A near-infrared colorimetric fluorescent chemodosimeter for the detection of glutathione in living cells

Article abstract of DOI:10.1039/c3cc48128j

A novel near-infrared (NIR) and colorimetric fluorescent molecular probe based on a dicyanomethylene-4H-pyran chromophore for the selective detection of glutathione in living cells has been developed. The fluorescence OFF-ON switch is triggered by cleavage of the 2,4-dinitrobenzensulfonyl (DNBS) unit by the interaction with GSH.

Full text of DOI:10.1039/c3cc48128j

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A near-infrared colorimetric fluorescent
chemodosimeter for the detection of
glutathione in living cells†
Cite this: Chem. Commun., 2014,
50, 1751
Received 23rd October 2013,
Accepted 3rd December 2013
Meng Li,a Xumeng Wu,b Yao Wang,b Yongsheng Li,b Weihong Zhu*b and
Tony D. James*ab
DOI: 10.1039/c3cc48128j
www.rsc.org/chemcomm
A novel near-infrared (NIR) and colorimetric fluorescent molecular
A number of sensing mechanisms have been employed for thiol
probe based on a dicyanomethylene-4H-pyran chromophore for the detection, including Michael addition,22 to –CHO attached fluoro-
selective detection of glutathione in living cells has been developed. phores,23 and deprotection of 2,4-dinitrobenzenesulfonyl (DNBS),24–26
The fluorescence OFF–ON switch is triggered by cleavage of the etc. Among them, the DNBS group is especially preferable as an
2,4-dinitrobenzensulfonyl (DNBS) unit by the interaction with GSH. efficient recognition unit for thiols due to its unique sensitivity and
high reactivity toward thiolate anions with OFF–ON signalling. With
Biothiols such as glutathione (GSH), cysteine (Cys), and homo- these in mind, we demonstrate the development of a colorimetric and
cysteine (HCys) play a crucial role in maintaining appropriate redox NIR fluorescence turn-on thiol probe 1 (Scheme 1) containing DCM as
homeostasis in biological systems.1,2 GSH, the most abundant cellular the fluorophore and DNBS as the fluorescence quencher and recogni-
thiol, is of great importance in cellular defense against toxins and free tion moiety. Specifically, we attached a benzene unit to a dicyanopyran
radicals.3 Changes in the level of GSH concentration have been moiety, which extended the conjugated system and produced an
correlated with various diseases, such as AIDS, leukocyte loss, liver emission band centred at about 690 nm, thus successfully making the
damage, cancer and neurodegenerative diseases.4 Therefore, much wavelength fall in the desired NIR region.27 Particularly, the developed
effort has been devoted to the development of efficient methods for probe 1 exhibits several meritorious features: (i) high sensitivity and
the detection of GSH, which is of significant interest in the fields of selectivity to sulfydryl-containing species; (ii) significant enhancement
chemical, environmental and biological sciences.5,6 Many fluorescent of NIR fluorescence upon removal of DNBS by GSH; (iii) excellent
sensors for the detection of GSH have been developed based on performance in live cell imaging.
various fluorophores, including BODIPY,7,8 coumarin,9,10 rhoda-
Probe 1 is synthesized by stirring 2-(2-(4-hydroxystyryl)-4H-
mine,11,12 squaraine,13 fluorescein14,15 and lanthanide based probes.16 chromen-4-ylidene)malononitrile and 2,4-dinitrobenzene-1-sulfonyl
However, the chromophore of dicyanomethylene-4H-pyran has sel- chloride in the presence of pyridine at room temperature, to give
1
dom been exploited in the detection of thiols.17,18 Due to their probe 1 in 45% yield, which was fully characterized by H NMR,
emission located at the red or near infra-red (NIR) region, (DCM)- 13C NMR, and HRMS, as shown in the ESI† (Fig. S3 and S8). The
type derivatives are particularly suitable for application in biological GSH-promoted specific O–S cleavage of probe 1 liberates DCPOÀ
samples, since they produce lower background fluorescence with less with an intramolecular charge transfer (ICT) emission band falling
scattering, penetrate much deeper into tissues than UV and visible in the desired NIR region at about 690 nm (Scheme 1).
light and cause little damage to living cells.19,20 The DCM fluorophore
Initially, UV-vis absorption of probe 1 was investigated.
is more photostable than comparable NIR fluorophores such as Probe 1 has an intense absorption at 414 nm (Fig. 1a) in a
squaraine and cyanine. Although our previously reported BODIPY
fluorophore21 is photostable it is much harder to synthesise than the
DCM chromophore described here.
a Department of Chemistry, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
E-mail: t.d.james@bath.ac.uk; Fax: +44 (0)1225 386231; Tel: +44 (0)1225 383810
b Shanghai Key Laboratory of Functional Materials Chemistry, Key Laboratory for
Advanced Materials and Institute of Fine Chemicals, East China University of
Science & Technology, Shanghai 200237, P. R. China.
E-mail: whzhu@ecust.edu.cn; Fax: +86-21-6425-2758
† Electronic supplementary information (ESI) available: Detailed procedures,
Scheme 1 Synthesis of probe 1 and GSH-promoted release of DCPOÀ.
characterization data, and additional plots. See DOI: 10.1039/c3cc48128j
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hand, the large Stokes shift of B130 nm is also conducive to decrease
the background fluorescence, enhancing the signal fidelity. Moreover,
the response rate of 1 to GSH was tested by time-course fluorescence
measurements. In the presence of GSH, the fluorescence intensity at
690 nm increases gradually and reaches a plateau after about 5 min
(Fig. S1†), indicating the completion of the reaction.
To evaluate the selectivity of the developed probe 1 for sulfydryl-
containing species, a series of amino acids were examined. Not
surprisingly, except for GSH, probe 1 shows similar response to other
sulfydryl-containing compounds such as cysteine, homocysteine,
dithiothreitol, while no measurable fluorescence enhancement could
be triggered by the treatment of other amino acids (Fig. 2). In fact, the
plausible disturbance of DTT, Cys and HCy can be neglected due to
their relatively low concentration in biological systems.28 To gain
insight into the sensing mechanism, mass spectral analysis was
further performed. The reaction between probe 1 and GSH that
occurred by the cleavage of O–S with the release of DCPOÀ was
confirmed by the appearance of the peak at 311.0828 in the
mass spectrum (Fig. S3†). Furthermore, the fluorescence inten-
sity at 690 nm shows a linear relationship (R = 0.98) with GSH
concentrations from 1 to 10 mM, and the detection limit was
evaluated to be 1.8 Â 10À8 M, indicating that probe 1 is
particularly sensitive to the detection of GSH (Fig. S2†).
The effect of pH on the photophysical properties of DCPO and
probe 1 was then investigated (Fig. S4† and S6†). As illustrated, we
believe that the probe is quite stable in aqueous media up to pH 8,
whereas DCPO displays a corresponding fluorescence enhancement
in the pH range of 5.7–8.0, which clearly demonstrates that the
fluorescence response of probe 1 in the physiological pH range is
due to the presence of thiols and verifies the generation of DCPOÀ
after treatment with GSH. Therefore, we decided to employ the
probe for fluorescent imaging of cellular thiols. The pKa of DCPO
was calculated to be 8.70 (Fig. S5†).
Fig. 1 (a) Absorption spectra of probe 1 (1 Â 10À5 M) at different con-
centrations of GSH (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.0, 2.0, 5.0 equiv.)
in a mixture of DMSO–water (50 : 50, v/v) with a PBS buffer solution
(10 mM, pH = 7.4). Insets: colour changes of probe 1 upon addition of
GSH (5 equiv.). (b) Fluorescence spectra of probe 1 (1 Â 10À5 M) at different
concentrations of GSH when excited at 560 nm.
The ability of probe 1 to detect GSH in HeLa cells was examined
using confocal fluorescence microscopy (Fig. 3). HeLa cells were
DMSO–PBS buffer solution (pH = 7.4, 50/50, v/v) at 37 1C. Upon the
addition of GSH, the colour of the solution turned from slight yellow
to pink, which was clearly recognizable by the naked eyes. At the same
time, a concomitant increase of a new absorption band at 560 nm was
observed with an isosbestic point at 446 nm. As illustrated in Fig. 1a,
due to the specific O–S cleavage, a distinct 146 nm red shift in
absorbance was observed (Scheme 1). Since the phenolate group is a
much stronger electron donor than the sulfonate group, the ICT
efficiency should be significantly enhanced by the interaction of probe
1 with GSH and thus shifting the absorption to a longer wavelength.
Subsequently, the fluorescence spectra of probe 1 in the absence
and presence of GSH were recorded. The probe alone exhibits almost
no emission when excited at 560 nm and no distinct variations are
observed over time, suggesting that probe 1 is stable and has no
tendency to convert into DCPOÀ under the measurement conditions.
In the presence of GSH, however, fluorescence at 690 nm was
dramatically enhanced (Fig. 1b).‡ The departure of the electron-
withdrawing DNBS moiety via a GSH-induced O–S bond cleavage
releases DCPOÀ, which possesses strong ICT and induces a turn-on
NIR fluorescence response with a high off/on ratio. On the other
Fig. 2 Fluorescence spectra of probe 1 in the absence and presence of
various amino acids and GSH, DTT, Cys, HCys in a mixture of DMSO–water
(50 : 50, v/v) with a PBS buffer solution (10 mM, pH = 7.4). amino acids are
arginine, threonine, serine, isoleucine, asparaginic acid, sarcosine, valine,
glutamine, tryptophan, glutamic acid, proline, leucine, histidine, glycine,
phenylalanine, alanine, asparagine, methionine.
1752 | Chem. Commun., 2014, 50, 1751--1753
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(CASE) network is thanked for research exchange opportunities.
W.H.Z. is grateful for financial support from the Oriental
Scholarship, and the Fundamental Research Funds for the Central
Universities (WK1013002). T.D.J thanks ECUST for a guest
professorship. T.D.J. also thanks the Royal Society for support.
Notes and references
‡ When probe 1 is excited at the isosbestic point (446 nm, Fig. S7†) a
fluorescence increase at 581 nm is observed which is ascribed to release
of the DNBS quenching group. Conversely, when probe 1 is excited at
560 nm the large observed increase in fluorescence at 690 nm is due
to the fluorescence of the DCPOÀ produced upon cleavage of DNBS
from probe 1.
Fig. 3 Confocal fluorescence images in HeLa cells: (top) (A–C) cell
incubated without probe 1; (bottom) (D–F) cell incubated with probe 1
(20 mM) for 0.5 h. Emission was collected at 660–740 nm upon excitation
at 488 nm. Bright field (A and D), fluorescence (B and E) and overlap field (C
and F).
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