A highly selective ratiometric fluorescent probe for biothiol and imaging in live cells

Article abstract of DOI:10.1039/c6ra04564b

A new N-butyl-4-amino-1,8-naphthalimide-based colorimetric and ratiometric fluorescent probe for the detection of biothiols (cysteine, homocysteine, and glutathione) was designed and synthesized. The probe exhibited good selectivity and sensitivity toward biothiols over other non-thiol-bearing amino acids and common anions with significant changes in both color (from colorless to yellow) and fluorescence (from blue to yellow-green). The mechanism is based on cleavage of disulfide by thiols followed by an intramolecular cyclization to give the product, N-butyl-4-amino-1,8-naphthalimide, which showed outstanding intramolecular charge transfer (ICT). Therefore, ratiometric fluorescence signal was observed. Furthermore, the probes were successfully applied for visualizing endogenous thiols in living cells.

Full text of DOI:10.1039/c6ra04564b

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: B. Gao, L. Cui, Y.
pan, Y. Zhou, G. M. zhang, C. zhang, S. Shuang and C. Dong, RSC Adv., 2016, DOI: 10.1039/C6RA04564B.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Please do not adjust margins
RSC Advances
Page 1 of 7
View Article Online
DOI: 10.1039/C6RA04564B
RSC Advances
ARTICLE
A highly selective ratiometric fluorescent probe for biothiol
and imaging in live cells
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
b
a
a
a,∗
Baozhen Gao , Lixia Cui , Yong Pan , Guomei Zhang , Ying Zhou , Caihong Zhang , Shaomin
a
a, ∗
Shuang , Chuan Dong
DOI: 10.1039/x0xx00000x
A new Nꢀbutylꢀ4ꢀaminoꢀ1,8ꢀnaphthalimideꢀbased colorimetric and ratiometric fluorescent probe for the detection of
biothiols (cysteine, homocysteine, and glutathione) were designed and synthesized. The probe exhibited good selectivity and
sensitivity toward biothiols over other nonꢀthiolꢀbearing amino acids and common anions with significant changes in both
color (from colorless to yellow) and fluorescence (from blue to yellowꢀgreen). The mechanism is based on cleavage of
disulfide by thiols followed by an intramolecular cyclization to give the product, Nꢀbutylꢀ4ꢀaminoꢀ1,8ꢀnaphthalimide, which
showed outstanding intramolecular charge transfer (ICT). Therefore, ratiometric fluorescence signal was observed.
Furthermore, the probes were successfully applied for visualizing endogenous thiols in living cells.
www.rsc.org/
actively developed. A number of excellent disulfide probes for
biothiols have been exploited such as Pullela et al. tethered
fluorescein and rhodamine by disulfid containing linker [27];
1
Introduction
Biological thiols such as glutathione (GSH), cysteine (Cys), and
homocysteine (Hcy) have similar structure and play essential roles
in many physiological processes. The abnormal levels of
intracellular biothiols will initiate many diseases [1-3]. Among
these agents, GSH is the most abundant intracellular nonprotein
thiol (about 0.1~10 mM) [4], and play a critical role in maintaining
redox homeostasis of the cell and is directly linked with many
diseases including cancer, Parkinson’s diease, Alzheimer disease,
etc [5-8]. Thus, it is important to develop efficient methods for the
detection and quantification of biothiols in physiological media for
academic research and for the diagnosis of the related diseases.
Currently, among the major methods for biothiols detection,
fluorescent probes have been recognized as the efficient
molecular tools due to their high selectivity, low cost and ability to
detect and image the intracellular biothiols [9], in particular,
ratiometric fluorescent probes have attracted increasing attention
which based on the ratio of emission intensity from two different
wavelengths can eliminate the influence of probe concentration,
environment and excitation intensity [10-13]. To date, a variety of
ratiometric fluorescent biothiols probes have been reported based
on different mechanisms: 1) thiol cleavage reaction [14-17], 2)
thiol nucleophilic reaction [18-21], 3) metal complexes-displace
coordination and others [22-26]. Among the various strategies, the
probes based on cleavage of disulfide by thiols have been most
Karuso et al. jioned two molecules of fluorescein by a short
disulfide-containing linker [28]; Chmielewski et al. synthesized a
new rhodamine derivative bearing disulfide unit [29]. Some
reports use GSH-triggered disulfide-based cleavable reactions for
targeted drug delivery [30-35]. Although many promising probes
have been reported for thiols detection in recent years, it still
remains a challenge to improve methodology for this purpose.
In this work, we report a new ratiometric fluorescent probe for
the detection of biothiols based on the nucleophilic cleavage-
cyclization of disulfides. N-butyl-4-amino-1,8-naphthalimide was
chosen as the fluorophore due to its desirable optical properties,
such as high photostability, large Stokes’ shift, insensitivity to pH
[
36–39]. 2-(pyridin-2-yldisulfanyl)ethanol owning to its sensitivity
and rapid cleavage by biothiols and an appropriate linker was
employed for reaction acceptor (Scheme 1). The probe showed
high selectivity for biothiols over other amino acids and anions. It
was successfully applied for the GSH bioimaging in live cells.
2
Experimental
2
.1 Chemicals
4
-Bromo-1,8-naphthalic
anhydride,
EDC
(1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), DMAP
4-dimethylaminopyridine) and other chemicals were purchased
(
from Sigma-Aldrich or Aldrich and were used as received. Solvents
were dried according to standard procedures. All biothiols
a.
Department of Chemistry and Chemical Engineering, Shanxi University, Taiyuan
30006, China. E-mail: chzhang@sxu.edu.cn; Fax: +86-351-7011688; Tel: +86-351-
(
including cysteine (Cys), homocysteine (Hcy), glutathione (GSH)),
0
b.7010588
amino acids and other anions were analytical grade and were
purchased from Beijing Experiment Reagent Co., Ltd (Beijing,
China).
State Key Laboratory of NBC Protection for Civilian, Research Institute of
Chemical Defense, Yangfang, Changping District, Beijing, 102205, China.
1
13
†Electronic supplementary information (ESI) available: H NMR, CNMR and MS
spectra of probe, ratiometric responses towards Cys and Hcy, time-dependent
fluorescence spectral changes of probe with biothinols, the effects of pH,
Determination of quantum yield.
2
.2 Apparatus
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
RSC Advances
Page 2 of 7
View Article Online
DOI: 10.1039/C6RA04564B
ARTICLE
Journal Name
1
13
H-NMR and C-NMR spectra were collected on a 300 MHz
spectrometer. Fluorescence excitation and emission spectra were
measured on a Cary Eclipse fluorescence spectrophotometer. High
resolution mass spectra were obtained on a Varian QFT-ESI mass
spectrometer. Fluorescence imaging was performed with a
fluorescence microscope with a GFP light cube for fluorescence
channel and 40×objectives.
v/v, PES buffer, pH 7.4) solutions. Fluorescence measurements
were carried out with a slit width of 5 nm (λex = 420nm).
2
.5 Cell imaging experiments with GSH
HeLa cells were cultured in Dulbecco’s modified Eagle’s Medium
DMEM, Cellgro company) supplemented with 10% fetal bovine
(
serum, 4mM glutamine, 100 IU/mL penicillin, and 100ug/mL
o
streptomycin at 37 C and with 5% CO for two days. Cells
2
.3 Preparation and characterization of probe
2
were transferred to 24-well plates one day before imaging. Before
use, the adherent cells were washed once with FBS-free DMEM.
Then the cell was treated with GSH (200 μM) in culture media for
Synthesis of probe (1) is summarized in Scheme 1. N-butyl-4-
amino-1,8-naphthalimide (2) [40, 41] and 2-(pyridine-2-
yldisulfanyl)ethanol (5) were synthesized by the literature
methods. Synthesis of probe 1 is described below [42].
3
0 min at 37°C and washed with phosphate-buffered saline (PBS).
After washing with PBS to remove the remaining GSH, the cells
were further incubated with the probe (5 μM) for 120min in PBS
buffer (pH 7.4, containing 100μM CTAB). Cell imaging was carried
out after washing the cells with PBS (pH 7.4). All of the microscopy
images were taken on a fluorescence microscope with blue and
green channels.
To a mixture of N-butyl-4-amino-1,8-naphthalimide 2 (60mg, 0.14
mmol) and DIPEA (67 mg, 0.52 mmol) in dry toluene (5 mL) was
added a solution of triphosgene (52 mg, 0.17 mmol) in toluene
dropwise in ice bath for 2h. Then the resulting solution was heated
to reflux for 3h. After cooling to room temperature, the reaction
mixture was flushed with nitrogen gas and removal of unreacted
2 2
phosgene gas. A solution was diluted with CH Cl (6 mL) and
added the 2-(pyridine-2-yldisulfanyl)ethanol (31.32 mg, 0.18 mmol)
and the solution was stirred at room temperature for overnight.
The reaction was then concentrated and purified by flash column
3 Results and discussion
3
.1 Design principle of the probe
2 2
chromatography (silica gel, 5:95 MeOH/CH Cl ) to give probe as a
The disulfide bond can undergo cleavage via reaction with the
thiols, many effective fluorescent probes were developed based
on it and proved to have high selectivity and sensitivity for
biothiols. In order to further improve the fluorescence properties,
and to use them for the quantitative detection of biothiols in
complex samples, development of ratiometric fluorescent probes
based on this strategy was required.
yellow solid (45 mg, 0.09 mmol, 40.5% yield).
1
3
H NMR (300 MHz, CDCl ) δ 8.57-8.64 (m, 2H), 8.45-8.48 (d, 1H, J =
8
4
2
.47), 8.24-8.33 (m, 2H,) 7.61-7.78 (m, 3H), 7.08-7.12 (m, 1H),
.52-4.56 (t, 2H, J = 4.54 Hz), 4.15-4.20 (t, 2H J = 4.17), 3.13-3.17 (t,
H, J = 3.15), 1.66-1.77 (m, 2H), 1.38-1.51 (m, 2H), 0.95-1.00 (t, 3H,
13
J = 0.97); C NMR (100 MHz, CDCl3) δ 164.4, 163.9, 159.8, 153.0,
150.0, 139.0, 137.4, 132.6, 131.5, 129.1, 126.9, 126.3, 123.6, 123.2,
Herein, we used N-butyl-4-amino-1,8-naphthalimide linked with
pyridine by disulfide-containing linker to prepare a ratiometric
fluorescent probe for biothiols (Scheme 2). Because carbamate
bond was formed, the electron-withdrawing group weakened the
ICT effect and resulted in a fluorescent emission wavelength blue
shift. Upon reacting with biothiols, the disulfide bond cleavage
generated the intermediate A, having a free -SH group. Then the
intramolecular cyclization of A should proceed to release the
byproduct, 1,3-oxathiolan-2-one and the fluorophore, N-butyl-4-
1
21.4, 120.6, 118.2, 117.0, 64.2, 40.5, 37.7, 30.4, 20.6, 14.1;
+
HRESI-MS: calcd for
C
24
23
H N
3
O
4
S
2
[M+H] 482.1203, found
482.11966.(Fig.S1)
amino-1,8-naphthalimide
B, which showed outstanding
intramolecular charge transfer (ICT). Therefore, ratiometric signal
was observed. This mechanism has been proved by reaction
between probe and GSH, and reaction product B was confirmed by
1
H NMR (Fig. S2).
Scheme 1 The synthesis route of probe.
2.4 General UV–vis and fluorescence spectra measurements
Stock solutions of probe 1 (0.25mM) were prepared in DMF (N, N-
Dimethylformamide). Biothiols solutions, amino acids solutions
and anion solution were also prepared using the deionized water.
UV–vis and fluorescence spectra were obtained in DMF: water (1:9
Scheme 2. Proposed detection mechanism of probe against biothiols.
This journal is © The Royal Society of Chemistry 20xx
2
| J. Name., 2012, 00, 1-3
Please do not adjust margins

Please do not adjust margins
RSC Advances
Page 3 of 7
View Article Online
DOI: 10.1039/C6RA04564B
Journal Name ARTICLE
.2 Ratiometric and colorimetric responses towards biothiols
3
With the probe in hand, we first investigated its fluorescence
spectroscopic properties in a mixture of DMF/buffer (1:9, 50 mM
PBS buffer, pH 7.4). The probe showed a wide fluorescence
emission band (420-650nm) with a peak at 482 nm. The
fluorescence quantum yield was 0.11.
The probe (1μM) was treated with GSH, Cys, and Hcy separately
(all at 1 mM), the fluorescence emission intensity of 482 nm
obviously decreases, accompanying appearance of a new emission
band (470−670 nm) with a peak at 540 nm, exhibiting a
ratiometric change with a large red-shift over 60 nm for the probe
as shown in Fig. 1. Meanwhile, the color of the solution changed
gradually from colorless to yellow, which was clearly recognizable
by naked eyes. We also noted that the solution’s fluorescence
color changed from blue to yellow-green under illumination with a
Fig.2. The emission spectra of probe (1 μM) in 50 mM PBS buffer (pH 7.4, 10%
DMF) with different concentrations of GSH (0, 20, 30, 40, 50, 60, 70, 80, 90, 100,
o
365 nm UV lamp. These results demonstrated that the probe was
200 μM) for 120 min at 25 C, λex=420 nm. Insert is the concentration of GSH
sensitive to these biothiols.
dependence of the ratiometric fluorescence signal (F540/F482).
3
.3 Time-dependence in the detection process of biothiols
We examined the timeꢀdependent fluorescence changes of the
probe (1ꢁM) towards GSH. It was found that in the presence of 1
mM GSH, the emission intensity at 482 nm was increased at first
(‘1ꢀ4’ in the Fig.3), the changes took place in 8min under these
experimental conditions, then decreased gradually, while the
emission at 540 nm increases (in the Fig.3). Within 120 min, the
fluorescence intensity reaches a plateau (insert fig.3).
Fig.1 The emission spectra of probe (1 μM) in 50 mM PBS buffer (pH 7.4, 10%
o
DMF) with Cys, Hcy and GSH (1 mM) for 120 min at 25 C, λex=420 nm. Inset:
the color changes of probe without and with addition of GSH. left: under
ambient lighting, right: under UV irradiation.
The fluorescence titrations were also investigated under the same
condition as shown in Fig. 2. Upon the addition of GSH, the
emission band at 482 nm decreased and a new band at 540 nm
gradually enhanced. The changes occur with an obvious isosbestic
point at 505 nm. It was found that with increased concentrations
of GSH (20 μM -200 μM), 0~6-fold enhancements of the
fluorescence intensity ratios could be observed. A good linearity (R
Fig.3. Time-dependent fluorescence response of the probe (1 uM) to GSH (1
mM) in 50 mM PBS buffer (pH 7.4, 10% DMF). Insert: ① Fluorescence intensity
of probe at 482 nm upon reaction with GSH. ② Fluorescence intensity of
probe at 540 nm upon reaction with GSH.
=
0.9961) between the fluorescence intensity ratio (F540/F482) and
the GSH concentration in the range of 20 μM -200 μM was
obtained, as depicted in insert Fig. 2. The detection limit for GSH,
based on the definition by IUPAC (CDL=3Sb/m), was calculated to
be 3.7 μM [43]. These results showed that the probe could be
applied for quantitative measurement of GSH concentrations.
Similar results were also observed upon the addition of Cys and
Hcy into the solution of probe (Fig. S3, S4).
The above changes are consistent with the mechanism of probe to
GSH. The increased emission intensity at 482 nm was assigned the
formation of intermediate A, which can formed readily as the result
of disulfide cleavage. Then A followed by slow intramolecular
cyclization and cleavage of a neighboring carbamate bond to give
B.
Corresponding, the emission intensity at 482 nm was
decreased and the emission at 540 nm was increased. Similar
results were achieved for Cys and Hcy (Fig.S5).
3
.4 pH effects
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
RSC Advances
Page 4 of 7
View Article Online
DOI: 10.1039/C6RA04564B
ARTICLE
Journal Name
The pH dependence of the thiol-mediated disulfide bond cleavage
step was also investigated and as seen in Figure S6, The probe
appeared to be stable in the pH range 4-10 as almost no
fluorescence changes were observed. In the presence of biothiols,
probe gave strong ratiometric fluorescence responses at pH (7-
increased cellular thiol concentration. When probe was incubated
with these cells, we could observe obvious blue fluorescence
decrease and green fluorescence increase (Fig. 5 d, e and f). In
another control experiment, HeLa cells were pretreated with 500
µM N-methylmaleimide (NMM) for 20 min to deplete intracellular
thiols, then the probe (5 µM) was applied to cells. The results were
shown in Fig.5 (g, h and i), the blue fluorescence were obvious and
green fluorescence were not observed, These results clearly
demonstrated that the probe is suitable for fluorescence imaging
of GSH in living cells.
10). In contrast, the probe did not show significant signals change
at acidic pH (4-6). Such findings showed that probe could be used
to detect cellular thiols in biological conditions.
3.5 Selectivity over biothiols
To assess the selectivity of the probe toward thiols, the probe
1μM) was treated with various thiol-bearing amino acids
including GSH, Cys, Hcy and other non-thiol-bearing amino acids
(
a
(
L-histidine (His), L-glutamic acid (Glu), L-asparagine (Asp), L-valine
Val), L-phenylalanine (Phe), L-tyrosine (Tyr), L-alanine (Ala), L-
(
serine (Ser), L-leucine (Leu), L-arginine (Arg), L-proline (Pro), L-
threonine (Thr), L-glutamine (Glu), L-tryptophan (Trp), L-isoleucine
(Ile), L-lysine (Lys)) (all at 1 mM). Fluorescence signals were
recorded after mixing for 120 min. As shown in Fig. 4 (a), the
fluorescence ratio (F540/F482) of probe exhibited a significant
increase upon reaction with GSH, Cys, Hcy, whereas little
fluorescence intensity changes were observed in the presence of
other non-thiol-bearing amino acid. Moreover, when biothiols
(including Cys, Hcy and GSH) co-existed with non-thiol-bearing
b
amino acid, the fluorescence ratio signal showed an obvious
increase. These results clearly demonstrated the excellent
selectivity of probe even when other non-thiol-bearing amino acid
coexisted with biothiols.
In order to check the influence of anions to the probe toward GSH,
the probe (1μM) was treated with some common anions such as
−
−
−
2-
2-
2-
2
−
−
2-
−
3
−
3 2 4 3 4 3 2 3 4
Cl , Br , I , SO , C O , CO , SO , NO , S O , Ac and PO . As
shown in Fig. 4 (b), these anions exhibited no noticeable
fluorescence and had no interference to the detection of
biothinols.
Fig.4(a) The ratiometric fluorescence intensity of probe (1 μM) in 50 mM PBS
buffer (pH 7.4, 10% DMF) upon the addition of 1 mM GSH in the presence of
3.6 Cellular imaging
1
mM non-thiol-bearing amino acids (His, Glu, Asp, Val, Phe, Tyr, Ala, Ser, Leu,
The cytotoxicity of the probe is an important consideration for
intracellular imaging applications. So the cytotoxicity of the probe
was evaluated using the HeLa cell line by the MTT assay (Fig.5).
From the results we can see that the probe exhibited little
cytotoxicity for cells, even at the concentration of 20 µM, the cell
viability is still more than 93%. The results demonstrated that the
probe has good biocompatibility and is appropriate for the
detection of biothiols in live cells.
Arg, Pro, Thr, Glu, Trp, Ile, Lys). λex=420 nm. Bars represent fluorescence
intensity ratio F540/F482. Black bars: probe + various non-thiol-bearing amino
acid. Red bar: probe + various non-thiol-bearing amino acid + GSH. λex = 420
nm. Each spectrum was acquired 120 min after various analytes addition at 25
o
C.
Fig.4(b) The ratiometric fluorescence intensity of probe (1 μM) in 50 mM PBS
buffer (pH 7.4, 10% DMF) upon the addition of 1 mM GSH in the presence of
1
-
-
-
2-
2-
2-
2-
-
2-
-
3-
3 2 4 3 4 3 2 3 4
mM common anions (Cl , I , Br , SO , C O , CO , SO , NO , S O ,Ac ,PO ).
λex=420 nm. Bars represent fluorescence intensity ratio F540/F482. Black bars:
probe + various anions. Red bar: probe + anion + GSH. λex = 420 nm. Each
spectrum was acquired 120 min after various analytes addition at 25 C.
o
It is well accepted that living cells maintain high concentrations of
biothiols, especially GSH. In order to evaluate the capability of
probe for selective fluorescence detection of intracellular GSH,
fluorescence imaging in live HeLa cells was carried out. The cells
were treated with 5μM probe for 120 min, after removal of excess
probe and washed. As shown in Fig.6 (a, b and c), we could
observe not only bule fluorescence ,but weakly green
fluorescence, it showed that the probe was able to penetrate the
cell membrane and react with intracellular thiols, resulting in
green fluorescence signals. In one control experiment, HeLa cells
were pre-treated with GSH (200 μM) for 30 min, leading to an
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
RSC Advances
Page 5 of 7
View Article Online
DOI: 10.1039/C6RA04564B
Journal Name ARTICLE
2
3
Z.A. Wood, E. Schroder, J. Robin Harris, L.B. Poole,
Trends. Biochem. Sci., 2003, 28, 32ꢀ40.
J. Liu, Y.Q. Sun, Y.Y. Huo, H.X. Zhang, L.F. Wang, P.
Zhang, D. Song, Y.W. Shi, W. Guo, J. Am. Chem. Soc.,
2
014, 136, 574ꢀ577.
4
M.H. Lee, J.H. Han, P. Kwon, S. Bhuniya, J.Y. Kim, J.L.
Sessler, C. Kang, J.S. Kim, J. Am. Chem. Soc., 2012, 134,
1
316ꢀ1322.
5
6
7
H.J. Forman, H.Q. Zhang, A. Rinna, Molecular Aspects of
Medicine, 2009, 30, 1ꢀ12.
D. Han, N. Hanawa, B. Saberi, N. Kaplowitz, Am. J.
Physiol., 2006, 291(1,Pt. 1), G1ꢀG7.
D. Ścibior, M. Skrzycki, M. Podsiad, H. Czeczot, Clin.
Biochem., 2008, 41, 852ꢀ858.
8
9
S.C. Lu, Curr. Top. Cell. Regul., 2000, 36, 95ꢀ116.
K. Wang, H.J. Peng, B.H. Wang, J. Cell. Biochem., 2014,
115, 1007ꢀ1022.
Fig.5 Vibility of HeLa cells after incubation with probe at difference
concentration (0, 3, 5,10 and 20 ꢁM) for 2h based on the results from three
separate MTT assays
1
1
1
1
1
0 W. Lin, L. Yuan, Z. Cao, Y. Feng, J. Song, Angew. Chem.
Int. Ed., 2010, 49, 375ꢀ379.
1 D. Srikun, E.W. Miller, D.W. Domaille, C.J. Chang, J. Am.
Chem. Soc., 2008, 130, 4596ꢀ4597.
2 J. Liu, Y.Q. Sun, J.Y. Zhang, T. Yang, J.B. Cao, L.S. Zhang,
W. Guo, Chem. Eur. J., 2013, 19, 4717ꢀ4722.
3 C.H. Zhang, B. Peng, W. Chen, S.M. Shuang, M. Xian, C.
Dong, Dyes and Pigments, 2015, 121, 299ꢀ304.
4 M.H. Lee, J.H. Han, P.S. Kwon, S. Bhuniya, J.Y. Kim, J.L.
Sessler, C. Kang, J.S. Kim, J. Am. Chem. Soc., 2012, 134,
1316ꢀ1322.
1
5 S.Q. Wang, Q.H. Wu, H.Y. Wang, X.X. Zheng, S.L. Shen,
Y.R. Zhang, J.Y. Miao, B.X. Zhao, Biosens. Bioelectron,
2014, 55, 386ꢀ390.
1
1
6 L.L. Long, W.Y. Lin, B.B. Chen, W.S. Gao, L. Yuan, Chem.
Commun., 2011, 47, 893ꢀ895.
7 C.H. Zhang, G.M. Zhang, L.H. Feng, J.F. Li, Sensors and
Actuators B, 2015, 216, 412ꢀ417.
18 W.Y. Lin, L.L. Long, L. Yuan, Z.M. Cao, B.B. Chen, W.
Tan, Org. Lett., 2008, 10, 5577ꢀ5580.
9 X.M. Liu, N. Xi, S.J. Liu, Y. Ma, H.R. Yang, H.R. Li, J.H.
Fig.6 Fluorescence images of living Hela cells: Hela cells on 24-well plate were
incubated with probe (5 μM) for 120 min: a) blue channel, b) green channel,
and c) bright-field transmission image; HeLa cells was treated with 200μM GSH
for 30min and further incubated probe (5μM) for 120 min: d) blue channel, e)
green channel, and f) bright-field transmission image. Cells were pre-incubated
with 500μM NMM for 20 min, washed, and then treated with probe (5μM) for
1
He, Q. Zhao, F.Y. Li, W. Huang, J. Mater. Chem., 2012, 22,
7894ꢀ7901.
2
0 X.Q. Chen, S.K. Ko, M.J. Kim, I. Shin, J. Yoon, Chem.
Commun., 2010, 46, 2751ꢀ2753.
1
20 min: g) blue channel, h) green channel, and i) bright-field transmission
21 V. Hong, A.A. Kislukhin, M.G. Finn, J. Am. Chem. Soc.,
2
009, 131, 9986ꢀ9994.
image.
2
2 Y.T. Yang, F.J. Huo, C.X. Yin, A.M. Zheng, J.B. Chao,
Y.Q. Li, Z.X. Nie, R. MartínezꢀMáñez, D.S. Liu, Biosensors
and Bioelectronics, 2013, 47, 300ꢀ306.
4
Conclusions
23 X.Q. Chen, Y. Zhou, X.J. Peng, J. Yoon, Chem. Soc. Rev.
010, 39, 2120ꢀ2135.
,
2
In conclusion, we have reported the design, synthesis, and
properties of a ratiometric fluorescent probe for biothinols
detection. The probe exhibited high selectivity towards biothinols
than other non-thiol-bearing amino acids and common anions. It
was successfully applied for the GSH bioimaging in living cells.
2
4 K.H. Hong, S.Y. Lim, M.Y. Yun, J.W. Lim, J.H. Woo, H.
Kwon, H.J. Kim, Tetrahedron Lett., 2013, 54, 3003ꢀ3006.
25 X.H. Cao, Y.Q. Wu, K.Y. Liu, X.D. Yu, B. Wu, H.Z. Wu,
Z.G. Gong, T. Yi, J. Mater. Chem., 2012, 22, 2650ꢀ2657.
6 L. Song, T. Jia, W.J. Lu, N.Q. Jia, W.B. Zhang, J.H. Qian,
Org. Biomol. Chem., 2014, 12, 8422ꢀ8427.
7 P.K. Pullela, T. Chiku, M.J. Carvan, D.S. Sem, Anal.
Biochem., 2006, 352, 265ꢀ273.
2
2
Acknowledgements
28 A.M. Piggott, P. Karuso, Anal. Chem., 2007, 79, 8769ꢀ8773.
2
3
9 M.M. Pires, J. Chmielewski, Org. Lett., 2008, 10, 837ꢀ840.
0 M.H. Lee, J.Y. Kim, J.H. Han, S. Bhuniya, J.L. Sessler, C.
Kang, J.S. Kim, J. Am. Chem. Soc., 2012, 134, 12668ꢀ
The work described in this paper was supported the National
Nature Science Foundation of China (No.21475080), the Nature
Science Foundation of Shanxi Province (2014011016ꢀ2), and
Postdoctoral Science Foundation of China (2013M530120).
1
2674.
.
3
3
1 M Lelle, S. Kaloyanova, C. Freidel, M. Theodoropoulou, M.
Musheev, C. Niehrs, G. Stalla, K. Peneva, Mol.
Pharmaceutics, 2015, 12, 4290ꢀ4300.
2 P. Liu, X. Wang, K. Hiltunen, Z. Chen, ACS Appl. Mater.
Interfaces, 2015, 7, 26811ꢀ26818.
Notes and references
1
T.P. Dalton, H.G. Shertzer, A. Puga, Annual Review of
Pharmacology and Toxicology, 1999, 39, 67ꢀ101.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
RSC Advances
Page 6 of 7
View Article Online
DOI: 10.1039/C6RA04564B
ARTICLE
Journal Name
3
3
3
3
3
3 A. Wierzba, M. Wojciechowska, J. Trylska, D. Gryko,
Bioconjugate Chem., 2016, 27, 189ꢀ197.
4 H. Yang, Q. Wang, S. Huang, A. Xiao, F. Li, L. Gan, X.
Yang, ACS Appl. Mater. Interfaces, 2016, 8, 7729ꢀ7738.
5 M.H. Lee, J.L. Sessler, J.S. Kim, Acc. Chem. Res., 2015, 48,
2935ꢀ2946.
6 R.M. Duke, E.B. Veale, F.M. Pfeffer, P.E. Kruger, T.
Gunnlaugsson, Chem. Soc. Rev., 2010, 39, 3936ꢀ3953.
7 Z.C. Xu, K.H. Baek, H.N. Kim, J.N. Cui, X.H. Qian, D.R.
Spring, I. Shin, J. Yoon, J. Am. Chem. Soc., 2009, 132, 601ꢀ
6
10.
8 Z.C. Xu, J. Yoon, D.R. Spring, Chem. Commun., 2010, 46,
563ꢀ2565.
3
3
4
4
4
4
2
9 B.C. Zhu, X.L. Zhang, Y.M. Li, P.F. Wang, H.Y. Zhang,
X.Q. Zhuang, Chem. Commun., 2010, 46, 5710ꢀ5712.
0 L.A. Montoya, M.D. Pluth, Chem. Commun., 2012, 48,
4767ꢀ4769.
1 J. Jiang, H. Jiang, W. Liu, X.L. Tang, X. Zhou, W.S. Liu,
R.T. Liu, Org. Lett., 2011, 13, 4922ꢀ1925.
2 X.L. Liu, X.J. Du, C.G. Dai, Q.H. Song, J. Org. Chem.,
2014, 79, 9481ꢀ9489.
3 Y.S. Xie, P.C. Wei, X. Li, T. Hong, K. Zhang, H. Furuta, J.
Am. Chem. Soc., 2013, 135, 19119ꢀ19122.
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 7
RSC Advances
View Article Online
DOI: 10.1039/C6RA04564B