A novel colorimetric and ratiometric NIR fluorescent sensor for glutathione based on dicyanomethylene-4H-pyran in living cells

Article abstract of DOI:10.1007/s11426-015-5490-y

Glutathione (GSH) plays a critical role in maintaining oxidation-reduction homeostasis in biological systems. Considering the detection of GSH by fluorescence sensors is limited by either the short wavelength emission or the poor photostability, a highly stable colorimetric and ratiometric NIR fluorescent sensor (DCM-S) for GSH detection has been constructed on the basis of dicyanomethylene-4H-pyran (DCM) chromophore. The specific disulfide bond is incorporated via a carbamate linker as the GSH responsive group, which simultaneously blue-shifts and quenches the fluorescence. Upon addition of GSH, DCM-S exhibits outstanding colorimetric (from yellow to red) and ratiometric fluorescent response with the 6-fold enhancement of NIR fluorescence at 665 nm in quantum yield. More importantly, the GSH-treated DCM-S (DCM-NH₂ actually) possesses 20-fold longer fluorescence half-life period as well as much better photostability than the FDA-approved ICG. Finally, the ratiometric detection of GSH is also successfully operated in the living cell imaging, exhibiting NIR fluorescence and large Stokes shift (215 nm) with nearly no background fluorescence interference. As a consequence, DCM-S can be utilized as colorimetric and ratiometric NIR fluorescent sensor for GSH, with a great potential in the development of GSH-induced drug delivery system.

Full text of DOI:10.1007/s11426-015-5490-y

SCIENCE CHINA
Chemistry
January 2016 Vol.59 No.1: 1–8
doi: 10.1007/s11426-015-5490-y
• ARTICLES •
· SPECIAL TOPIC · Chemical/Biological Sensors and Imaging
doi: 10.1007/s11426-015-5490-y
A novel colorimetric and ratiometric NIR fluorescent sensor for
glutathione based on dicyanomethylene-4H-pyran in living cells
Xumeng Wu, Andong Shao, Shiqin Zhu, Zhiqian Guo & Weihong Zhu*
Shanghai Key Laboratory of Functional Materials Chemistry; Key Laboratory for Advanced Materials and Institute of Fine Chemicals;
East China University of Science and Technology, Shanghai 200237, China
Received June 22, 2015; accepted July 15, 2015
Glutathione (GSH) plays a critical role in maintaining oxidation-reduction homeostasis in biological systems. Considering the
detection of GSH by fluorescence sensors is limited by either the short wavelength emission or the poor photostability, a high-
ly stable colorimetric and ratiometric NIR fluorescent sensor (DCM-S) for GSH detection has been constructed on the basis of
dicyanomethylene-4H-pyran (DCM) chromophore. The specific disulfide bond is incorporated via a carbamate linker as the
GSH responsive group, which simultaneously blue-shifts and quenches the fluorescence. Upon addition of GSH, DCM-S ex-
hibits outstanding colorimetric (from yellow to red) and ratiometric fluorescent response with the 6-fold enhancement of NIR
fluorescence at 665 nm in quantum yield. More importantly, the GSH-treated DCM-S (DCM-NH2 actually) possesses 20-fold
longer fluorescence half-life period as well as much better photostability than the FDA-approved ICG. Finally, the ratiometric
detection of GSH is also successfully operated in the living cell imaging, exhibiting NIR fluorescence and large Stokes shift
(215 nm) with nearly no background fluorescence interference. As a consequence, DCM-S can be utilized as colorimetric and
ratiometric NIR fluorescent sensor for GSH, with a great potential in the development of GSH-induced drug delivery system.
near-infrared, GSH, ratiometric, colorimetric, fluorescent sensor
Citation:
Wu XM, Shao AD, Zhu SQ, Guo ZQ, Zhu WH. A novel colorimetric and ratiometric NIR fluorescent sensor for glutathione based on dicyanometh-
ylene-4H-pyran in living cells. Sci China Chem, doi: 10.1007/s11426-015-5490-y
disease, Alzheimer’s disease [7,8]. In order to further ex-
1 Introduction
plore the function of GSH in biology, it is of great signifi-
cance to monitor GSH at the cell, tissue and organism level.
Accor- dingly, a variety of analytical methods for GSH de-
termination have been developed, such as high performance
liquid chromatography (HPLC) [9], electrophoresis-based
methods [10], flow-injection analysis [11] and voltammetry
[12]. However, these conventional detection technologies
generally suffer from severe drawbacks including either
complicated operation procedures or expensive equipments.
Recently, chemosensors based on fluorescence imaging
have attracted considerable attention because of the highly
sensitive and non-invasive imaging, and the avoidance of
sophisticated instrumentation or time-consuming sample
preparation [13–16]. Indeed, a variety of fluorescent sensors
Intracellular reducing thiol entities are well-known to be of
great importance in biology [1–3]. Among them, glutathi-
one (GSH) is the most abundant cellular thiol with concen-
tration of 1 to 15 mmol/L. It plays a crucial role in main-
taining oxidation-reduction (redox) homeostasis in biologi-
cal systems to avoid or repair oxidative damage leading to
dysfunction and cell death via the equilibrium between the
thiol form (GSH) and the disulfide form (GSSG) [4–6].
Imbalance of GSH/GSSG ratios in cells is directly associ-
ated with various diseases including cancer, Parkinson’s
*Corresponding author (email: whzhu@ecust.edu.cn)
© Science China Press and Springer-Verlag Berlin Heidelberg 2015
chem.scichina.com link.springer.com

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Wu et al. Sci China Chem January (2016) Vol.59 No.1
for GSH detection have been established based on fluoro-
phores such as naphthalimide [17], fluorescein [18], rhoda-
mine [19], BODIPY [20,21], coumarin [22] and squaraine
[23], and functional groups that respond to GSH. However,
the previously reported examples generally utilized fluores-
cence in the visible region as the response signal, which
results in the bottleneck of penetration depth and auto-
fluorescence background and hinders the practical applica-
tion of them in practical biological systems. In this respect,
fluorophores that possess intrinsic fluorescence in the
near-infrared (NIR) region are of particular interest, taking
advantage of the minimal interfering fluorescence from bi-
ological samples, low scattering, deep penetration into tis-
sues and ignorable damage to living cells [24–28]. Never-
theless, the application of the most frequently used NIR
fluorophore such as ICG (indocyanine green), which has
been approved for clinical application by US Food and Drug
Administration (FDA), still has the thorny hurdle of the poor
photo-stability [29–32]. Accordingly, it is particularly de-
sirable to develop fluorescent sensors for detecting GSH with
high selectivity and sensitivity, utilizing NIR emissive and
highly photo-stable fluorophores as the signaling subunit.
Dicyanomethylene-4H-pyran (DCM), as typical donor-
-acceptor (D--A) featured chromophore, has been inten-
sively focused in fields of non-linear optic materials [33],
logic gates [34,35] and photovoltaic sensitizers [36] due to
its long wavelength emission in the NIR region, large
Stokes shift resulted from the ultra-fast intramolecular
charge transfer (ICT) and fairly high photo-stability [37,38].
Nonetheless, it has rarely been applied in the detection of
GSH in living cells [39,40]. Bearing all these in mind, a
novel DCM derivative, named DCM-S, was constructed as
colorimetric and ratiometric NIR fluorescent sensor for
GSH with high selectivity and sensitivity. Enlightened by
the disulfide linker based drug delivery systems [41,42], a
disulfide bond is strategically conjugated with DCM-NH2
(highly NIR fluorescent) via a carbamate linker which sim-
ultaneously blue-shifts and quenches the fluorescence. It is
proposed to undergo a chemical transformation to regener-
ate the free DCM-NH2 under the interaction with GSH. Ex-
citingly, the novel GSH fluorescent sensor DCM-S features
several meritorious characteristics: (1) colorimetric and out-
standing ratiometric fluorescent detection of GSH with the
enhancement of NIR fluorescence at 665 nm; (2) 20-fold
longer fluorescence half-life as well as much better photo-
stability than ICG under continuous illumination; and (3)
successful application in ratiometric fluorescent detection of
cellular GSH. Notably, compared with fluorescence
intensity-based measurements, which may be disturbed by
the probe concentration, auto-fluorescence background and
equipment factor, the ratiometric approach could eliminate
the disadvantages to achieve more precise detection by cal-
culating the ratio of fluorescence intensities at two different
wavelengths.
2 Results and discussion
The synthetic routes for DCM-S and DCM-C are depicted
in Scheme 1 and Scheme S1 (Supporting Information
online). The intermediate compound 2-(2-methyl-4H-
chromen-4-ylidene)malononitrile was synthesized accord-
ing to the established procedures, utilizing 2-acetyl-phenol
as the starting material [43,44]. DCM-NH2 was generated
by Knoevenagel condensation of DCM and 4-acetami-
dobenzaldehyde in toluene followed by the deprotection of
amino unit with concentrated hydrochloric acid in ethanol.
Subsequently, DCM-NH2 was reacted with triphosgene
Scheme 1 Synthetic route from DCM-NH2 to DCM-S or DCM-C, and molecular structure of reference ICG.

Wu et al. Sci China Chem January (2016) Vol.59 No.1
3
in the presence of N,N′-diisopropylethylamine (DIEA) and
treated with 2,2′-dithiodiethanol to afford DCM-S in toluene
[45,46]. The GSH-cleavable disulfide bond and the carba-
mate linker were anticipated as the GSH-response site and
fluorescence quencher, respectively. Meanwhile, the control
compound DCM-C was synthesized via the same route us-
ing 1,6-hexanediol instead of 2,2′-dithiodiethanol. Their
comparatively strong NIR fluorescence at 665 nm (Figure
S1, Supporting Information online). The distinctly different
emission property and significant red-shift in both absorp-
tion and fluorescence spectra of DCM-NH2 relative to
DCM-S and DCM-C could be dominantly attributed to the
formation of the amide structure that precludes the electron-
donating capacity of the nitrogen atom. Moreover, the limit
of detection of GSH by DCM-S was calculated to be
2.4×105 mol/L via linear fitting of the plot ((RRmin)/
(RmaxRmin) vs. log[GSH]), where the R is the fluorescence
intensity ratio of 665 to 568 nm.
Subsequently, to further inspect and compare the GSH
response of DCM-S and DCM-C, they were both allowed to
be treated with GSH in the DMSO/PBS solution at 37 °C
for 1 h. Obviously, upon the treatment with GSH, a 57 nm
red-shift in the absorption spectra of DCM-S was observed,
along with a distinct yellow-to-red change in solution color
observed by the naked eyes (Figure 1(c)), presenting a
slightly ambiguous isobestic point at 450 nm and allowing
the colorimetric detection of GSH. More excitingly, utiliz-
ing the isobestic point (450 nm) as the excitation wave-
length, the fluorescence of DCM-S was simultaneously
red-shifted to 665 nm in the NIR region with a clear isos-
bestic point at 594 nm, and significantly enhanced (Figure
molecular structures were fully characterized by 1H and 13
C
NMR, and by HRMS (see the Supporting Information
online).
Initially, the photophysical properties of DCM-S and
DCM-C in the absence and presence of GSH were investi-
gated to verify the feasibility of GSH response. The 1:1
mixed system of dimethyl sulfoxide (DMSO) and PBS
buffer solution (pH 7.4, 10 mmol/L) was exploited as the
solvent system to simulate the physiological environment
while guaranteeing sufficient solubility. As shown in Figure
1, DCM-S and DCM-C exhibit similar photophysical prop-
erties: typical broad absorption band of ICT-featured fluor-
ophores that locate at 435 nm (=2.20×104 L/(mol cm)) and
448 nm (=2.17×104 L/(mol cm)) with fairly faint fluores-
cence centered at 568 and 566 nm, respectively. Meanwhile,
under the same condition, DCM-NH2 displays a broad ab-
sorption band at 492 nm (=2.26×104 L/(mol cm)) with
Figure 1 (a, b) Absorption (a) and fluorescence spectra (b) of DCM-S and DCM-C (10 μmol/L) in DMSO/PBS (v/v=50:50, pH 7.4, 10 mmol/L) with and
without 250 equiv. of GSH; (c) absorption spectra of DCM-S (10 μmol/L) in DMSO/PBS (v/v=50:50, pH 7.4, 10 mmol/L) with 0, 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 50, 100 equiv. of GSH, each spectra were recorded 1 h after exposure at 37 °C (Inset: color change of solutions of DCM-S); (d) fluorescence spectra
of DCM-S (10 μmol/L) in DMSO/PBS (v/v=50:50, pH 7.4, 10 mmol/L) with 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 50, 100 equiv. of GSH, excitation wave-
length at 450 nm (Inset: fluorescence observed from solutions of DCM-S with and without GSH). Each spectral curve was recorded 1 h after exposure at
37 °C. (color online)

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Wu et al. Sci China Chem January (2016) Vol.59 No.1
1(d)). Accordingly, the fluorescence quantum yield of
GSH-treated DCM-S is more than 6-fold higher than free
DCM-S. Moreover, a linear relationship between the fluo-
rescence intensity ratio of 665 to 568 nm and the GSH con-
centration up to 200 μmol/L was observed. The linear re-
gression equation, y=–0.122+1.3108x, was obtained with
the linear correlation coefficient is 0.9952, where y is the
calculated fluorescence intensity ratio and x is the equiva-
lent of GSH (Figure 2). Conversely, upon the treatment by
GSH, the reference compound DCM-C exhibited nearly no
alteration in either absorption or fluorescence spectra (Fig-
ure 1(a, b)), clearly demonstrating the crucial role played by
the disulfide bond in the GSH response capacity of DCM-S.
Conclusively, DCM-S could act as highly qualified colori-
metric and ratiometric NIR fluorescent probe for GSH.
In order to validate the reaction mechanism of DCM-S
with GSH, spectroscopic properties of DCM-NH2, which is
the anticipated product of the GSH-induced reaction, were
compared with that of GSH-treated DCM-S. Clearly, after
normalization, high coincidence of their absorption and flu-
orescence spectra is observed. The identical peak positions
and shapes convince us to believe that, after cleavage of the
disulfide bond by GSH, an intramolecular cyclization gen-
erates DCM-NH2 as both the reaction product and NIR flu-
orescence emitter (Figure S2). Moreover, by comparing the
HPLC retention time (Figure S3) of the reaction mixture
with independently measured DCM-S (retention time 7.14
min) and DCM-NH2 (retention time 6.15 min), the genera-
tion of DCM-NH2 as the direct result of the two-step reac-
tion, was proved again (Scheme 2). Therefore, the red-shift
(to NIR region) and significant enhancement of the fluores-
cence of DCM-S could be reasonably ascribed to the gener-
ation of amino unit (DCM-NH2) which is a much stronger
electron donor than the amide group. Importantly, the fluo-
rescence of DCM-NH2 locates in the NIR region (650–900
nm), which is especially preferred in bioimaging because of
the outstanding penetration depth and low background auto-
fluorescence in vivo. Furthermore, the extremely large
Stokes shift (215 nm), derived from the ICT process in the
Figure 2 Ratiometric calibration curve I665 nm/I568
for DCM-S (10
nm
μmol/L, DMSO/PBS=1:1, pH 7.4, 10 mmol/L) as a function of equivalent
of GSH, excitation wavelength at 450 nm, each spectral curve was record-
ed 1 h after exposure at 37 °C.
D--A featured structure, benefits for high quality fluores-
cence imaging because of the improvement in the signal
fidelity [47–49].
As one of the most severe barriers in the practical appli-
cation of organic contrast agent [50], photostability of
DCM-S was subsequently evaluated with respect to the
commercially available ICG (the FDA approved contrast
agent) by monitoring the fluorescence spectra upon contin-
uous irradiation (Hamamatsu, LC8 Lightningcure, 300 W).
Merely after illumination for 2 min, the fluorescence of ICG
was quenched almost completely to the baseline, indicative
of the rapid photo-degradation of the scaffold of ICG (Fig-
ure 3(a)). In contrast, undergoing the same irradiation,
DCM-S maintained more than 80% of the initial fluores-
cence (Figure 3(a)). Estimated directly from the time-course
fluorescence measurements, the fluorescence half-life peri-
od of GSH-treated DCM-S (~500 s) is 20-fold longer than
that of ICG (~25 s). Clearly, the photostability of GSH-
treated DCM-S, DCM-NH2 in practice, is much better than
Scheme 2 Reaction mechanism of DCM-S with GSH.

Wu et al. Sci China Chem January (2016) Vol.59 No.1
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Figure 3 (a) Time dependence of the fluorescence intensity of ICG (10 μmol/L in DMSO/PBS=1:1, monitored at 812 nm and excited at 780 nm), DCM-S
(10 μmol/L in DMSO/PBS=1:1, monitored at 665 nm and excited at 450 nm) and DCM-NH2 (10 μmol/L in DMSO/PBS=1:1, monitored at 665 nm and
excited at 450 nm) under illumination, treated with GSH for 1 h at 37 °C before illumination; (b) time dependence of fluorescence intensity at 665 nm of
DCM-S and DCM-C (10 μmol/L) in DMSO/PBS solution (v/v=50:50, pH 7.4, 10 mmol/L) with or without various equivalents of GSH (excitation wave-
length at 450 nm; data was recorded every 0.5 s). (color online)
the FDA-approved ICG, making DCM-S more qualified
GSH probe for bioimaging. On the other hand, the time-
dependent fluorescence measurements of DCM-S and
DCM-C in the absence and presence of GSH were also in-
vestigated. Apparently, the fairly weak fluorescence of free
DCM-S has no variation over time, suggesting that DCM-S
is stable and has no tendency to convert into DCM-NH2
spontaneously. Moreover, the fluorescence intensity at
665 nm, proved to be resulted from DCM-NH2, gradually
increased to a plateau within 20 min which is mildly accel-
erated along with addition of more GSH (Figure 3(b)). Oth-
erwise, in the case of DCM-C, even in the presence of GSH,
the constant and fairly weak fluorescence intensity effec-
tively confirmed the specific reaction between the disulfide
bond and GSH again.
tioned. Not surprisingly, except for GSH, DCM-S exhibits
similar spectroscopic response upon the treatment with
other thiol-containing species including 1,4-dithiothreitol
(DTT), cysteine (Cys) and homocysteine (Hcy). Meanwhile,
all the other analytes including non-thiol amino acid and
biologically abundant metal ions could hardly induce the
fluorescence enhancement, establishing the key status of
thiol as the functional group in the reaction with disulfide
bond (Figure 4(a)). Actually, because of the relatively high
physiological concentration of GSH in cytoplasm (1–15
mmol/ L), the seeming interference of DTT, Cys and Hcy
could be sufficiently neglected. In accordance with the
aforementioned reaction mechanism, DCM-C with the more
stable “C–C” bond exhibits no response to all the amino
acids and metal ions, remaining the weak fluorescence at
566 nm (Figure 4(b)). Moreover, the pH-dependence of the
fluorescence turn-on response of DCM-S to GSH was also
evaluated. Firstly, in the whole testing range of pH (3.5–9),
DCM-S remains stable as well as non-fluorescent and pos-
sesses intact aforementioned NIR fluorescence off-on
response to GSH in the pH range from 6 to 9 which is coin-
To further investigate the potential application of DCM-S
in detecting cellular GSH, the performance of DCM-S in the
presence of biological interference such as amino acids and
abundant metal ions should be determined as the precondi-
tion. The probe DCM-S was treated with various amino
acids and metal ions under the same condition as aforemen-
Figure 4 Fluorescence of DCM-S (a, 10 μmol/L) and DCM-C (b, 10 μmol/L) in DMSO/PBS (v/v=50:50, pH 7.4, 10 mmol/L) upon addition of GSH, DTT,
Cys, Hcy, K+, Ca2+, Na+, Mg2+ and other amino acids including Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Phe, Pro, Ser, Thr, Tro, Tyr, Val (2.5
mmol/L). Excitation wavelength 450 nm, each spectral curve was recorded 1 h after exposure at 37 °C. (color online)

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Wu et al. Sci China Chem January (2016) Vol.59 No.1
cide with the biological environment (Figure S4). Therefore,
DCM-S is equipped with the promising potential to be uti-
lized as GSH probe in real biological environment with se-
lectivity over various biological interference including
amino acids and metal ions.
ratiometric mode (Figure 5(d, h)).
Finally, in order to provide further evidence of the GSH-
induced cleavage of the disulfide bond, DCM-S was incu-
bated with HeLa cells that were pretreated with 10 mmol/L
of strong thiol reacting agent N-ethylmalemide (NEM) or
not. Obviously, the cells successively treated with NEM and
DCM-S exhibit similar fluorescence with the case of cells
incubated with DCM-C, of which the fluorescence ratio of
the red channel to green channel was less than 1 (Figure
6(e–h)). What is the same with previous results is that the
cells treated with only DCM-S display bright NIR fluores-
cence at the red channel with weak fluorescence at the green
channel (Figure 6(a–d)). The above results demonstrate the
essential role played by intracellular GSH in the change of
photophysical properties of DCM-S by the cleavage of the
disulfide bond.
Subsequently, the novel GSH probe DCM-S was utilized
to HeLa cells (human cervical cancer cell) with the help of a
confocal fluorescence microscopy to explore its practical
application in bioimaging and compared with DCM-C. Dis-
tinctly, after incubation with 20 μmol/L of DCM-S for 40
min at 37 °C, sufficient intracellular fluorescence signal can
already be detected within the channel of 660–750 nm (red
channel, Figure 5(c)). Meanwhile, the fluorescence intensity
obtained at 480–520 nm (green channel, Figure 5(b)) was
fairly weak. The overlay of fluorescence and bright-field
images reveals that the fluorescence signals were localized
in the perinuclear area of the cytosol, reflecting a subcellu-
lar distribution of GSH (Figure 5(a–c)). This result is well
in line with the spectroscopic results and demonstrates that
DCM-S is living cell membrane permeable and could react
with intracellular GSH, resulting in strong NIR fluorescence
emission. In the control experiment, cells incubated with
DCM-C instead of DCM-S exhibited nearly no intracellular
fluorescence at the NIR channel (660–750 nm) with slightly
stronger fluorescence at the green channel (Figure 5(e–g)).
Given similar fluorescence property of free DCM-S and
DCM-C, the much stronger fluorescence at the red channel
of DCM-S treated cells undoubtedly stemmed from the
GSH-induced generation of DCM-NH2. Otherwise, bright-
field measurements confirm that the cells after treatment
with both DCM-S and DCM-C were viable throughout the
imaging experiments. The fluorescence ratio images of the
Hela cells co-cultured with DCM-S and DCM-C illustrated
the ability of DCM-S to image GSH in living cells in a
3 Conclusions
In summary, a novel colorimetric and ratiometric NIR fluo-
rescent sensor for GSH using dicyanomethylene-4H-pyran
as the NIR fluorophore was constructed. Disulfide bond
which has specific response to GSH was incorporated by a
carbamate linker to simultaneously blue-shift and quench
the fluorescence. Upon the interaction of GSH, the disulfide
bond in DCM-S could be cleaved and further leaves via an
intramolecular cyclization, presenting distinct colorimetric
(yellow to red) and ratiometric fluorescence response with a
6-fold enhancement of NIR fluorescence at 665 nm. Com-
pared to the previously reported GSH sensors which gener-
ally have the bottleneck of poor photostability, the
GSH-treated DCM-S (DCM-NH2 in fact) exhibits excellent
photostability: 20-fold longer fluorescence half-life period
Figure 5 Confocal laser scanning microscopy images of HeLa cells incubated with DCM-S (20 μmol/L) (a–d) and DCM-C (20 μmol/L) (e–h) for 40 min.
Images were taken from the red channel (660–750 nm) (c, g), green channel (480–520 nm) (b, f) and the overlapped image (a, e), excitation wavelength at
488 nm, and (d, h) was the ratio of fluorescence intensity of red channel to green channel. (color online)

Wu et al. Sci China Chem January (2016) Vol.59 No.1
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Figure 6 Confocal laser scanning microscopy images of HeLa cells incubated with DCM-S (20 μmol/L) for 40 min with (e–h) or without (a–d) pretreat-
ment of NEM (10 mmol/L) for 40 min. Images were taken from the red channel (660–750 nm) (c, g), green channel (480–520 nm) (b, f), the overlapped
image (a, e), excitation wavelength 488 nm, and (d, h) was the ratio of fluorescence intensity of red channel to green channel. (color online)
333: 19–39
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