NOF-OH using B3LYP method on the basis of 6-31G
S2-) other than thiophenol may be due to the local soft and (d). The energies for the frontier MOs as well as their
hard acid-base principle. The local softness of the sulfur atom corresponding highest occupied molecular orbital (HOMO) and
in these nucleophiles are different, thus respond differently at lowest unoccupied molecular orbital (LUMO) gaps are listed in
different strengths to the probe NOF. Moreover, the selectivity Table S2 (ESI†), and the HOMO-LUMO diagram is shown in Fig.
of NOF for PhSH over metal ions was also evaluated. As shown 5 (More details can also be found in Fig. S7, ESI†). According to
in Fig S5, the coexistent metal cations had a negligible the data, the HOMO of NOF was located in the fluorophore,
interfering effect on the fluorescence intensity of NOF upon while its LUMO and LUMO+1 were located in the whole
addition of PhSH. These data suggest that the probe NOF has molecule and the 2,4-dinitrobenyl group, respectively. The
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high selectivity towards thiophenol.
lowest-energy transition of NOF was mainly the result of the
orbital transitions from HOMO to LUMO and HOMO to
LUMO+1. The latter orbital transition indicates that an
electron was transferred from the excited state of fluorophore
to the LUMO of the electron acceptor 2,4-dinitrobenyl moiety,
which in turn caused the fluorescence quenching of NOF.
Fig. 3 (a) Absorption spectra and (b) fluorescence spectra of probe NOF (10 μM)
-
2-, NO
-,
treated with various analytes (DTT, GSH, Hcy, Cys, Phe, Trp, Leu, ONOO , S
2
O
8
3
-
2-
-
-
2
NO , S , ClO , SCN , and PhSH), λex = 670 nm, λem max= 740 nm.
3
.4 Thiophenol-sensing mechanism
Fig. 4 HR-MS of (a) NOF and (b) product of NOF+PhSH.
To investigate the mechanism of the probe NOF in sensing
thiophenol, we first carried out a reaction between the probe
NOF (10 μM) and thiophenol (PhSH) and then separated the
reaction product (NOF+PhSH) by silica gel chromatography.
We subsequently measured the UV-vis absorption and
fluorescence spectra of the product NOF+PhSH and compared
them with those of NOF-OH, from which we observed that the
UV-vis absorption and fluorescence spectra of NOF-OH were
similar to those of NOF+PhSH (Fig. S6). We also examined the
change of the molecular weight of the probe NOF before and
after reacting with thiophenol by high-resolution mass
spectrometry (HR-MS). As shown in Fig. 4a, the peak of the
+
probe NOF appeared at m/z = 646.2186 (calculated M
6
46.2184). As shown in Fig. 4b, after reacting with thiophenol, Fig. 5 Representations of the frontier molecular orbitals (MOs) for the S
0
geometry of
NOF as determined at the DFT//B3LYP/6-31G* level.
a new peak corresponding to the molecular weight of NOF-OH
+
appeared at m/z = 480.2165 (calculated M 480.2169). These
3
.5 Analysis of thiophenol in real samples by the probe NOF
results demonstrate that the probe NOF senses thiophenol by
first reacting with it and then generating NOF-OH.
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The ability of the probe NOF to detect thiophenol in real water time and in vivo, thus may be used to further inVvieewstAirgtiaclteeOntlhinee
samples, including tap water and mineral water, was toxicological effects of thiophenol. NoneDthOeI:le10ss.1,0t3h9e/Ds0eNaJr0e33o7n0lGy
investigated. All water samples’ pH value was adjusted to 7.4 the preliminary results showing the applicability of the probe
using PBS buffer (10 mM) containing 1 mM CTAB. The water NOF; the detection of thiophenol by the probe NOF in other
samples were spiked with different concentrations of animals or tissues should be further carried out in future
thiophenol (0, 5, 10, and 15 μM) before adding the probe NOF; studies.
after that, the fluorescence intensity of NOF in the water
samples was measured. As shown in Table 1, the recovery of
thiophenol in tap water and mineral water were good with
values ranging from 91.0% to 101.0%. These results indicate
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that the probe NOF can be used as a reliable tool for analysis
of thiophenol in real water samples.
Table 1. Determination of thiophenol concentrations in real water samples.
Sample
Thiophenol
spiked (μM)
0.0
Thiophenol
recovered (μM)
Not detected
4.61±0.37
Recovery (%)
Tap water
－
92.2
99.5
98.1
－
5
.0
Fig. 6 Confocal luminescence images of living MCF-7 cells. MCF-7 cells were
incubated with (a) PhSH (50 μM) for 1 h and then incubated NOF (5 μM); (b)
MCF-7 cells were incubated NOF (5 μM). Emission was collected by a NIR channel
at 650−750 nm, under excitation with 543 nm laser, scale bar = 20 μm.
1
1
0.0
5.0
9.95±0.23
14.72±0.32
Not detected
4.55±0.42
10.10±0.33
15.12±0.21
Mineral water
0.0
.0
5
91.0
101.0
100.8
1
1
0.0
5.0
3.6 Cytotoxicity and imaging of thiophenol in cells
We first determined the cytotoxicity of the probe NOF by MTT
assay. The results showed that viability of cells incubated with
the probe NOF at various concentrations (5-20 μM) for 24 h
was high (> 90% cell viability at 10 μM; Fig. S8, ESI†), indicating
that the probe NOF has low cytotoxicity. This finding indicates Fig. 7 NIR luminescence images of living mice. Mouse was subcutaneously injected in
that the probe NOF is biocompatible, thus is suitable for bio-
abdomen with 50 μL normal saline + NOF (20 μM, 50 μL) (green circle, left groin) and
PhSH (50 μL, 50 μM) + NOF (20 μM, 50 μL) (red circle, right groin), the images was
captured after 20 min. Emissions were collected at > 650 nm as detection signals, upon
imaging.
We then examined the ability of the probe NOF to detect
thiophenols in living cells (MCF-7 cells) by laser confocal
fluorescence microscopy. As illustrated in Fig. 6, the cells
incubated with thiophenol (50 μM) and the probe NOF (10 μM) 4. Conclusions
irradiation at 635 nm laser.
exhibited a significant fluorescence enhancement. By contrast,
In summary, we successfully designed and synthesized an ‘off-
the cells incubated only with the probe NOF exhibited a weak
on’ NIR fluorescent probe (NOF) for detection of thiophenol in
fluorescent signal. This result indicates that the probe NOF can
vitro and in vivo. The probe had various remarkable
detect intracellular thiophenol.
advantages including high sensitivity, high selectivity and fast
response towards thiophenol. It could recover thiophenol in
3
.7 NIR imaging of PhSH in living animal
In addition to its biocompatibility and high selectivity towards real water samples with high recovery rates of 91.0%–101.0%.
thiophenol, another unique characteristic of the probe NOF is In addition, the probe NOF had low toxicity and good cell
its emission wavelength in the NIR region. Therefore, we permeability, thus was successfully employed to image
carried out NIR imaging of thiophenol using the probe NOF in intracellular thiophenol. Finally, the probe was successfully
living animals, using mice as an animal model. Mice were applied in the fluorescence imaging of thiophenol in living
subcutaneously injected with the probe NOF, followed by mice. In future studies, the developed probe NOF ought to be
thiophenol. Thiophenol was injected into the right groin of applied to detect and image thiophenols in other
mice in the experimental group (red circle), but saline was environmental and biological samples.
injected into the left groin of mice in the control group (green
circle). Fluorescence images of mice (Fig. 7) showed that the
fluorescence signal of mice in the experimental group was Conflicts of interest
significantly higher than that of mice in the control group. This
There are no conflicts to declare.
indicates that the probe NOF can detect thiophenol in real
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Acknowledgements
2
, 599-605.
DOI: 10.1039/D0NJ03370G
This work was financially supported by the National Natural 28 Y. Geng, H. Tian, L. Yang, X. Liu and X. Song, Sens. Actuators B
Chem., 2018, 273, 1670-1675.
Science Foundation of China (21967001), the Natural Science
Foundation of Jiangxi Province (20192BAB213004), the open
research fund of Jiangsu Key Laboratory for Biosensors
29 F. Wu, H. Wang, J. Xu, H.-Q. Yuan, L. Zeng and G.-M. Bao, Sens.
Actuators B Chem., 2018, 254, 21-29.
0 S. Zhou, Y. Rong, H. Wang, X. Liu, L. Wei and X. Song, Sens.
3
(JKLB201909), and the Training Plan for Innovation and
Actuators B Chem., 2018, 276, 136-141.
Enterprise of Gannan Normal University (201910418004).
31 Y. Li, W. Su, Z. Zhou, Z. Huang, C. Wu, P. Yin, H. Li and Y.
Zhang, Talanta, 2019, 199, 355-360.
32 Q. Liu, A. Li, X. Li, B. Li, Y. Zhang, J. Li and Y. Guo, Sens.
Actuators B Chem., 2019, 283, 820-830.
3 T. Xu, S. Zhao, X. Wu, L. Zeng and M. Lan, ACS Sustain. Chem.
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DOI: 10.1039/D0NJ03370G
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A novel near-infrared xanthene-based fluorescent probe for detection of thiophenol in
living cells and mice.