A new turn-on fluorescent probe for selective detection of glutathione and cysteine in living cells

Article abstract of DOI:10.1039/c3cc39045d

A fluorescent probe (N-(4-methyl-2-oxo-2H-chromen-7-yl)-2,4- dinitrobenzenesulfonamide), which exhibits high selectivity to glutathione and cysteine among amino acids including sulphur-containing methionine and metal ions, was synthesized. The experiments demonstrate that the fluorescent probe is a reliable and specific probe for glutathione and cysteine in living cells. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc39045d

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A new turn-on fluorescent probe for selective
detection of glutathione and cysteine in living cells†
Cite this: Chem. Commun., 2013,
49, 4640
Mingjie Wei, Peng Yin, Youming Shen, Lingli Zhang, Jianhui Deng, Shanyan Xue,
Haitao Li, Bin Guo, Youyu Zhang* and Shouzhuo Yao
Received 18th December 2012,
Accepted 27th March 2013
DOI: 10.1039/c3cc39045d
www.rsc.org/chemcomm
A fluorescent probe (N-(4-methyl-2-oxo-2H-chromen-7-yl)-2,4-dinitro-
Initially, we proposed the optical probe (N-(4-methyl-2-oxo-
benzenesulfonamide), which exhibits high selectivity to glutathione 2H-chromen-7-yl)-2,4-dinitrobenzenesulfonamide) (NDS), with
and cysteine among amino acids including sulphur-containing the coumarin moiety and a strong electron-withdrawing group
methionine and metal ions, was synthesized. The experiments that could be applied for the detection of such a reactive
demonstrate that the fluorescent probe is a reliable and specific nucleophile as GSH or Cys. The coumarin moiety is a fluores-
probe for glutathione and cysteine in living cells.
cence signaling unit, and the intramolecular charge-transfer
(ICT) process occurs in NDS molecule because of the introduc-
The development of detection methods for thiols has attracted tion of the strong electron-withdrawing group, 2,4-dinitro-
continuing interest because of their important roles in biological benzenesulfonyl12 (Scheme 1). When electrons are diverted
and pharmacological processes. There are several analytical methods from 7-amino-4-methylcoumarin to 2,4-dinitrobenzenesulfo-
reported for the analysis of thiols in biological samples, such as nyl, the fluorescence of NDS becomes very weak. However,
high performance liquid chromatography,1 electrophoresis-based upon removal of the electron-withdrawing 2,4-dinitrobenzene-
methods,2 flow-injection analysis,3 spectrophotometry,4 voltam- sulfonyl, the fluorescence is turned on. The sulphur anion
metry5 as well as electrospray ionization-mass spectrometry.6 (RSÀ) is the nucleophilic center in biothiols (RSH). When
However, these conventional methods are complex and expensive. compounds containing sulfhydryl such as GSH and Cys coexist
In recent years, some fluorescence detection methods have with NDS under appropriate conditions, the removal of 2,4-
been reported.7 Qu’s group reported a facile, label-free, highly dinitrobenzenesulfonyl from NDS will occur, resulting in a high
sensitive sensor for detection of biothiols based on silver fluorescent signal. NDS was synthesized by a nucleophilic
metallization engineered conformational transition of the reaction of aniline-appended coumarin with 2,4-dinitrobenze-
G-quadruplex.8 Ma and colleagues developed a luminescent nesulfonyl chloride. The synthesis processes and structure
G-quadruplex switch-on probe for selective and tunable detec- characterization (using 1H NMR, 13C NMR, MS and IR) of
tion of cysteine (Cys) and glutathione (GSH).9 Hepel and Xu NDS are provided in the ESI.†
reported a fluorescence turn-on ‘‘molecular beacon’’ for detec-
The fluorescence of NDS in the absence and presence of
tion of GSH and Cys.10 Moreover, the fluorescent probes have GSH/Cys was investigated. As shown in Fig. 1, in the absence
become an important diagnostic tool due to their versatile of GSH/Cys, the fluorescence of NDS was very weak (curve a
applications in chemical, biological and environmental in Fig. 1). The fluorescence intensity was dramatically
fields.11 However, most of the previously reported fluorescent enhanced in the presence of GSH/Cys (curve b in Fig. 1),
sensors for GSH and Cys also have marked responses to resulting from the effective cleavage of the electron-withdrawing
homocysteine (Hcy) and have low selectivity to GSH and Cys.
Some of them have a relatively low sensitivity. There is an
urgent need to develop novel reliable analytical methods.
Herein, we report a new and simple strategy for the determina-
tion of GSH and Cys both in vivo and vitro.
Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research
(Ministry of Education), College of Chemistry and Chemical Engineering,
Hunan Normal University, Changsha 410081, PR China.
E-mail: zhangyy@hunnu.edu.cn; Fax: +86 73188872531; Tel: +86 75188865515
† Electronic supplementary information (ESI) available: Synthesis of the probe
NDS and experimental details. See DOI: 10.1039/c3cc39045d
Scheme 1 The principle for the detection of GSH/Cys with NDS.
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Fig. 1 Fluorescence emission spectra (lex = 353 nm) of NDS before (a) and after
the addition of GSH (A) or Cys (B) in phosphate buffer (pH 9.0, 0.01 M). The
concentrations of NDS and GSH/Cys were 20 mM and 30 mM, respectively. The
insets show the photos of samples illuminated by UV light of 365 nm.
2,4-dinitrobenzenesulfonyl moiety from the weakly fluorescent
probe NDS.13 The reaction of GSH with NDS was investigated
and described in the ESI.† As expected, the electron-withdrawing
2,4-dinitrobenzene-sulfonyl moiety was effectively dropped from
DNS after the reaction, and compound 1 and compound 2 were
generated, which were confirmed using NMR (Fig. S5–S8 in
ESI†).14 As shown in the inset of Fig. 1, the probe emitted bright
blue light under UV light excitation in the presence of GSH and
Cys, suggesting that the fluorescence product was generated in
the test system.
Fig. 2 Fluorescence spectra of NDS in the presence of various concentrations of
GSH (A) and Cys (C). A linear relationship of the fluorescence intensity vs. the
concentrations of GSH (B) and Cys (D). The concentrations of GSH and Cys were
from 30 nM to 60 mM. The error bars represent the standard deviation of three
independent measurements.
regression equation for GSH, F = 0.0189 + 0.0161x, was
obtained, where F refers to the measured fluorescence intensity
and x refers to the concentration of GSH. The linear correlation
coefficient was 0.9966. For Cys detection, the linear regression
equation was F = 0.0225 + 0.0169x, with a linear correlation
coefficient of 0.9956.
To evaluate the selectivity of the developed method for
GSH/Cys, a series of metal ions, amino acids and bovine serum
albumin (BSA) were detected. The results are shown in Fig. 3.
Only GSH and Cys can cause remarkable fluorescence responses.
Because no thiol anion was produced in this test system, other
amino acids including sulfur-containing methionine and metal
ions had no apparent interference in the detection. 50 mM Hcy
only caused a little fluorescence response. Under the experi-
mental conditions, the capability of GSH, Cys and Hcy to form
RSÀ is of the order GSH > Cys > Hcy (their corresponding pKa
are 9.12, 10.29 and 10.86, respectively).16 Theoretically, the
response of GSH should be greater than Cys. But due to the
greater steric hindrance of GSH, the response of GSH was in
Considering the factors affecting the reaction of NDS with
GSH/Cys, pH, incubation temperature and time were optimized
(Fig. S9, ESI†). As shown in Fig. S9A (ESI†), the fluorescence of
NDS increased with increasing pH value and reached the
maximum value at pH 9.0. This fact reflects the influence of
the pKa value of aliphatic thiols on the reaction.15 In a reaction
medium, GSH/Cys can be ionized to a reactive nucleophilic
neutral form (RSÀ). The degree of dissociation of the thiol
group in GSH/Cys depends on the pH of the medium. The
concentration of RSÀ increases with the increasing in the pH
value. As expected, a dramatic enhancement of the fluorescence
intensity was observed as a result of the effective cleavage of the
electron-withdrawing 2,4-dinitrobenzene-sulfonyl moiety from
the weakly fluorescent probe NDS by GSH/Cys in an alkaline
solution. Therefore, a pH 9.0 phosphate buffer was selected as
the working buffer in the following experiments. Furthermore,
the effects of incubation temperature and time were investi-
gated. The experimental result suggested that the optimum
temperature was 45 1C (Fig. S9B in ESI†). Therefore, 45 1C was
chosen as the favorable incubation temperature for the detec-
tion of GSH/Cys. As shown in Fig. S9C (ESI†), the fluorescence
intensity at 450 nm increased sharply upon increasing the
incubation time from 0 h to 2 h and then reached a constant
value after 2 h. This demonstrates that the interaction of NDS
with GSH/Cys reached equilibrium within 2 h. Thus, the
incubation time was controlled at 2 h. Furthermore, no appar-
ent hydrolysis of NDS was detected in the above experimental
conditions.
Under the optimal conditions, we evaluated the capability of
the proposed method for quantitative detection of GSH/Cys.
The fluorescence responses of NDS with different concentra-
tions of GSH and Cys are shown in Fig. 2. A linear relationship
between the fluorescence intensity and the GSH/Cys concen-
Fig. 3 Fluorescence responses of NDS to different analytes. The concentration of
NDS was 20 mM. GSH, Cys and BSA were used at 20 mM, Hcy at 50 mM and other
tration ranging from 30 nM to 60 mM was obtained. And, a linear analytes were used at 1 mM.
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NDS, is a reactive probe of incision-type and can be used to
detect GSH and Cys both in vivo and in vitro. The probe has a
low background signal and good sensitivity to GSH and Cys.
The developed method exhibits a good selectivity for GSH/Cys
over a series of amino acids, metal ions and protein. The
application of the probe for the detection of GSH/Cys in living
cells has been well-demonstrated, which shows potential appli-
cations in disease diagnosis.
This work was supported by the National Natural Science
Foundation of China (21275051, 20975037, 21005037), Scientific
Research Fund of Hunan Provincial Education Department
(12A084) and Aid program for Science and Technology Inno-
vative Research Team in Higher Educational Institutions of
Hunan Province.
Fig. 4 Fluorescence microscopy images of Hela cells. (b) Images of cells pre-
treated with N-methylmaleimide (0.5 mM) for 1 h at 37 1C and then incubated
with NDS (30 mM) overnight at 37 1C. (d) Images of cells incubated with NDS
(30 mM) overnight at 37 1C. (A) and (C) represent the bright-field images of (B)
and (D), respectively.
fact slightly smaller than Cys. Hcy should be the least negatively
charged among the three analytes because of its highest pKa
value, so the response of Hcy is much lower than GSH/Cys. The
reaction of NDS with BSA did not show any increase in the
fluorescence intensity. This can be explained by the fact that
most of the cysteines in BSA participate in disulfide bonds and
BSA has a large steric hindrance.17 These results clearly demon-
strate that our proposed sensing system is specific for GSH
and Cys.
In order to evaluate the function of NDS for biological
applications, and clarify whether it is sensitive enough to detect
the physiologically relevant level of GSH/Cys in living cells, we
performed an assay to detect intracellular GSH/Cys in living
cells. Hela cells were incubated with a solution of NDS (30 mM,
1 : 99 DMSO–PBS v/v, pH 7.4) overnight at 37 1C. The results of
fluorescence microscopic observations are shown in Fig. 4.
It was found that NDS was cell-permeable and could react with
intracellular thiols, resulting in strong fluorescence emission
(Fig. 4D). The results clearly demonstrate that NDS was able to
sense the thiols in living cells. In a control experiment, cells
were pretreated with an excess of the thiol-reactive N-methyl-
maleimide to consume all of the free thiols within the cell, and
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The results confirm that the fluorescence changes in the
Hela cells really resulted from the change of the intracellular
thiol level. The bright-field images (Fig. 4A and C) confirm that
the cells were viable throughout the imaging experiments.
These results demonstrate that NDS is a specific probe for
detection of thiols in living cells. The method described here
might provide a simple way to monitor alterations of thiol
concentration in living cells.
In summary, we synthesized a novel fluorescent probe NDS
for selective detection of GSH/Cys. The new synthesized probe,
c
4642 Chem. Commun., 2013, 49, 4640--4642
This journal is The Royal Society of Chemistry 2013