A “turn-on” near-infrared fluorescent probe with high sensitivity for detecting reduced glutathione based on red shift in vitro and in vivo

Article abstract of DOI:10.1016/j.dyepig.2019.107837

Fluorescence imaging has become a powerful tool for detecting reduced glutathione (GSH) to comprehend the physiological and pathological roles of GSH and the potential clinical diagnosis of GSH-related diseases, such as AIDS, liver damage, cancer, and leu

Full text of DOI:10.1016/j.dyepig.2019.107837

Journal Pre-proof
A “turn-on” near-infrared fluorescent probe with high sensitivity for detecting reduced
glutathione based on red shift in vitro and in vivo
Kaiping Wang, Gang Nie, Siqi Ran, Huiling Wang, Xiqiu Liu, Ziming Zheng, Yu Zhang
PII:
S0143-7208(19)31643-2
DOI:
https://doi.org/10.1016/j.dyepig.2019.107837
DYPI 107837
Reference:
To appear in: Dyes and Pigments
Received Date: 16 July 2019
Revised Date: 24 August 2019
Accepted Date: 24 August 2019
Please cite this article as: Wang K, Nie G, Ran S, Wang H, Liu X, Zheng Z, Zhang Y, A “turn-on” near-
infrared fluorescent probe with high sensitivity for detecting reduced glutathione based on red shift in
vitro and in vivo, Dyes and Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.107837.
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In this manuscript, a “turn-on” near-infrared fluorescent probe was developed
and applied to monitor glutathione (GSH) with excellent sensitivity and selectivity in
vitro. The novel probe HCG was successfully applied to detect endogenous and
exogenous GSH in three living cells and the bio-distribution of GSH in BALB/c mice.

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A “turn-on” near-infrared fluorescent probe with high sensitivity for detecting
reduced glutathione based on red shift in vitro and in vivo.
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Kaiping Wanga, Gang Niea, Siqi Rana, Huiling Wangc, Xiqiu Liua, Ziming Zhengb and
Yu Zhangb,*
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aHubei Key Laboratory of Nature Medicinal Chemistry and Resource Evaluation,
Tongji Medical College of Pharmacy, Huazhong University of Science and
Technology, Wuhan, China
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bUnion Hospital of Tongji Medical College, Huazhong University of Science and
Technology, Wuhan, China
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cKey Laboratory of Pesticide and Chemical Biology, Ministry of Education, Chemical
Biology Center, College of Chemistry, and International Joint Research Center for
Intelligent Biosensing Technology and Health, Central China Normal University,
Wuhan, China
*Corresponding author: Dr. and Prof. Yu Zhang, Email: zhangwkp@163.com
Abstract
Fluorescence imaging has become a powerful tool for detecting reduced
glutathione (GSH) to comprehend the physiological and pathological roles of GSH
and the potential clinical diagnosis of GSH-related diseases, such as AIDS, liver
damage, cancer, and leucocyte loss. High sensitivity and high selectivity remain
challenges for near-infrared fluorescent probes to monitor GSH. Herein, a turn-on
near-infrared fluorescent probe (HCG) was designed and developed in a convenient
synthetic procedure, which had high sensitivity and selectivity to detect GSH based on
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the red shift of Schiff base. HCG could discriminate against amino acid that resemble
GSH to monitor GSH in vitro. The fluorescence emission intensity linearly increased
with an increasing concentration of GSH in the range of 0-16 µM, with a limit
detection of 252 nM. HCG exhibited a diminutive detection limit (0.5 µM in actual
experiments), a fast response time (30 s) and low cytotoxicity. The detection
mechanism was confirmed by HPLC and HRMS spectra. Furthermore, HCG
exhibited an excellent capacity for fluorescence imaging and has been successfully
applied to detect endogenous GSH in three living cells. Finally, the bio-distribution of
GSH in BALB/c mice was studied by using HCG. The results suggest that HCG has
potential utility in biological science research.
Keywords: Near-infrared fluorescent probe; Reduced glutathione detection; High
sensitivity and selectivity; Schiff base’s red shift; Fluorescence imaging; Facile
synthetic procedures.
1. Introduction
Intracellular biothiols, including reduced glutathione (GSH), cysteine (Cys), and
homocysteione (Hcy), act as significant roles in many biological processes [1-3]. It is
deserved to be mentioned that GSH is widely distributed in living organisms and can
serve as a biomarker in the biological system among intracellular bio-thiols [4]. As
reported in literatures [5-8], GSH can contribute to an improvement in immunity and
anti-aging among physiological processes and GSH is a great scavenger for reactive
oxygen to maintain the appropriate physiological redox state . However, aberrant GSH
levels have been associated with a variety of diseases, such as AIDS, liver damage,
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cancer, and leucocyte loss [9-11]. Based on these disease features, traditional
analytical method has been developed for detecting GSH by high-performance liquid
chromatography and mass spectrometry [5]. However, it is difficult to prepare
biological samples and monitor GSH in real time. Thus, many new strategies have
been gradually used to monitor GSH in biological models [12-15]. Among these
strategies, fluorescence imaging has been paid great attention for the detection of
GSH in biological systems [16-18]. Different kinds of fluorescent probes have been
developed for detecting GSH on the basis of different reaction mechanisms, such as
Michael addition [19, 20], nucleophilic substitution [21], cyclization with an aldehyde
[22], Se-N cleavage by thiols [23], the elimination of disulfide [24] and others [25-27].
In the reported fluorescent probes for the detection of GSH, there are strong
interference from Cys and Hcy, due to their similar molecular structures and
nucleophilicity. So, it is still a challenge to develop a highly specific fluorescent probe
for detecting GSH. [28-31].
In recent years, near-infrared fluorescent probes have increasingly been applied
to detect GSH, due to the reduced biological damage and deeper tissue penetration of
the light in this wavelength region [21, 28, 31-34]. Near-infrared fluorescent probes
contain low photon absorption and can provide high-resolution fluorescence intensity
to produce effective fluorescent signals in vitro and in vivo without fluorescent
interference. Compared with non-near-infrared fluorescent probes, near-infrared
fluorescent probes have greater sensitivity and more extensive applications [35].
Taking advantage of these near-infrared fluorescent probes, several turn-on and
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ratiometric near-infrared fluorescent probes have been developed for detecting GSH
[32-34]. The detection moieties of these near-infrared fluorescent probes still utilize
the nucleophilicity of GSH to lead to low selectivity for discriminating against amino
acids that resemble GSH. Moreover, these probes exhibited slow response time and
their synthetic routes were complicated. Based on the shortcomings of the existing
fluorescent probes, it is still of great importance to develop a more sensitive and
selective near-infrared probe to detect intracellular GSH through a convenient
synthetic procedure.
As reported in the literature [36-41], non-N-alkylated cyanine dyes include a
protonatable amino group with an indole group. When the indole nitrogen atom
deprotonates, it absorbs in the short wavelength region. The dye will have a strong red
fluorescence when the indole nitrogen atom has a proton. This change occurs over a
very short time, which means that a more sensitive probe with an indole nitrogen atom
moiety may be designed to detect GSH, and suggests an excellent spectroscopic
window to monitor the turning off or on of a visual optical signal (Scheme 1). This
strategy offers a new way to design near-infrared fluorescent probes. Encouraged by
this mechanism, we hope to develop a highly sensitive and selective near-infrared
fluorescent probe with a similar structure to detect GSH.
Herein, making use of this shift strategy, we report the turn-on hemi-cyanine
fluorescent probe HCG with excellent sensitivity and selectivity based on a Schiff
base redshift reaction for the detection of GSH in vitro. Moreover, HCG was
successfully applied to three living cells and the BALB/c mice to detect GSH by
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fluorescence imaging. The fluorescent images showed that using HCG to detect GSH
is helpful to understand the possible biogenesis approaches of endogenous GSH in
vivo. The results indicated that HCG was an effective tool for monitoring GSH in
bioscience research.
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Scheme 1. Schiff base redshift
2. Experimental
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2.1 Reagents and apparatus
All commercial chemicals were of analytical reagent grade and used without
further purification. All reactions were monitored by TLC and the TLC plate was
detected by ultraviolet light (254 nm or 365 nm). Silica gel column chromatography
was used to purify the compound. A reduced glutathione (GSH) assay kit was
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obtained from Jiancheng Bioengineering Institute (Nanjing, China). H-NMR and
13C-NMR spectra were acquired on a Bruker NMR 400MHz spectrometer. The high
resolution mass spectra of all compounds were recorded by FT-MS (Bruker Daltonics
SolariX 7.0T). The UV spectra were obtained with UV/VIS (Jena，Specord 210)
spectrophotometer in 1 cm quartz cells. Fluorescence excitation and emission spectra
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were measured on a Hitachi F-4600 fluorescence spectrophotometer. Fluorescent
images of cells were obtained with a confocal laser scanning microscope (Nikon A1).
Fluorescent images of BALB/c were recorded by a small-animal in vivo imaging
system (IVIS).
2.2 Synthesis of HCG
The synthesis procedures of the other compounds are shown in Scheme S1. Cy7
(300 mg, 1.037 mmol) and 2-nitrobenzene-1,4-diamine (190 mg, 1.244 mmol) were
dissolved in 5 mL of anhydrous DMSO. Then, N,N-diisopropylethylamine (100 µL)
o
was added to the reaction mixture. The reaction was kept at 60 C for 24 h under
nitrogen atmosphere. Next, the solvent was removed under reduced pressure to obtain
the crude product, which was purified by column chromatography on silica gel with a
gradient of ethyl acetate ; petroleum ether from 10:1 to 3:1 (v/v), and the black solid
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HCG was finally obtained (153.3 mg, 32%). H-NMR (400 MHz, CDCl3) δ 8.91 (s,
1H), 8.02 (d, J = 2.4 Hz, 1H), 7.65 (d, J = 12.7 Hz, 1H), 7.41 (dd, J = 8.8, 2.4 Hz, 1H),
7.26 – 7.18 (m, 2H), 6.95 – 6.83 (m, 2H), 6.70 (d, J = 7.8 Hz, 1H), 6.13 (s, 2H), 5.47
(d, J = 12.7 Hz, 1H), 3.22 (s, 3H), 2.77 (t, J = 6.1 Hz, 2H), 2.67 – 2.60 (m, 2H), 1.91 –
1.83 (m, 2H), 1.69 (s, 6H). 13C-NMR (100 MHz, CDCl3) δ 160.8 (s), 158.5 (s), 144.8
(s), 142.8 (s), 142.8 (s), 141.9 (s), 139.1 (s), 132.2 (s), 130.8 (s), 128.9 (s), 127.8 (s),
124.5 (s), 121.7 (s), 120.3 (s), 119.3 (s), 117.1 (s), 106.3 (s), 92.9 (s), 46.1 (s), 29.3 (s),
28.3 (s), 26.7 (s), 26.6 (s), 21.4 (s). HRMS (ESI+): m/z found [M+H]+ 463.1889.
+
molecular formula C26H28ClN4O2 , requires [M+H]+ 463.1895.
2.3 Cell culture and fluorescent imaging
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MCF-7, HT-29 and HepG2 cells were incubated in DMEM supplemented with
10 % (v/v) FBS, penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37°C under
a humidified atmosphere containing 5 % CO2. For fluorescence imaging, cells were
pretreated with varying concentrations of GSH or other analytes in 12-well plates (1×
106 cells/well) at 37 for the appropriate time. Then, the cells were washed with
HEPES (10 mM, pH=7.4) and incubated with a mixture of fresh DMEM and an
administered the probe HCG (10 µM) at 37 ºC for 15 min. Fluorescence images were
acquired with a confocal laser scanning microscope. The fluorescence detection
setting were kept constant throughout all imaging experiments.
2.4 General flow cytometry methods
In general, for flow cytometry, the cells were incubated with different
concentrations of GSH, and GSH scavengers in 12-well plates (1× 106 cells/well) at
37 for a certain amount of time. Then, the cells were washed with HEPES (10 mM,
pH=7.4) and incubated with a mixture of fresh DMEM without fetal bovine serum
and administered HCG (10 µM) at 37º C for 15 min. Subsequently, the cells with
HEPES were analyzed by flow cytometry (BD, AccuriTM C6) equipped with 640 nm
excitation laser light line source.
2.5 Fluorescence imaging in BALB/c mice
BALB/c mice were divided into two groups. The control group mice were
pre-injected with saline for 5 min and GSH group mice were pre-injected with
concentration of GSH (1 mM) for 5 min. Next, all mice were injected with HCG (10
µM, 100 µL in 1:99 DMSO/saline, v/v). The probe was incubated in BALB/c mice for
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30 min and the fluorescent images of the BALB/c mice were obtained by a
small-animal in vivo imaging system (IVIS).
2.6 High-performance liquid chromatography (HPLC) analysis
The HPLC technique was employed in the reaction mechanism experiment.
HPLC was used to monitor the process by which the probe HCG (10 µM) reacted
with 500 µM GSH for 15 min. The retention time of each compound was confirmed
so that a better understanding of the reaction mechanism could be obtained.
3. Results and discussion
3.1 Design and synthesis of probe HCG
Because protonatable amino groups can undergo a red shift, we hoped to design
a similar structure. The Schiff base also contains a protonatable nitrogen atom and can
produce a red emission optical signal. Meanwhile, the Schiff base moiety can be
synthesized conveniently. Thus, for the purpose of constructing a turn on fluorescent
probe,
1,2,3,3-tetramethyl-3H-indole
and
(E)-2-chloro-cyclohex-1-ene-1-carbaldehyde were synthesized by known conditions
initially. Next, compound S1 reacted with compound S2 under aldol condensation
conditions to give Cyanine 7. The probe HCG was obtained in one step by the
combination compound Cy7 and 2-nitrobenzene-1,4-diamine [42, 43]. HCG contains
a Schiff base structure, which is the core of the probe and can serve as the recognition
group for GSH. Moreover, the withdrawing group of probe HCG is beneficial to
produce a large red shift emission in the fluorescence spectrum signal. Compared with
traditional synthetic approaches, this procedure was more convenient. (Scheme 2).
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Scheme 2. The synthetic procedure of probe HCG
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3.2 Spectroscopic properties and sensitivity of the probe HCG
Our studies began by examining the UV/Vis absorption and fluorescence spectra
of HCG and 10 µM HCG reacted with 500 µM GSH. The results indicated that the
maximum absorption wavelength of HCG was located at 450 nm. Interestingly, after
reacting with GSH, its maximum absorption wavelength was observed in the 653 nm
region (Fig 1a). In the fluorescence spectra, there is a weak fluorescence emission
from HCG at 720 nm. After 500 µM GSH was added followed by incubation for 1
min, the fluorescence intensity increased 8-fold at 720 nm (Fig 1b). This result
indicated that HCG is a turn on near-infrared fluorescent probe and has a short
detection time. The fluorescent quantum yield of HCG reacted with GSH was 17%
relative to Oxazine 1 (Фoxz=0.14 in EtOH) [44]. To test the sensitivity of HCG, the
fluorescence spectra of HCG (10 µM) reacted with varying concentrations of GSH
(0-1000 µM) were recorded by fluorescence spectrophotometry. The results showed
that the fluorescence intensity increased and reached a gradual plateau with increasing
concentration of GSH (Fig 1c and S1c). Moreover, it was found that the fluorescence
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emission intensity linearly increased with the concentration of GSH in the range of
0-16 µM and the detection limit (DL) of HCG was calculated to be 252 nM
(signal-to-noise ratio (S/N) = 3). In the real detection experiment, the DL of HCG was
0.5 µM (Fig 1d). There is a reduced glutathione (GSH) assay kit from Jiancheng
Bioengineering Institute (Nanjing, China), whose DL is 451 nM and the real detection
limit is 1.5 µM (Fig S1). Compared with this commercial kit, HCG has higher
sensitivity.
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Fig 1. The UV spectra and fluorescence emission spectra of HCG in response to GSH. a)
Black: Absorbance spectrum of HCG (10 µM) in DMSO/HEPES buffer (10 mM, 1:10, v/v, pH 7.4)
at 25 °C, Red: Absorbance spectrum of 10 µM HCG reacted with 500 µM GSH in DMSO/HEPES
buffer (10 mM, 1:10, v/v, pH 7.4) at 25 °C for 15 min; b) Fluorescence emission spectra of HCG,
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and 10 µM HCG with 500 µM GSH in DMSO/HEPES buffer (10 mM, 1:10, v/v, pH 7.4) at
37 °C for 1 min; c) Fluorescence emission spectra of HCG (10 µM) with various concentrations of
GSH (0, 10, 20, 40, 60, 80, 100, 200, 500, 1000 µM) at 37 for 15 min; d) Linear correlation of
fluorescence emission intensity towards GSH concentration and the real experimental detection
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limit. (λex = 653 nm).
3.3 Selectivity of the probe HCG
To examine the selectivity of HCG, different amino acids and other species were
introduced to HCG, such as Cys, GSH, Ala, Pro, Lys, Thr, His, Val, Tyr, Ser, Phe, Arg,
Trp, Met, Leu, Asp, Gly, Ile, Gln, Hcy and others. As shown in Fig 2a, only GSH can
be monitored by HCG among all the amino acids tested. It is worth mentioning that
the fluorescence intensity of the reaction of HCG with Cys, Hcy and GSH showed a
large difference, indicating that there is good anti-interference and high selectivity for
HCG to react with GSH compared with similar amino acids. In addition, the
time-dependence was measured to explore the time required for HCG to respond to
GSH. Excitingly, when GSH was added into the HCG buffer solution, the
fluorescence intensity increased immediately and achieved a balance within 30 s (Fig
2b), suggesting that HCG has a great response to GSH and exhibits excellent
sensitivity. A possible interpretation is that the Schiff base redshift emission leads to a
rapid response to GSH.
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Fig 2. The selectivity and kinetics of the HCG response to GSH. a) Fluorescence responses of
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10 µM HCG to 500 µM of biologically relevant species in DMSO/HEPES buffer (10 mM, 1:10,
v/v, pH 7.4) at 37 °C for 15 min: (1) probe, (2) GSH, (3) Cys, (4) Ala, (5) Pro, (6) Lys, (7) Thr, (8)
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His, (9) Val, (10) Tyr, (11) Ser, (12) Phe, (13) Arg, (14) Cystine, (15) Trp, (16) Met, (17) Leu, (18)
-
2-
Asp, (19) Gly, (20) Ile, (21) Gln, (22) HSO3 , (23) SO3 , (24) H2O2, (25) Hcy; b) Kinetics of 500
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µM GSH detected by 10 µM HCG at 37°C in HEPES (pH 7.4) (λex = 653 nm).
3.4 Flow cytometry analysis of GSH and confocal microscopy images in living cells
using HCG
Based on the good fluorescent properties of HCG in buffer, HCG was applied to
detect GSH in living cells. The cytotoxicity of HCG was established firstly, using
MTT assays with MCF-7, HT-29 and HepG2 cells. The cell viabilities exceeded 92%
when incubated with 10 µM HCG. In the range of 0-100 µM HCG, the cell viabilities
were still over 82%, demonstrating the low cytotoxicity of the probe HCG in living
cells (Fig S2). Furthermore, the fluorescence intensity of HCG was weak and stable in
the range of pH 5-10. When added GSH into the solution of HCG, the fluorescence
intensity of HCG response to GSH had a significant improvement in the range of pH
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5-10 (Fig S3). As shown in Fig S2 and S3, the results above demonstrated that HCG
was stable and suitable for cell imaging under biological conditions. On the basis of
these results, flow cytometry was used to analyze of the fluorescence intensity of
HCG in response to GSH in MCF-7, HT-29 and HepG2 cells. HT-29 cells were
pretreated with different concentrations of GSH (0, 200, 500, 1000 µM) for 30 min,
followed by, incubation with 10 µM HCG for 15 min and analysis by flow cytometry.
As shown in Fig 3a, the fluorescence intensity of the HCG response to GSH improved
with increasing GSH concentration. N-ethylmaleimide (NEM, 2 mM), a well-known
scavenger of biothiols [45, 46], was pretreated with HT-29 cells for 30 min, and then
subsequently, incubated with 10 µM HCG for 15 min and analyzed by flow cytometry.
The flow cytometry data showed that the fluorescence intensity of HT-29 cells
incubated with NEM decreased to half that of the control group (Fig 3b). The flow
cytometry results in MCF-7 cells and HepG2 cells were analogous to those in HT-29
cells (Fig S4). Encouraged by the results above, confocal microscopy imaging was
applied to HT-29, MCF-7 and HepG2 cells. HT-29 cells were incubated with different
concentrations of GSH (0, 200, 500, 1000 µM) for 30 min. Next, the cells were
treated with 10 µM HCG for additional 15 min, and fluorescent images were acquired
by confocal microscopy. As shown in Fig 3c, the fluorescence intensity of the HCG
response to GSH gradually increased in a GSH concentration dependent manner.
When the control group only was incubated with 10 µM HCG, it was obvious that a
red fluorescence was observed in HT-29 cells. This result indicated that HCG could
detect endogenous GSH in living cells. When 2mM NEM was introduced in HT-29
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cells prior to, and all other procedures were consistent with the above mentioned
conditions, only weak red fluorescence was observed the confocal microscopy image
of HT-29 cells. These results were consistent with the flow cytometry results. Similar
results were achieved with MCF-7 cells and HepG2 cells (Fig S5). Therefore,
according to these results, it has been suggested that the probe HCG can be an
effective tool for detecting GSH in living cells.
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Fig 3. Flow cytometry analysis of GSH and confocal microscopy images of GSH in HT-29
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cells using HCG. a) Cells were pretreated with GSH (0, 0.2, 0.5, 1.0 mM) at 37 for 30 min and
then stained with 10 µM HCG for 15 min before flow cytometry analysis; b) Normalized
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fluorescence intensity of flow cytometry data; c) Cells were pretreated with varying
concentrations of GSH or NEM at 37 for 30 min, and then treated with 10 µM HCG for 15 min
before obtaining the confocal microscopy images. Scale bar: 20 µm. Representative images from
replicate experiments (n = 5) are shown.
3.5 The plausible mechanism
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To gain insights into the mechanism of the HCG fluorescent response to GSH,
HCG (10 µM) was reacted with GSH (500 µM) in 5 mL of methanol. The reaction
mixture was measured by high-performance liquid chromatography (HPLC). The
retention time of the HCG, GSH and the reaction product were recorded. As it is
shown in Fig 4, the retention time of the HCG was 8.0 min (Fig 4a), and the retention
time of GSH was 2.5 min (Fig 4b). However, the reaction product has a strong signal
at 6.0 min (Fig 4c). Additionally, the reaction mixture was surveyed with a
high-resolution liquid chromatograph mass spectrometer (HR-LC-MS). In positive ion
mode of the HRMS analysis, the thiol-imine Michael adduct was found to have a m/z
of 770.27025 (Fig S6) and HCGS was found to have a m/z of 463.19006 in the mass
spectra. However, in negative ion mode of the HRMS analysis, the HCGS wasn’t
found in mass spectra and the probe HCG could be found to have a m/z of 461.17547
in the mass spectra. This indicated that HCG reacted with GSH to form a thiol-imine
adduct and then the thiol-imine adduct decomposed into a protonatable Schiff base,
which couldn’t be measured in negative ion mode on the mass spectrometer (Fig S6
and Fig 5). The plausible mechanism was speculated that firstly, the non-red
fluorescent HCG reacted with GSH to gain a thiol-imine adduct [47]. Because the
thiol-imine Michael addition were unstable, the thiol-imine adduct was easily
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decomposed into a protonatable Schiff base of HCG. The protonatable Schiff base of
HCG was generated in the reaction mixture and led to a red shift in the optical signal
(Scheme 3).
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Fig 4. The reaction between 500 µM GSH and 10 µM HCG was tracked by HPLC. a) the
retention time of HCG; b) the retention time of the GSH; c) the retention time of the reaction
product.
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Fig 5. High resolution mass spectrum of 10 µM HCG reacted with 500 µM GSH. a) the
positive ion mode of reaction product; b) the negative ion mode of probe HCG; c) the negative ion
mode of the reaction product.
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Scheme 3. Proposed detection mechanism of probe HCG for GSH.
3.6 The application of HCG in vivo
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To investigate the application of HCG to respond to GSH in vivo, the BALB/c
mice were pretreated with GSH (1 mM) and saline for 5 min, and then injected with
HCG (10 µM, 100 µL in 1:99 DMSO/saline, v/v). After 30 min, fluorescence images
were obtained by a small-animal in vivo imaging system (IVIS). There was a
fluorescence signal in the control group. GSH was introduced into the mice ahead of
time, and the fluorescence signal was greatly improved in the liver of the mice (Fig 6),
indicating that the probe HCG can detect endogenous GSH by the blood circulation in
mice. Because GSH is gathered and metabolized first in the liver of the mice by the
blood circulation [48-50], the probe HCG enters the liver and detects GSH to lead to a
fluorescence signal in vivo. In GSH group mice, the liver had a more obvious
fluorescence signal than the other organs, because the liver contains abundant GSH.
In this regard, HCG is an effective probe to detect changes in the concentrations of
GSH in vivo and can provide a visual signal from IVIS.
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Fig 6. Fluorescence imaging of BALB/c mice. a) HCG was applied to a control group and a GSH
group with IVIS; b) Relative fluorescence intensity of the control group and GSH group; c) Ex
vivo fluorescence imaging: the separated organs from the sacrificed GSH group mice; d) Relative
fluorescence intensity of the separated organs from the GSH group.
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4. Conclusions
In summary, the near-infrared fluorescent probe HCG was designed and
developed successfully based on the Schiff base red shift for the detection of GSH.
HCG shows high selectivity to discriminate between GSH and similar amino acids,
shows a fast response time (30 s) and has a low actual detection limit of 0.5 µM in
vitro. Due to the low cytotoxicity and great near-infrared properties, HCG was
successfully applied to the fluorescence imaging of endogenous GSH in HT-29,
MCF-7 and HepG2 cells. Last, we studied the bio-distribution of GSH in BALB/c
mice through fluorescence imaging of HCG. With facile preparations, this strategy
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can provide an effective tool to detect GSH levels. The results suggest that HCG has
potential utility in biological science research.
Acknowledgements
We appreciate the staff from the Analysis and Testing Center of Huazhong
University of Science and Technology for their technical assistance. The research was
supported by grants from the National Natural Science Foundation of China (No.
81373300) and Hubei Provincial Natural Science Foundation of China.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://xxxxxxxxxxxxx.
References
[1] Sedgwick AC, Han HH, Gardiner JE, Bull SD, He XP, James TD. The
development of a novel AND logic based fluorescence probe for the detection of
peroxynitrite and GSH. Chem Sci 2018; 9: 3672-6.
[2] Chen X, Zhou Y, Peng X, Yoon J. Fluorescent and colorimetric probes for
detection of thiols. Chem Soc Rev 2010; 39: 2120-35.
[3] Weerapana E, Wang C, Simon GM, Richter F, Khare S, Dillon MB, Bachovchin
DA, Mowen K, Baker D, Cravatt BF. Quantitative reactivity profiling predicts
functional cysteines in proteomes. Nature 2010; 468: 790-5.
[4] Townsend DM, Tew KD, Tapiero H. The importance of glutathione in human
disease. Biomed Pharmacother 2003; 57: 145-55.
19

358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
[5] Saito C, Zwingmann C, Jaeschke H. Novel mechanisms of protection against
acetaminophen hepatotoxicity in mice by glutathione and N-acetylcysteine.
Hepatology 2010; 51: 246-54.
[6] Homma T, Fujii J. Application of glutathione as anti-oxidative and anti-aging
drugs. Curr Drug Metab 2015; 16: 560-71.
[7] Tapiero H, Townsend DM, Tew KD. The antioxidant role of selenium and
seleno-compounds. Biomed Pharmacother 2003; 57: 134-44.
[8] Enami S, Hoffmann MR, Colussi AJ. OH-radical specific addition to glutathione
S-atom at the air–water interface: relevance to the redox balance of the lung
epithelial lining fluid. J Phys Chem Lett 2015; 6: 3935-43.
[9] Pittet MJ, Weissleder R. Intravital imaging. Cell 2011; 147: 983-91.
[10]James ML, Gambhir SS. A molecular imaging primer: modalities, imaging agents,
and applications. Physiol Rev 2012; 92: 897-965.
[11]Yin C, Huo F, Zhang J, Martínez-Máñez R, Yang Y, Lv H, Li S. Thiol-addition
reactions and their applications in thiol recognition. Chem Soc Rev 2013; 42:
6032-59.
[12]Niu LY, Chen YZ, Zheng HR, Wu LZ, Tung CH, Yang QZ. Design strategies of
fluorescent probes for selective detection among biothiols. Chem Soc Rev 2015;
44: 6143-60.
[13]Huang Y, Zhou J, Feng H, Zheng J, Ma HM, Liu W, Tang C, Ao H, Zhao M, Qian
Z. A dual-channel fluorescent chemosensor for discriminative detection of
glutathione based on functionalized carbon quantum dots. Biosens Bioelectron
20

380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
2016; 86: 748-55.
[14]Li Y, Liu W, Zhang P, Zhang H, Wu J, Ge J, Wang P. A fluorescent probe for the
efficient discrimination of Cys, Hcy and GSH based on different cascade
reactions. Biosens Bioelectron 2017; 90: 117-24.
[15]Fu ZH, Han X, Shao Y, Fang J, Zhang ZH, Wang YW, Peng Y. Fluorescein-based
chromogenic and ratiometric fluorescence probe for highly selective detection of
cysteine and its application in bioimaging. Anal Chem 2017; 89: 1937-44.
[16]Jung HS, Chen X, Kim JS, Yoon J. Recent progress in luminescent and
colorimetric chemosensors for detection of thiols. Chem Soc Rev 2013; 42:
6019-31.
[17]Lu J, Song Y, Shi W, Li X, Ma H. A long-wavelength fluorescent probe for
imaging reduced glutathione in live cells. Sens Actuators B Chem. 2012; 161:
615-20.
[18]Lee S, Li J, Zhou X, Yin J, Yoon J. Recent progress on the development of
glutathione (GSH) selective fluorescent and colorimetric probes. Coord Chem
Rev 2018; 366: 29-68.
[19]Umezawa K, Yoshida M, Kamiya M, Yamasoba T, Urano Y. Rational design of
reversible fluorescent probes for live-cell imaging and quantification of fast
glutathione dynamics. Nat Chem 2017; 9: 279-86.
[20]Yi L, Li H, Sun L, Liu L, Zhang C, Xi Z. A highly sensitive fluorescence probe
for fast thiol quantification assay of glutathione reductase. Angew Chem Int Ed
2009; 121: 4094-97.
21

402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
[21]Yin J, Kwon Y, Kim D, Lee D, Kim G, Hu Y, Ryu JH, Yoon J. Cyanine-based
fluorescent probe for highly selective detection of glutathione in cell cultures and
live mouse tissues. J Am Chem Soc 2014; 136: 5351-58.
[22]Chen H, Zhao Q, Wu Y, Li F, Yang H, Yi T, Huang C. Selective phosphorescence
chemosensor for homocysteine based on an Iridium(III) complex. Inorg Chem
2007; 46: 11075-81.
[23]Tang B, Xing Y, Li P, Zhang N, Yu F, Yang G. A rhodamine-based fluorescent
probe containing a Se-N bond for detecting thiols and its application in living
cells. J Am Chem Soc 2007; 129: 11666-7.
[24]Lim CS, Masanta G, Kim HJ, Han JH, Kim HM, Cho BR. Ratiometric detection
of mitochondrial thiols with a two-photon fluorescent probe. J Am Chem Soc
2011; 133: 11132-5.
[25]Ji S, Yang J, Yang Q, Liu S, Chen M, Zhao J. Tuning the intramolecular charge
transfer of alkynylpyrenes: effect on photophysical properties and its application
in design of OFF−ON fluorescent thiol probes. J Org Chem 2009; 74: 4855-65.
[26]Han B, Yuan J, Wang E. Sensitive and selective sensor for biothiols in the cell
based on the recovered fluorescence of the CdTe quantum Dots−Hg(II) system.
Anal Chem 2009; 81: 5569-73.
[27]Xia Y, Zhang H, Zhu X, Fang M, Yang M, Zhang Q, Li X, Zhou H, Yang X, Tian
Y. Fluorescent probes for detecting glutathione: Bio-imaging and two reaction
mechanisms. Dyes Pigments 2019; 163: 441-6.
[28]Lim SY, Hong KH, Kim DI, Kwon H, Kim HJ. Tunable heptamethine-azo dye
22

424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
conjugate as an NIR fluorescent probe for the selective detection of mitochondrial
glutathione over cysteine and homocysteine. J Am Chem Soc 2014; 136:
7018-25.
[29]Kim Y, Mulay S, Choi M, Yu SB, Jon S, Churchill D. Exceptional time response,
stability and selectivity in doubly-activated phenyl selenium-based
glutathione-selective platform. Chem Sci 2015; 6: 5435-39.
[30]Zhang H, Liu R, Liu J, Li L, Wang P, Yao SQ, Xu Z, Sun H. A minimalist
fluorescent probe for differentiating Cys, Hcy and GSH in live cells. Chem Sci
2016; 7: 256-60.
[31]Mulay SV, Kim Y, Choi M, Lee DY, Choi J, Lee Y, Jon S, Churchill D. Enhanced
doubly activated dual emission fluorescent probes for selective imaging of
glutathione or cysteine in living systems. Anal Chem 2018; 90: 2648-54.
[32]Zhang J, Bao X, Zhou J, Peng F, Ren H, Dong X, Zhao W. A
mitochondria-targeted turn-on fluorescent probe for the detection of glutathione
in living cells. Biosens Bioelectron 2016; 85: 164-70.
[33]Gong D, Han S, Iqbal A, Qian J, Cao T, Liu W, Liu W, Qin W, Guo H. Fast and
selective two-stage ratiometric fluorescent probes for imaging of glutathione in
living cells. Anal Chem 2017; 89: 13112-9.
[34]Gong D, Ru J, Cao T, Qian J, Liu W, Iqbal A, Liu W, Qin W, Guo H. Two-stage
ratiometric fluorescent responsive probe for rapid glutathione detection based on
BODIPY thiol-halogen nucleophilic mono- or disubstitution. Sens Actuators B
Chem 2018; 258: 72-9.
23

446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
[35]Mizukami S. Targetable fluorescent sensors for advanced cell function analysis. J
Photochem Photobiol C Photochem Rev 2017; 30: 24-35.
[36]Sun W, Guo S, Hu C, Fan J, Peng X. Recent Development of Chemosensors
Based on Cyanine Platforms. Chem Rev 2016; 116: 7768-817.
[37]Hoischen D, Steinmuller S, Gartner W, Buss V, Martin HD. Merocyanines as
Extremely Bathochromically Absorbing Chromophores in the Halobacterial
Membrane Protein Bacteriorhodopsin. Angew Chem Int Ed 1997; 36: 1630-3.
[38]Yapici I, Lee KSS, Berbasova T, Nosrati M, Jia X, Vasileiou C. “Turn-on” protein
fluorescence: in situ formation of cyanine dyes. J Am Chem Soc 2015; 137:
1073-80.
[39]Oshikawa Y, Ojida A. PET-dependent fluorescence sensing of enzyme reactions
using the large and tunable pKa shift of aliphatic amines. Chem Commun (Camb)
2013; 49: 11373-5.
[40]Puyol M, Encinas C, Rivera L, Miltsov S, Alonso J. Characterisation of new
norcyanine dyes and their application as pH chromoionophores in optical sensors.
Dyes Pigments 2007; 73: 383-9.
[41]Zhang S, Wang Q, Liu X, Zhang J, Yang XF, Li Z, Li H. Sensitive and selective
fluorescent probe for selenol in living cells designed via a pKa shift strategy. Anal
Chem 2018; 90: 4119-25.
[42]Zhang J, Wang J, Liu J, Ning L, Zhu X, Yu B, Liu X, Yao X, Zhang H.
Near-infrared and naked-eye fluorescence probe for direct and highly selective
detection of cysteine and its application in living cells. Anal Chem 2015; 87:
24

468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
4856-63.
[43]Rajasekaran K, Sarathi A, Ramalakshmi S. Micellar catalysis in the
retro-Knoevenagel reaction of ethyl-α-cyanocinnamates. J Chem Sci 2008; 120:
475-80.
[44]Rurack K, Spieles M, Fluorescence quantum yields of a series of red and
near-infrared dyes emitting at 600-1000 nm. Anal Chem 2011; 83: 1232-42.
[45]Becker PS, Cohen CM, Lux SE. The effect of mild diamide oxidation on the
structure and function of human erythrocyte spectrin. J Biol Chem 1986; 261:
4620-8.
[46]Yellaturu CR, Bhanoori M, Neeli I, Rao GN. N-Ethylmaleimide inhibits
platelet-derived growth factor BB-stimulated Akt phosphorylation via activation
of protein phosphatase 2A. J Biol Chem 2002; 277: 40148-55.
[47]Stacy GW, Day RI, Morath RJ. Schiff bases and related substances. II. reactions
of thiols with N-benzylideneaniline and N-benzylideneanthranilic acid. J Am
Chem Soc 1955; 77: 3869-73.
[48]Liang M, Zheng X, Tu L, Ma Z, Wang Z, Yan D, Shi Z. The liver-targeting study
of the N-galactosylated chitosan in vivo and in vitro, Artif Cells Nanomed
Biotechnol 2014; 42: 423-8.
[49]Triger DR. The liver as an immunological organ. Hepatology 1976; 71: 162-4.
[50]Zhang Y, Zheng Z, Yang X, Pan X, Yin L, Huang X, Li Q, Shu Y, Zhang Q, Wang
K. A sensitive and rapid radiolabelling method for the in vivo pharmacokinetic
study of lentinan. Food Funct 2018; 9: 3114-25.
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1
2
Highlights
ò A turn-on near-infrared fluorescent probe was developed for monitoring
reduced glutathione (GSH) by facile synthetic procedures
ò The probe can detect GSH based on the red shift of Schiff base, which had
high sensitivity, high selectivity, low detection limit and fast response time in
vitro.
3
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ò The probe served as bioimging tool for detecting endogenous and exogenous
GSH in three living cells.
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ò The bio-distribution of GSH in the BALB/c mice was studied through
fluorescent imaging of HCG
10