An ESIPT-based fluorescent probe for highly selective detection of glutathione in aqueous solution and living cells

Article abstract of DOI:10.1016/j.dyepig.2016.02.027

In this paper, highly sensitive and selective fluorescent probe 1 for GSH is designed and synthesized based on modulation of the excited-state intramolecular proton transfer (ESIPT) process of 2-(2′-hydroxy-3′-ethoxyphenyl)benzothiazole. Upon introducing

Full text of DOI:10.1016/j.dyepig.2016.02.027

Dyes and Pigments 129 (2016) 156e162
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
An ESIPT-based fluorescent probe for highly selective detection of
glutathione in aqueous solution and living cells
Xintong Ren, Fang Wang, Jing Lv, Tingwen Wei, Wei Zhang, Yong Wang, Xiaoqiang Chen*
State Key Laboratory of Materials-Oriented Chemical Engineering College of Chemistry and Chemical Engineering, Nanjing Tech University, Nanjing, 210009,
PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper, highly sensitive and selective fluorescent probe 1 for GSH is designed and synthesized
based on modulation of the excited-state intramolecular proton transfer (ESIPT) process of 2-(20-hy-
droxy-30-ethoxyphenyl)benzothiazole. Upon introducing GSH in neutral solution containing CTAB mi-
celles, dinitrophenyl, the protecting group of the probe, is removed via the nucleophilic substitution,
thereby retrieving the ESIPT process of 2-(20-hydroxy-30-ethoxyphenyl)benzothiazole, which results in a
fluorescence enhancement at 485 nm. Additionally, the fluorescence intensity of the probe is linearly
Received 19 January 2016
Received in revised form
21 February 2016
Accepted 25 February 2016
Available online 3 March 2016
proportional to GSH concentration ranging from 0 to 100
mM and the obtained detection limit is as low as
Keywords:
0.81 M. The enhanced selectivity toward GSH over Cys and Hcy is also attributed to one more carboxyl
m
Fluorescent probe
Glutathione
ESIPT
group in GSH. Importantly, the probe is successfully utilized for monitoring GSH in living HeLa cells.
© 2016 Elsevier Ltd. All rights reserved.
Benzothiazole
Dinitrophenyl
1. Introduction
the reactivity, the target for distinguishing GSH from Cys and Hcy is
still arduous to achieve. Nowadays, a number of excellent fluores-
Much attention has been focussed in the last two decades on
detection of biological thiols, such as cysteine (Cys), homocysteine
(Hcy) and glutathione (GSH) [1]. GSH plays various roles in
reversible redox reactions and has vital cellular functions in living
cells [2e4]. It is noteworthy that the abnormal concentration levels
of GSH are more likely to bring about AIDS, cancer and Alzheimer's
disease [5e7]. Hence, selective and sensitive detection of GSH, in
biological systems particularly, is very important for monitoring
these diseases.
Recently, extensive efforts have been made to develop fluores-
cent probe owing to its unique advantages such as high sensitivity,
good specificity, real-time determination and simple operation. So
far, many probes have been synthesized to detect S-containing
compounds, especially for H2S [8e10] and thiols [11e14]. Only a
few fluorescent probes are available to detect GSH, Cys and Hcy, and
the familiar detection mechanisms include Michael addition reac-
tion [15e17], nucleophilic substitution [18,19], cleavage reaction
[20e23] and ligand displacement of metal complexes [24e26].
Nevertheless, because of the similarities in both the structure and
cent probes have been developed for Cys and Hcy over GSH
[27e31], while there are few probes that can efficiently tell GSH
apart from Cys and Hcy. Thus, the development of probes with great
selectivity to GSH is still in need.
2-(20-hydroxyphenyl)benzothiazole and its derivatives have
gained wide attention in recent years because they can undergo an
excited-state intramolecular photon transfer (ESIPT) process upon
photoexcitation [32e34]. Furthermore, the dinitrophenyl ether can
be introduced to a fluorophore easily to get a new probe, which has
very low background fluorescence due to the strong quenching
effect of the nitro group [35,36]. Keeping these in mind, we
designed and synthesized a highly sensitive and selective fluores-
cent probe 1 (Scheme 1). It can differentiate GSH from other thiols
within 30 min in aqueous solution containing CTAB micelles.
Moreover, it was also been successfully applied to monitoring GSH
in living cells.
2. Experimental
2.1. Materials
2-Aminothiophenol was purchased from Aladdin Co., Ltd.
(Shanghai, China). Lys-Cys-Gly and Glu-Cys-Glu were purchased
* Corresponding author.
E-mail address: chenxq@njtech.edu.cn (X. Chen).
http://dx.doi.org/10.1016/j.dyepig.2016.02.027
0143-7208/© 2016 Elsevier Ltd. All rights reserved.

X. Ren et al. / Dyes and Pigments 129 (2016) 156e162
157
O
HO
O
HO
SH
H
O
Na2S2O5
DMF
S
N
+
NH2
2
e
n
e
z
n
e
b
o
r
o
F
u
l
f
M
D
o
O2N
r
t
i
,
n
i
3
d
O
-
C
2
4
,
2
K
NO2
O
O
S
N
1
Scheme 1. Synthesis of probe 1.
from Sangon Biotech (Shanghai, China). Cetyltrimethylammonium
bromide (CTAB) was purchased from Lingfeng Chemical Reagent
Co., Ltd. (Shanghai, China). 3-Ethoxysalicylaldehyde, 2,4-
dinitrofluorobenzene and other reagents of analytical reagent
grade were purchased from Sigma-aldrich Co., Ltd. (Shanghai,
China). All reagents were directly used for the following experi-
ments without further purification. Unless otherwise noted, the
aqueous solutions were prepared with deionized water. Chroma-
tography was implemented on silica gel 60 (230e400 mesh ASTM).
d, J ¼ 7.92 Hz), 7.51 (1H, t, J ¼ 7.41 Hz), 7.41 (1H, t, J ¼ 7.49 Hz), 7.32
(1H, d, J ¼ 7.29 Hz), 6.98 (1H, d, J ¼ 7.38 Hz), 6.89(1H, t, J ¼ 7.95 Hz),
4.21e4.14 (2H, m), 1.52 (3H, t, J ¼ 6.98 Hz); 13C NMR (CDCl3,
100 MHz) d(ppm): 169.43, 151.76, 148.56, 148.28, 132.70, 126.69,
125.54, 122.23, 121.49, 120.05, 119.10, 116.93, 115.65, 64.75, 14.92.
Compound 1: 1H NMR (CDCl3, 400 MHz)
d(ppm): 8.94 (1H, d,
J ¼ 2.72 Hz), 8.25e8.22(1H, m), 8.12e8.05 (2H, m), 7.88e7.86(1H,
m), 7.52e7.37 (3H, m), 7.15e7.12 (1H, m), 6.93e6.91 (1H, m),
4.07e4.01(2H, m), 1.19 (3H, t, J ¼ 6.96 Hz); 13C NMR (CDCl3,
100 MHz) d(ppm): 160.96, 155.73, 152.53, 150.75, 141.60, 139.77,
138.92,135.73,128.63,127.98,127.64,126.50,125.66,123.42,122.04,
121.71, 121.54, 117.30, 115.79, 65.05, 14.49. LC-MS (ESIþ): m/
z ¼ 438.0692 [M þ H]þ, calc. for C21H16N3O6S ¼ 438.0760; m/
z ¼ 460.0511 [M þ Na]þ, calc. for C21H15N3NaO6S ¼ 460.0579.
2.2. Equipments and instruments
1H NMR and 13C NMR spectra were obtained using Bruker
spectroscopy. Mass spectra were recorded on Agilent HPLC-MS.
Ultravioletevisible light (UVevis) absorption spectra were carried
out on
a
ꢀ1860A UV/vis spectrophotometer. Fluorescence emission
2.4. Crystal growth and X-ray structure determination
spectra were collected using RF-5301/PC fluorophotometer. Single
crystal X ray diffraction measurements were carried out on a Bruker
Single crystals of compound 1 suitable for X-ray analysis were
grown from CH3CN by slow evaporation at room temperature for a
week. The well-shaped single crystals of 1 were selected for lattice
parameter determination and collection of intensity data at 296 K
on a Bruker SMART CCD diffractometer with a detector distance of
SMART CCD diffractometer using a Mo Ka radiation (
l
¼ 0.71073 Å).
The cells were imaged by confocal laser scanning microscopy
(Leica, TCS sp5 II).
5
cm and frame exposure time of 10
s using a graphite-
2.3. Synthesis
monochromated Mo Ka
(l
¼ 0.71073 Å) radiation. The structures
were all solved by direct methods and refined on F2 by full-matrix
least squares procedures using SHELXTL software [38]. All non-
hydrogen atoms were anisotropically refined. All H atoms were
located from a difference map and refined isotropically.
Compound 2 was synthesized according to previous typical
literature [37]. 2-aminothiophenol (0.391 g, 3.12 mmol) and 3-
ethoxysalicylaldehyde (0.525 g, 3.16 mmol) were mixing together
in 10 mL N,N-dimethylformamide (DMF). Then, sodium meta-
bisulfite (Na2S2O5, 0.61 g) was added to the stirring mixture. The
reaction mixture was heated to 75 ꢁC for 2 h under N2 atmosphere
and the progress of the reaction was monitored by TLC. After
completion of the reaction, the mixture was cooled to room tem-
perature. 20 mL of H2O was added and the product which precip-
itated as a yellow solid was collected. Recrystallization from
methanol afforded pure product 2 (0.761 g, 2.81 mmol) in high
yield (90%). Then, N,N- dimethylformamide (DMF) solution (10 mL)
2.5. Absorption and fluorescence measurements
The probe 1 (1 mM) was dissolved in acetonitrile and main-
tained at room temperature. Stock solutions (10 mM) of amino
acids including glutathione (GSH), homocysteine (Hcy), cysteine
(Cys), alanine (Ala), arginine (Arg), glycine (Gly), lysine (Lys), serine
(Ser), phenylalanine (Phe), glutamic acid (Glu), histidine (His),
glutamine (Gln), methionine (Met) and tyrosine (Tyr) were pre-
pared in deionized water. Test solutions were prepared by placing
of compound
2 (0.271 g, 1 mmol) was mixed with 2,4-
dinitrofluorobenzene (0.223 g, 1.2 mmol) in the present of K2CO3
to obtain compound 1. The mixture was heated to 80 ꢁC under N2
atmosphere with monitoring by TLC in the progress of the reaction.
After 3 h, 20 mL water was added and pale yellow sediment
appeared. The sediment was then collected and dried in vacuo.
Chromatography of the crude product on silica gel using CH2Cl2 as
eluent afforded 1 (0.363 g, yield 83%). Compound 2: 1H NMR (CDCl3,
30 mL of the probe stock solution into a test tube, diluting the so-
lution to 3 mL with HEPES buffer (20 mM, pH ¼ 7.4) containing
CTAB (1 mM) and then different analytes of corresponding con-
centrations were added. All UVevis absorption and fluorescence
measurements were measured at room temperature. For fluores-
cence study, the samples were excited at 385 nm and the fluores-
cence emission ranged from 400 nm to 650 nm. Both the excitation
300 MHz)
d(ppm): 12.75 (1H, s), 7.98 (1H, d, J ¼ 8.07 Hz), 7.89 (1H,

158
X. Ren et al. / Dyes and Pigments 129 (2016) 156e162
Fig. 1. (a) Absorption and (b) fluorescence spectra of 1 (10
m
M) upon adding various
Fig. 2. (a) Fluorescence titration spectra of 1(10 mM) with various concentrations of
analytes including GSH, Cys, Hcy, Phe, Ser, Glu, Arg, Ala, His, Lys, Gln, Gly, Tyr and Met
(100
M) in the HEPES buffer (20 mM, pH 7.4) solution. (lex ¼ 385 nm, slit: 5 nm/
5 nm).
GSH in HEPES solution (20 mM, pH 7.4) and (b) the linear relationship between
fluorescence intensity at 485 nm and GSH concentration. (lex ¼ 385 nm, slit: 5 nm/
5 nm).
m
and emission slit widths were 5 nm. After the analyte adding into
the tube, it would take 30 min for reaction.
After 3 h, 2 mg NaClO4 was added to the reaction mixture. White
precipitates came out gradually. Removing the precipitates, the
product without CTAB was obtained for mass checking.
2.6. Dynamic assays
2.8. Fluorescence imaging of GSH in living cells
Glu-Cys-Glu, Lys-Cys-Gly, GSH, Cys and Hcy were stocked in
deionized water. All the stock solutions (10 mM) were preserved at
4
HeLa cells used in this study were purchased from Cobioer
Biosciences Co., Ltd. (Nanjing, China). The cell lines were cultured in
DMEM medium supplemented with 10% (v/v) calf serum, penicillin
(100 UmLꢀ1), and streptomycin (100 mg/mL). The cells were seeded
in laser confocal fluorescence microscope (LCFM) culture dishes
and maintained at 37 ꢁC in a humidified atmosphere containing 5%
CO2. When the whole cells took up 60%e70% space of culture
dishes, the cells were treated without or with 1 mM N-ethyl-
maleimide (NEM) in culture media for 30 min at 37 ꢁC. After
washing with phosphate buffered saline (PBS) to remove the
ꢁC. Test solutions were prepared by placing 30
mL of the probe
stock solution into a test tube, diluting the solution to 3 mL with
HEPES buffer (20 mM, pH ¼ 7.4) containing CTAB (1 mM) and
adding 30 mL of each analyte stock. Excitation and emission wave-
lengths were chosen at 385 nm and 485 nm, respectively. The
excitation and emission slit widths were both 5 nm.
2.7. Mass checking
1 (43 mg), GSH (60 mg) were dissolved in 5 mL acetonitrile-
HEPES buffer (20 mM, pH ¼ 7.4, 1:4 v/v) containing CTAB (1 mM).
remaining NEM, the cells were further incubated with 50
mM of 1
for 30 min at 37 ꢁC. After treating with NEM and incubating with 1,

X. Ren et al. / Dyes and Pigments 129 (2016) 156e162
159
HO
O
O2N
NH2
O
O
OH
NO2
O
HO
O
GSH
NH
S
N
O
O
N
H
CTAB
S
S
N
O2N
fluorophore
1
ESIPT ON
ESIPT OFF
NO2
2,4-dinitrobenzene-GSH
Scheme 2. Proposed signaling mechanism of probe 1 to GSH.
Fig. 3. ESI-MS spectra of the mixture of 1 and GSH. (A, B and C denote the compounds 2-(20-hydroxy-30-ethoxyphenyl)benzothiazole, GSH and 2,4-dinitrobenzene-GSH,
respectively).
Fig. 4. Pictorial visualization of GSH attacking CTAB micelles and reacting with probe 1.
the cells were subsequently treated with GSH (500
were imaged by confocal laser scanning microscopy.
m
M). The cells
obtained with a yield of 83% and characterized by X-ray crystal-
lography, 1H NMR, 13C NMR and high resolution mass spectroscopy
(Figs. S4eS9).
3. Results and discussion
3.2. Spectral properties of probe 1 towards GSH
3.1. Synthesis
To study the selectivity of probe 1, we examined 13 different
amino acids including Cys, Hcy, Gly, Phe, Ser, Glu, Lys, Arg, His, Ala,
Gln, Met and Tyr as the alternatives of GSH. First of all, we tested the
The chemical structure of the target probe 1 and its synthetic
process were depicted in Scheme 1. After purification, probe 1 was

160
X. Ren et al. / Dyes and Pigments 129 (2016) 156e162
absorption spectral properties of probe 1 in 20 mM HEPES buffer
solutions (pH ¼ 7.4) containing 1 mM CTAB. As shown in Fig. 1a, an
obvious change at ~380 nm was observed only after 10 equiv. GSH
was added into probe 1 for 30 min. We next investigated the
fluorescence spectra of the probe 1. Notably, a sharp fluorescence
intensity increase (about 15-fold enhancement) occurred at
485 nm when the probe 1 was incubated with 10 equiv. GSH for
30 min (Fig. 1b). Such cyan fluorescence could be detected by the
naked-eye under UV light (Fig. S1).
The good selectivity inspired us to explore the concentration-
dependent emission response of probe 1 toward GSH. We incu-
bated our probe with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 equiv. GSH in
20 mM HEPES buffer (pH 7.4) containing 1 mM CTAB for 30 min. As
shown in Fig. 2a, the fluorescence at 485 nm enhanced gradually
with the increase of GSH concentration. A good fitted regression
line was presented as well (Fig. 2b), and the detection limit was
calculated as low as 0.81
mM (S/N ¼ 3).
The influence of pH on the fluorescence process was further
investigated. The fluorescence intensities at 485 nm of initial probe
1 in the absence and presence of GSH were collected, respectively.
As seen from Fig. S2, the fluorescence spectra of probe 1 with or
without GSH had no change in acidic condition. On the contrary, in
Fig. 5. Time-dependent fluorescence intensity of 1 (10
m
M) at 485 nm in the presence
M). (lex ¼ 385 nm, slit:
of thiols (100
5 nm/5 nm).
mM) and the two short SH-bearing peptides (100 m
Fig. 6. Confocal fluorescence images of HeLa cells. (a) Cells incubated with 1 (50
with 50 M of 1 for 30 min. (g) Subsequent treatment of the cells with GSH (500
images).
m
M) for 30 min (d) HeLa cells were pre-incubated with 500
mM NEM for 30 min and then treated
m
mM) for 30 min (a, d and g, phase contrast images; b, e and h, fluorescence images; c, f and i, merged

X. Ren et al. / Dyes and Pigments 129 (2016) 156e162
161
neutral and alkaline conditions, fluorescence intensity of the so-
4. Conclusion
lution containing probe and GSH had a significant enhancement,
which indicated that the probe could be used in neutral and basic
conditions. Time-dependent fluorescence intensity assay revealed
that the reaction between probe 1 and GSH was completed on the
whole within 2 h (Fig. S3).
In summary, we have successfully developed probe 1 for specific
detection of GSH over Cys and Hcy based on ESIPT. Additionally,
probe 1 exhibits excellent sensitivity toward GSH in aqueous so-
lution under physiological pH condition. The addition of GSH to the
probe solution induces 15-fold fluorescence intensity enhancement
and the detection limit is calculated as low as 0.81 mM. One more
3.3. Proposed mechanism
carboxyl group in GSH is the reason for the enhanced selectivity to
GSH over Cys and Hcy. What's more, probe 1 is demonstrated with
good application in the study of GSH in living cells and shows po-
tential biological significance.
The fluorescence enhancement at 485 nm was attributed to the
release of 2-(20- hydroxy-30-ethoxyphenyl)benzothiazole via the
nucleophilic substitution reaction between probe 1 and GSH
(Scheme 2). Once 2-(20-hydroxy-30-ethoxyphenyl)benzo-thiazole
acted as fluorophore was free, an ESIPT process would come true,
which ensured the strong fluorescence enhancement. To elucidate
the details, an ESI-MS analysis of the reaction mixture of probe 1
and GSH was performed. It was noted that three distinct signals at
m/z ¼ 288.0391 [A þ OH]ꢀ, 306.0492 [BeH]ꢀ and 472.0450 [CeH]ꢀ
appeared in the mass spectrum, where A, B and C denoted the
compounds 2-(20-hydroxy-30-ethoxyphenyl)benzothiazole, GSH
and 2,4-dinitrobenzene-GSH, respectively (Fig. 3). It was obvious
that the probe 1 developed in this work provided a special signaling
mechanism, which may contribute to the development of new
fluorescent sensors for detection of GSH.
In this reaction system, CTAB micelles used as catalyst improved
the reaction rate, which could be explained by the synchronous
existences of electrostatic and hydrophobic interactions. GSH was
negatively charged, so it was capable of closely binding with and
breezily penetrating into positively charged CTAB micelles with
hydrophobic substrate. In addition, the number of carboxyl group
in thiols was considered to be another important reason for supe-
rior selectivity of probe 1 to GSH over Cys and Hcy. Compared to Cys
and Hcy, GSH bearing two carboxyl groups which enhanced the
ability of generating negative charge was more easily to have a
strong affinity to positively charged CTAB micelles (Fig. 4). Thus,
GSH was apt to attack probe substrate inside of the micellar ag-
gregates, leading to the release of fluorophore. From the standpoint
of the positive effect of carboxyl groups on nucleophilic substitu-
tion catalyzed by CTAB, we processed the reactions between probe
1 and thiols including GSH, Cys, Hcy and the other two short SH-
bearing peptides (Glu-Cys-Glu and Lys-Cys-Gly). As we can see in
Fig. 5, the reactivity of probe 1 to various thiols was manifested in
the following order: Glu-Cys-Glu > GSH » Hcy, Cys and Lys-Cys-Gly.
Obviously, Glu-Cys-Glu with three carboxyl groups demonstrated
the highest reactivity to sensor 1, while Hcy, Cys and Lys-Cys-Gly
with only one carboxyl group showed little reactivity, which
proved the validity of our original vision.
Appendix A. Supplementary data
CCDC 1454726 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.
uk/data_request/cif. Supplementary data associated with this
article can be found in the online version.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (21376117, 21406109), the Jiangsu Natural
Science Funds for Distinguished Young Scholars (BK20140043), the
Natural Science Foundation of the Jiangsu Higher Education In-
stitutions of China (14KJA150005), the Qing Lan Project and the
Project of Priority Academic Program Development of Jiangsu
Higher Education Institutions (PAPD). Thank Prof. Dunru Zhu for
the help of X-ray structure determination.
Appendix B. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2016.02.027.
References
[1] Refsum H, Smith AD, Ueland PM, Nexo E, Clarke R, McPartlin J, et al. Facts and
recommendations about total homocysteine determinations: an expert
opinion. Clin Chem 2004;50:3e32.
[2] Rusin O, St Luce NN, Agbaria RA, Escobedo JO, Jiang S, Warner IM, et al. Visual
detection of cysteine and homocysteine. J Am Chem Soc 2004;126:438e9.
[3] Wang WH, Escobedo JO, Lawrence CM, Strongin RM. Direct detection of ho-
mocysteine. J Am Chem Soc 2004;126:3400e1.
[4] Forman HJ, Zhang H, Rinna A. Glutathione: overview of its protective roles,
measurement, and biosynthesis. Mol Asp Med 2009;30:1e12.
[5] Biswas SK, Rahman I. Environmental toxicity, redox signaling and lung
inflammation: the role of glutathione. Mol Asp Med 2009;30:60e76.
[6] Zhang Y, Li Y, Yan XP. Photoactivated CdTe/CdSe quantum dots as a near
infrared fluorescent probe for detecting biothiols in biological fluids. Anal
Chem 2009;81:5001e7.
[7] Liu J, Sun YQ, Huo YY, Zhang HX, Wang LF, Zhang P, et al. Simultaneous
fluorescence sensing of Cys and GSH from different emission channels. J Am
Chem Soc 2014;136:574e7.
3.4. Cell imaging experiments
In consideration of the excellent performance of probe 1 under
physiological pH, confocal microscopy experiments were carried
out to further investigate the permeability and the real-time
monitor of GSH in living cells. Once HeLa cells were incubated
[8] Li DW, Qu LL, Hu K, Long YT, Tian H. Monitoring of endogenous hydrogen
sulfide in living cells using surface-enhanced Raman scattering. Angew Chem
Int Ed 2015;54:12758e61.
€
[9] Zhao CC, Zhang XL, Li KB, Zhu SJ, Guo ZQ, Zhang LL, et al. Forster resonance
energy transfer switchable self-assembled micellar nanoprobe: ratiometric
fluorescent trapping of endogenous H2S generation via fluvastatin-stimulated
upregulation. J Am Chem Soc 2015;137:8490e8.
with 1 (50 mM), strong fluorescence was observed inside the cells
due to the high level concentration of thiols in living cells (Fig. 6b).
In the control experiment, when cells were pre-treated with excess
N-ethylmaleimide (NEM), a trapping reagent for thiol species, fol-
lowed by treatment with 1 (50 mM), the confocal microscopic image
showed a negligible fluorescence signal (Fig. 6e). That verified the
specificity of probe 1 for thiols over other analytes in living cells.
Subsequently, with addition of 500 mM GSH to NEM-treated cells
for 30 min, the fluorescence signal was restored (Fig. 6h), which
indicated the rapid response of probe 1 toward GSH in living cells.
[10] Li HD, Yao QC, Fan JL, Jiang N, Wang JY, Xia J, et al. A fluorescent probe for H2S
in vivo with fast response and high sensitivity. Chem Commun 2015;51:
16225e8.
[11] Chen X, Zhou Y, Peng XJ, Yoon J. Fluorescent and colorimetric probes for
detection of thiols. Chem Soc Rev 2010;39:2120e35.
[12] Yin J, Kwon Y, Kim D, Lee D, Kim G, Hu Y, et al. Cyanine-based fluorescent
probe for highly selective detection of glutathione in cell cultures and live
mouse tissues. J Am Chem Soc 2014;136:5351e8.
[13] Jung HS, Chen X, Kim JS, Yoon J. Recent progress in luminescent and colori-
metric chemosensors for detection of thiols. Chem Soc Rev 2013;42:6019e31.

162
X. Ren et al. / Dyes and Pigments 129 (2016) 156e162
[14] Balkrishna SJ, Hodage AS, Kumar S, Panini P, Kumar S. Sensitive and regen-
erable organo-chalcogen probes for the colorimetric detection of thiols. RSC
Adv 2014;4:11535e8.
[15] Chen X, Ko SK, Kim MJ, Shin I, Yoon J. A thiol-specific fluorescent probe and its
applica- tion for bioimaging. Chem Commun 2010;46:2751e3.
selective detection of thiols. Sensors Actuators B 2013;176:698e703.
[27] Wang H, Zhou G, Gai H, Chen X. A fluorescein-based probe with high selec-
tivity to cysteine over homocysteine and glutathione. Chem Commun
2012;48:8341e3.
[28] Yang X, Guo Y, Strongin RM. Conjugate addition/cyclization sequence enables
selective and simultaneous fluorescence detection of cysteine and homocys-
teine. Angew Chem Int Ed 2011;50:10690e3.
[16] McMahon BK, Gunnlaugsson T. Selective detection of the reduced form of
glutathione (GSH) over the oxidized (GSSG) form using a combination of
glutathione reductase and
a
Tb(III)-cyclen maleimide based lanthanide
[29] Guo Z, Nam S, Park S, Yoon J. A highly selective ratiometric near-infrared
fluorescent cyanine sensor for cysteine with remarkable shift and its appli-
cation in bioimaging. Chem Sci 2012;3:2760e5.
[30] Jung HS, Pradhana T, Han JH, Heo KJ, Lee JH, Kang C, et al. Molecular modu-
lated cysteine-selective fluorescent probe. Biomaterials 2012;33:8495e502.
[31] Shiu HY, Wong MK, Che CM. “Turn-on” FRET-based luminescent iridium(III)
probes for the detection of cysteine and homocysteine. Chem Commun
2011;47:4367e9.
[32] Majumdar P, Zhao JZ. 2-(2-Hydroxyphenyl)-benzothiazole (HBT)-rhodamine
dyad: acid-switchable absorption and fluorescence of excited-state intra-
molecular proton transfer (ESIPT). J Phys Chem B 2015;119:2384e94.
[33] Sahana S, Mishra G, Sivakumar S, Bharadwaj PK. A 2-(20-hydroxyphenyl)
luminescent ‘switch on’ assay. J Am Chem Soc 2012;134:10725e8.
[17] Kim GJ, Yoon DH, Yun MY, Kwon H, Ha HJ, Kim HJ. Ratiometric fluorescence
probes based on a Michael acceptor type of coumarin and their application for
the multichannel imaging of in vivo glutathione. RSC Adv 2014;4:18731e6.
[18] Niu LY, Guan YS, Chen YZ, Wu LZ, Tung CH, Yang QZ. BODIPY-based ratio-
metric fluorescent sensor for highly selective detection of glutathione over
cysteine and homocysteine. J Am Chem Soc 2012;134:18928e31.
[19] Yin J, Kwon Y, Kim D, Lee D, Kim G, Hu Y, et al. Preparation of a cyanine-based
fluorescent probe for highly selective detection of glutathione and its use in
living cells and tissues of mice. Nat Protoc 2015;10:1742e54.
[20] Long L, Lin W, Chen B, Gao W, Yuan L. Construction of a FRET-based ratio-
metric fluorescent thiol probe. Chem Commun 2011;47:893e5.
[21] Shao J, Sun H, Guo H, Ji S, Zhao J, Wu W, et al. A highly selective red-emitting
FRET fluorescent molecular probe derived from BODIPY for the detection of
cysteine and homocysteine: an experimental and theoretical study. Chem Sci
2012;3. 1049e 61.
[22] Cao X, Lin W, Yu Q. A ratiometric fluorescent probe for thiols based on a
tetrakis(4-hydroxyphenyl)porphyrin-coumarin Scaffold. J Org Chem 2011;76:
7423e30.
[23] Lee MH, Han JH, Kwon PS, Bhuniya S, Kim JY, Sessler JL, et al. Hepatocyte-
targeting single galactose-appended naphthalimide: a tool for intracellular
thiol imaging in vivo. J Am Chem Soc 2012;134:1316e22.
[24] Zhou L, Lin Y, Huang Z, Ren J, Qu X. Carbon nanodots as fluorescence probes
for rapid, sensitive, and label-free detection of Hg2þ and biothiols in complex
matrices. Chem Commun 2012;48:1147e9.
benzo- thiazole (HBT)equinoline conjugate:
a highly specific fluorescent
probe for Hg2þ based on ESIPT and its application in bioimaging. Dalton Trans
2015;44:20139e46.
[34] Huang Q, Yang XF, Li H. A ratiometric fluorescent probe for hydrogen sulfide
based on an excited-state intramolecular proton transfer mechanism. Dyes
Pigments 2013;99:871e7.
[35] Huang Z, Ding S, Yu D, Huang F, Feng G. Aldehyde group assisted thiolysis of
dinitrophenyl ether: a new promising approach for efficient hydrogen sulfide
probes. Chem Commun 2014;50:9185e7.
[36] Wang L, Chen H, Wang H, Wang F, Kambam S, Wang Y, et al. A fluorescent
probe with high selectivity to glutathione over cysteine and homocysteine
based on positive effect of carboxyl on nucleophilic substitution in CTAB.
Sensors Actuators B 2014;192:708e13.
[37] Khan KM, Rahim F, Halim SA, Taha M, Khan M, Perveen S, et al. Synthesis of
[25] Liang W, Zhao Z, Zhang Y, Wang Q, Zhao X, Ouyang J. Highly selective
novel inhibitors of b-glucuronidase based on benzothiazole skeleton and
study of their binding affinity by molecular docking. Bioorgan Med Chem
2011;19:4286e94.
detection of gluta- thione using
a
NIP/Cu2þ complex fluorescent probe.
J Lumin 2012;132:1160e5.
[26] Wang H, Zhou G, Chen X. An iminofluorescein-Cu2þ ensemble probe for
[38] Sheldrick GM. A short history of SHELX. Acta Crystallogr A 2008;64:112e22.