Twisted intramolecular charge transfer and aggregation-enhanced emission characteristics based quinoxaline luminogen: photophysical properties and a turn-on fluorescent probe for glutathione

Article abstract of DOI:10.1039/c8tc05360j

Developing solid and aggregate state emitters with large Stokes shifts has long been a significant challenge in the detection and bioimaging fields. In this study, a novel quinoxaline-based luminogen (QUPY) with twisted intramolecular charge transfer (TICT) and aggregation-enhanced emission (AEE) characteristics has been designed and synthesized. Its pyridine salt derivative (QUPY-S) was also AEE-active and could be utilized as a fluorescent “turn-on” probe for the specific detection of glutathione (GSH). The cleavage of dinitrophenyl ether from QUPY-S by GSH generated AEE-active and less water-soluble QUPY, which exhibited a “turn-on” fluorescence response at around 516 nm. This reaction-based probe showed a large Stokes shift (131 nm), low detection limit (434 nM), fast response time, and low toxicity. QUPY-S was successfully applied for the detection of GSH in bovine serum samples with recoveries ranging from 91.9% to 100.7%. Additionally, QUPY-S could detect GSH in HeLa cells by confocal laser scanning microscopy.

Full text of DOI:10.1039/c8tc05360j

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Nguyên T. K. Thanh, Xiaodi Su et al.
Fine-tuning of gold nanorod dimensions and plasmonic properties using
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Twisted intramolecular charge transfer plus aggregatDiOoI:n10-.1039/C8TC05360J
enhanced emission active based quinoxaline luminogen:
photophysical properties and a light-up fluorescent probe
for glutathione
Mingming Cui,∥ Wenting Li,∥ Lingyun Wang,* Lingshan Gong, Hao Tang, Derong Cao
Key Laboratory of Functional Molecular Engineering of Guangdong Province, School of Chemistry
and Chemical Engineering, South China University of Technology, Guangzhou, China, 510641
∥M.M. Cui and W.T. Li contributed equally.
*Corresponding author: Tel. +86 20 87110245; fax: +86 20 87110245. E-mail: lingyun@scut.edu.cn
1

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Abstract
Developing solid and aggregate state emitters with large Stokes shift has long been a
significant challenge in detection and bioimaging field. In this work, a novel
quinoxaline-based luminogen (QUPY) with twisted intramolecular charge transfer
(TICT) and aggregation-enhanced emission (AEE) characteristics has been designed
and synthesized. Its pyridine salt derivative (QUPY-S) is also AEE-active and can be
utilized as a fluorescent “turn-on” probe for the specific detection of GSH. The cleavage
of dinitrophenyl ether of QUPY-S by GSH generated AEE-active and less water-
soluble QUPY, which in turn opened “turn-on” fluorescence response around 516 nm.
This reaction-based probe showed a large Stokes shift (131 nm), low detection limit
(434 nM), fast response time, and low toxicity. QUPY-S was successfully applied to
the detection of GSH in bovine serum samples with recoveries ranging from 91.9% to
100.7%. Additionally, QUPY-S can detect GSH in HeLa cells by confocal laser
scanning micrographs.
Keywords: Aggregation-enhanced emission (AEE); GSH detection; Quinoxaline;
Bioimaging
2

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1 Introduction
As a “power house” to produce ATP, mitochondria participate in reactive oxygen
species (ROS)-induced apoptosis. For instance, there are a large amount of free radical
scavengers in mitochondria to protect cells from the oxidative stress. Intracellular thiols
like glutathione (GSH), cysteine (Cys) and homocysteine (Hcy) can regulate the
homeostasis of ROS through the respiratory chain of mitochondria [1-5]. As a critical
antioxidant reservoir, mitochondrial GSH pool within cells plays important roles to
keep oxidation-reduction equilibrium [6-7]. Moreover, abnormal levels of GSH may
result in liver damage and neurogenic diseases and even cancer [8-12]. Therefore, it is
urgent and important to develop a mitochondrial GSH probe for understanding
mitochondrial associated diseases.
By taking advantage of the unique nucleophilicity of thiol group, discrimination of
biothiols from other amino acids can be successfully achieved. However, identification
of GSH from Cys/ Hcy was much more difficult due to bulkiness of GSH and its weak
nucleophilicity [13-32]. Only very limited fluorescent probes targeted to detect GSH in
mitochondria [33-40]. Unfortunately, most of reported sensors suffered from aggregation
caused quenching (ACQ) effect and possessed small Stokes shifts. Especially, when
they accumulated in cells, their fluorescence emission was much weaker than that in
solutions and limited their use in vivo. Thus, it is of great interest to develop GSH
probes with large Stokes shift and anti- ACQ effect for in vivo application.
Opposite to the photophysical phenomenon of ACQ, aggregation-induced emission
(AIE) active fluorophores possessing strong fluorescence in their solid or aggregate
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states attract more attentions. By manipulating aggregation/disaggregation process,
various fluorescence turn-on probes based on AIE-active luminophores have been
fabricated for sensitive detection of different analytes [41-42]. Moreover, because the
nonplanar conformations of AIE molecules often lead to a loose packing in the solid
state, linking a highly sterically bulky AIE moiety to a dye is expected to provide
efficient solid-state emission. On the other hand, dyes with twisted intramolecular
charge transfer (TICT) characteristics usually possess Stokes shifts larger than 100 nm
[43-45]. In polar solvents, luminogens turn from the locally excited (LE) state at short
wavelength to the TICT state at long wavelength through intramolecular rotation and
result in large Stokes shift. During this process, charge separation between the donor
and acceptor units occurs. Quinoxaline with tunable photophysical properties receives
more attentions, because it is easy to construct donor and acceptor type structured
luminogens through intramolecular charge transfer process [46-50]
.
Inspired by this design strategy, we covalently linked a popular AIE group
(triphenylethylene) and pyridine moiety to quinoxaline core, resulting in a novel
compound (QUPY) with AEE characteristics and large Stokes shifts through TICT
process. Moreover, when the pyridinium group was functionalized by a dinitrophenoxy
benzyl moiety to yield anotherAEE-active compound (QUPY-S), which can be utilized
as a light-up probe for GSH detection. In the presence of GSH, the dinitrophenoxy
benzyl group in QUPY-S was cleaved and regenerated QUPY with poor water
solubility (Scheme 1). As a result, turn-on fluorescent probe for the specific detection
of GSH was developed.
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2 Experimental
2.1 Chemicals and instruments
Compound 1 and compound 3 were synthesized by previous reported methods,
respectively [50, 39]
.
NMR spectra were recorded on Bruker advance III 400 MHz and chemical shifts
were expressed in ppm using TMS as an internal standard. The UV-vis absorption
spectra were recorded using a Helios Alpha UV-vis scanning spectraphotometer.
Fluorescence spectra were obtained with a Hitachi F-4500 FL spectrophotometer with
quartz cuvette (path length = 1 cm).
Absolute fluorescence quantum yield was measured by Hamamatsu spectrometer
C11347, it consists of an excitation light source based on a xenon arc lamp and a high-
sensitivity multichannel detector. The emitted light is collected by the integrating
spheres. The use of integrating spheres has usually required a laser as the excitation
source in combination with a fibre coupled CCD camera or a calibrated photodiode as
the luminescence detectors.
The solutions of amino acids and anions were prepared from the corresponding
nitrate salts or hydrochloride salts in deionized water. QUPY-S was dissolved in DMSO
at room temperature to afford the stock solution (10-3 M). The PBS buffer were prepared
with deionized water. The resulting solution was shaken well before recording the
absorption and emission spectra.
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3. Results and discussions
3.1 Design and synthesis of QUPY-S
The synthetic route to QUPY-S is shown in Scheme S1 (Supporting information).
The palladium-catalyzed Suzuki coupling reaction of 5,8-dibromo-2,3-bis(4-
methoxyphenyl)quinoxaline (compound 1) and 1-boric acid ester -1,2,2-
triphenylethylene afforded compound 2. Further Suzuki coupling reaction of compound
2 with 4-pyridinylboronic acid produced compound QUPY. When treated QUPY in
toluene under reflux with compound 3, QUPY-S was obtained in 60% yield. The
1
chemical structure of QUPY-S was confirmed by H NMR, 13C NMR and HRMS
(Figures S1-S3, Supporting Information).
3.2 Photophysical properties of QUPY and QUPY-S
According to literatures [51], luminogens with a rotatable donor–acceptor (D-A)
structure usually possess two excited states: a locally excited (LE) state and a TICT
state. Meanwhile, increasing the polarity of the solvent can always bring the
luminogens from the LE state to the TICT state, resulting in a large bathochromic shift
in the emission wavelength and a dramatic decrease in the emission efficiency [52]. In
our case, QUPY has D-A structure and AIE group (triphenylethylene). The
photophysical properties of QUPY in DMSO–water mixtures were investigated with
different water fractions, which is a combination of both strong AEE and TICT
characteristics, as shown in Figure 1a. The locally-excited (LE) state at 473 nm is
emissive in pure DMSO but its emission decreases as the solvent polarity is increased
by adding water. At the 30% water fraction (fw), the AEE state starts to dominate and
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the emission is further enhanced. The emission wavelength of QUPY is
bathochromically shifted from 473 to 519 nm during the solvent induced aggregation
process due to the synergetic TICT-AEE effects. The fluorescence quantum yields of
QUPY also increased from 4.3% in DMSO to 15.2% in DMSO/water (90:10, v/v).
Meanwhile, distinct emission color changes from dark blue to bright green can be found.
The aggregation of the QUPY in high water fraction was investigated by UV-vis
spectrum and dynamic light scattering (DLS) measurements. As compared with QUPY
in pure DMSO, the UV-vis spectrum of QUPY in 90% fw was broader with level-off
tails and baseline drifted, which in accordance of characteristics of nanoparticles due to
Mie effect (Figure S4a). DLS measurements further indicated nanoparticles with 100
nm size were formed in presence of 90% fw (Figure S4b).
In THF-water and CH3CN-water mixtures, similar PL behaviors as that in DMSO–
water mixtures were found (Figures 1b-1c). In addition, the remarkable 131 ~134 nm
Stokes shift of QUPY was an advantage, facilitating its efficient signal in bioimaging
and detection in living cells. In solid state, QUPY powder emitted bright green color
(λem =517 nm) with 29.3% quantum efficiency (Figure 1d). Evidently, QUPY was both
TICT and AEE characteristics.
As shown in Figure 2a, QUPY-S behaved differently. QUPY-S only showed a weak
emission in DMSO (λem =566 nm) and almost had no change until fw reached 40%.
Afterward, the emission intensified swiftly. The emission intensity was boosted to the
maximum when fw was 70%, which was 15.5-fold higher than that in DMSO. With
regard as fluorescence quantum yields, QUPY-S showed 10.4-fold enhancement from
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2.6% in DMSO to 27.2% in DMSO/water (3/7, v/v). When fw was beyond 70%, the
emission intensity began to decrease. It is possible that the molecules might
agglomerate quickly to form small and tight aggregates in presence of high fw, which
was verified by DLS data for 705 nm and 243 nm in presence of 70% and 90% fw,
respectively (Figures S5a,5b). The decrease in the emission intensity of QUPY-S when
the water fraction is increased from 70% to 90% may be attributed to the change in the
packing mode of the molecules in the aggregates. In the mixture with the ‘‘low’’ water
content, the molecules may steadily assemble in an ordered fashion to form more
emissive crystalline aggregates. At water fractions above 70%, the molecules may
quickly agglomerate in a random way to form less emissive amorphous particles with
small size. Similar results have been reported by others [53]
.
In addition, the emission of QUPY-S in DMSO/ glycerol mixture with various
viscosity was carried out (Figure 2b). The highest fluorescence intensity was observed
in 90% glycerol fraction, where 9.3-fold emission enhancement was present than that
in DMSO. Moreover, the fluorescence spectrum of QUPY-S in the solid state were
recorded and shown in Figure 2c, where emission peak at 584 nm with 18.9% quantum
efficiency was present. Thus, QUPY-S feature the unique AEE characteristics.
3.3 Spectral Properties of QUPY-S with GSH
To demonstrate that QUPY-S can be used as a light-up probe, we measured
fluorescence response of QUPY-S in H2O/DMSO (1:1, v/v) after incubation with 10
equiv. GSH at 37 °C for 90 min (Figure 3). Before incubating with GSH, QUPY-S was
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weakly emissive at 566 nm. However, the emission at 516 nm emerged after incubation
with GSH. The fluorescence intensity at 516 nm increased by 12.5 times when the
concentration of GSH reached 100 μM. Meanwhile, such fluorescence enhancement
can be distinguished with naked-eye as shown in the inset of Figure 3, where the color
changed from dark orange to bright green before and after incubation with GSH under
UV light illumination. This experiment showed that QUPY-S was potential to be used
as a fluorescent light-up probe.
The quantitative determinations were measured with QUPY-S by incremental
addition of GSH and the turn-on response at 516 nm was measured. As shown in Figure
4a, it can be seen that the fluorescence intensity of the QUPY-S gradually increases
upon gradual increase of GSH level. Moreover, a good linear relationship with R2 of
0.996 between the fluorescent intensity at 516 nm in the range of 0 to 10 μM can be
found (Figure 4b). The limits of detection (LOD) were determined from the equation
LOD =3σ/K, where σ is the standard deviation of the blank solution and K is the slope
of the calibration curve. The limits detection of QUPY-S was determined to be 434 nM
for GSH detection, which was at the nearly same level with other results (Table S1).
3.4 Selectivity and pH effect of QUPY-S for detecting GSH
Since GSH is the most predominant biological thiol in cells and mimicking typical
concentration in blood, GSH (1 mM), Cys (80 μM), Hcy (80 μM) and NaSH (50 μM)
were used to evaluate the selectivity QUPY-S. Figure 5a showed time-dependent of
fluorescence intensity at 516 nm of QUPY-S in DMSO/PBS buffer (1:1, v/v, pH 7.4)
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at 37 C in presence of these analytes. GSH induced 12.1-fold fluorescence
enhancement while Cys, Hcy and NaSH only showed no more than 2.1-fold
enhancement within 30 min. If we shortened incubation time to 9 min, selectivity of
QUPY-S for GSH detection was improved, where GSH intensified 8.3-fold emission
strength while others caused slight fluorescent enhancement (Figure 5b). In addition,
different kinds of amino acids (Ala, Asp, Glu, Gly, Asr, Leu, Lys, Met, Phe, Ser, Thr,
Trp, Tyr, Val), some physiological related neurochemical transition metal ions (Fe3+,
-
-
2-
Cu2+, Mn2+, Zn2+), reactive nitrogen species NO2 , reactive sulfur species (HSO3 , SO3 ,
2-
S2O5 ), anions and other possible interferences were chosen to measure the spectral
response. As shown in Figure S6-S7, no obvious fluorescence response was displayed,
which confirmed that QUPY-S exhibited high selectivity for detecting GSH.
As shown in Figure S8, the reaction capacity of QUPY-S to GSH was pH-dependent.
The higher fluorescence “Turn On” observation along with increased pH value can be
found. This can be explained by higher concentration of thiolate species in high pH
environment. In this work, pH of 7.4 with DMSO/PBS buffer (1/1) was selected as the
standard sensing conditions.
3.5 Mechanism of GSH Sensing
As we know, dinitrophenyl nitro group can be utilized as the GSH-selective reaction
unit [54-58]. The fluorescence enhancement observed for QUPY-S in presence of GSH
can be attributed to the transformation of QUPY-S into AEE-active QUPY, which
easily aggregated in detection solution, leading to fluorescence enhancement. This
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assumption was supported by the following results: (i) the absorption and fluorescence
spectra of QUPY-S after incubation with GSH overlaps well with that of QUPY in the
same solution (Figure S9); (ii) the mass signal at m/z = 674.419, corresponding to the
molecular weight of [QUPY +H]+, was found after the reaction solution of QUPY-S
and GSH (Figure S10). According to Scheme S2, compound 5 originated from active
quinone-methide species attacked by GSH as well as compound 4 would generate
simultaneously. In fact, two signals at m/z = 399.1058, 496.1762 corresponding to the
molecular weight of [5+H]+, [4+Na]+ were observed, respectively (Figure S11). As
displayed in Figure S12, QUPY-S in DMSO/PBS buffer (v/v, 1/1) formed aggregates
of ca. 106.4 nm and possessed +93.9 mV Zata potential. After incubation with GSH
(100.0 μM), larger aggregates of ca. 251.2 nm were present, supporting the
transformation of QUPY-S into more hydrophobic QUPY after reaction with GSH
(Figure S12). At the same time, Zata potential was also reduced to +34.6 mV.
Compared with Cys and Hcy, GSH has highest pKa, largest steric bulkiness and
lowest reactivity, why did QUPY-S show highest reactivity to GSH? The proposed
pyridinium modulated SNAr reaction mechanism for selective recognition of GSH was
illustrated in Scheme S2. Some possibilities can be included: (1) the steric fit of the
carboxylate group in three biothiols is optimal in GSH, and (2) the pyridinium cation
in QUPY-S may have the strongest electrostatic interaction with carboxylate group in
GSH, thus might modulate the SNAr attack of the thiol group in GSH against
dinitrophenoxy moiety. Such electrostatic interaction may accelerate the reaction rate
of GSH for SNAr attack significantly. In contrast, Cys and Hcy are not affected much.
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Since Cys and Hcy are shorter than GSH (HS- carboxylate separation: 3 bondsDfOoIr: 1C0.1y0s3,9/C8TC05360J
4 bonds for Hcy, 8 bonds for GSH), they cannot generate strong electrostatic interaction
with cationic pyridinium and thiol groups in Cys and Hcy failed to reach the reactive
site of dinitrophenoxy moiety. Some similar selective GSH fluorescent probes have
been reported [59]
.
3.6 The Density Functional Theory Investigations
The theoretical calculations with a B3LYP/6-31G (d) basis set was performed to gain
more insight into molecular orbital and charge distribution of QUPY-S and QUPY. As
shown in Figure S13, the electrons on the HOMO and LUMO of QUPY-S were
distributed on the two marginal sides of triphenylethene groups and dinitrophenoxy
benzyl pyridinium moiety, respectively. This might cause the possible photoinduced
electron transfer (PET) process in QUPY-S and weak fluorescence. However, the
electron densities of the HOMO of QUPY was mainly located on the quinoxaline core
and triphenylethene units, LUMO is mostly localized on the central quinoxaline core
and pyridine ring, which indicated that there was partial electronic conjugation between
them. In presence of GSH, QUPY-S converted to QUPY by removal dinitrophenoxy
benzyl moiety and the energy gap (Eg) value also increased from 2.54 eV to 3.40 eV,
which was in good accordance with the fluorescence “turn on” response.
3.7 Detection applications in Complex Biological Samples
The practical application of QUPY-S was examined by quantitative determining
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GSH fluorometrically in complicated biological samples - bovine serum albumin
(BSA). When serum sample was added into QUPY-S (10 μM), the emission intensity
at 516 nm was enhanced. On the basis of the linear relationship of between the
fluorescence intensity at 516 nm and concentration of GSH in Figure 4b, the
concentration of GSH in the bovine serum albumin (BSA) sample was determined to
be 1.67 μM. In order to confirm the observed emission enhancement at 516 nm was due
to presence of GSH, a known concentration of GSH was added into the same solution.
It can be found more intense emission peak at 516 nm was present. As shown in Table
S2, the recovery of the spiked samples ranged between 91.9% and 100.7%, indicating
the practicability of the proposed sensing platform. These results confirmed presence
of GSH in blood serum sample, thus resulting in fluorescence enhancement. So, QUPY-
S can be used for the determination of GSH in real samples.
3.8 Bioimaging of probe QUPY-S in living cells
The photostability of QUPY-S in DMSO/PBS buffer (v/v, 1/1) under continuous 365
nm irradiation was investigated. With increasing irradiation time, the maximal
absorbance of the QUPY-S at 411 nm showed only a negligible decrease of absorbance
(Figure S14). These results indicated that QUPY-S had good photostability.
Biocompatibility is important to be taken into account in an intracellular probe for
bioimaging. As shown Figure S15, QUPY-S showed in vitro low toxicity toward
cultured HeLa cell lines under the experimental conditions through the CCK-8 assay.
Similarly, QUPY showed no apparent toxicity to the cells. So, QUPY-S and QUPY
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were favorable for imaging in living cells due to excellent biocompatibility.
Confocal microscopy experiments were carried out to bioimage and monitor of GSH
in living cells with QUPY-S. As shown in Figure 6a, when HeLa cells were incubated
with QUPY-S (0.05 mM), strong yellow fluorescence was observed inside the cells,
which can be ascribed to the presence of endogenous GSH reacting with QUPY-S.
However, if HeLa cells were pre-treated with excess N-ethylmaleimide (NEM), a
trapping reagent for thiol species, followed by treatment with QUPY-S (0.5 mM), only
a negligible fluorescence signal was present (Figure 6c). The further addition of 200
mM of an exogenous GSH also led to an obvious fluorescence enhancement (Figure
6e). These results demonstrating the ability of QUPY-S to detect GSH in HeLa cells.
The yellow-fluorescence image in HeLa cells is further supported by Lambda mode of
confocal microscope and corresponding spectrum (Figures S16-S17), which can be
ascribed to interactions between QUPY and multiple factors such as weak acidity,
protein, ions and complex physiological environment of cells.
4 Conclusions
A triphenylethylene-containing quinoxaline pyridinium compound (QUPY) and its
salt (QUPY-S) were synthesized and investigated. QUPY showed both TICT and AEE
characteristics with large Stoke shifts. QUPY-S was also AEE-active. As a new
selective fluorescence turn-on detection of GSH, QUPY-S can be transformed into
QUPY by removal of 2,4-dinitrophenyl group in presence of GSH in aqueous solution,
and the subsequent aggregation of QUPY turns on the emission with the detection limit
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of 434 nM. The electrostatic interaction to modulate the attack of GSH is belDieOvI: e10d.10t3o9/C8TC05360J
play significant role for high selectivity. QUPY-S has been successfully used to detect
in complex biological samples and mitochondrial GSH in HeLa cells.
Acknowledgements
The supports by the Natural Science Foundation of Guangdong Province
(2015A030313209, 2016A030311034) and the Fundamental Research Funds for the
Central Universities (2017ZD075) are gratefully acknowledged.
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H3CO
OCH3
H3CO
OCH3
GSH
N
N
N
N
Br
NO2
O
SG
N
N
NO2
O
NO2
,
NO2
QUPY-S
QUPY
Scheme 1 Schematic representation of light-up sensing of GSH by QUPY-S.
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519 nm
Water (%)
(a)
200
150
100
50
0
10
30
50
70
90
473 nm
0
450
500
550
600
Wavelength (nm)
519 nm
Water (%)
(b)
THF-H2O mixture
120
90
60
30
0
0
10
30
50
70
90
99
459 nm
450
500
550
600
Wavelength (nm)
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150
120
90
60
30
0
519 nm
Water (%)
0
(c)
CH3CN-H2O mixture
10
30
50
70
90
99
467 nm
450
500
550
600
Wavelength (nm)
10000
7500
5000
2500
0
(d)
(b)
517 nm
450
500
550
600
650
Wavelength (nm)
Figure 1 (a) Emission spectra of QUPY (10 μM) in (a) DMSO/water, (b) THF/water
and (c) CH3CN/water mixtures with different fraction of water. Inset: emission
photographs of QUPY (10 μM) in DMSO and DMSO/water (1/9, v/v). (d) Solid-state
emission spectrum of QUPY. inset: photo images of the powder solids for QUPY (λex
= 365 nm).
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15
100 Water (%)
10
5
0
(a)
10
20
30
40
50
60
70
80
90
80
60
40
20
0
0
0
20
40
60
80
Water fraction (%)
500
550
600
650
Wavelength (nm)
70
60
50
40
30
20
10
0
10
8
Glyerol (%) (b)
0
579 nm
6
10
20
30
40
50
60
70
80
90
4
2
0
0
20
40
60
80
Glyerol fraction (%)
589nm
500
550
600
650
700
Wavelength (nm)
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500
400
300
200
100
0
584nm
(c)
450
500
550
600
650
Wavelength (nm)
Figure 2 (a) Emission spectra of QUPY-S (10 μM) in DMSO/water mixtures with
different fraction of water. (a) Emission spectra of QUPY-S (10 μM) in DMSO/
glycerol mixtures with different fraction of glycerol. Inset: fluorescence intensity ratio
and emission photographs. (c) Solid-state emission spectrum of QUPY-S. inset: photo
images of the powder solids for QUPY-S (λex = 365 nm).
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400
300
200
100
0
516 nm
QUPY-S
QUPY-S + GSH
450
500
550
600
650
Wavelength (nm)
Figure 3 Emission spectra of QUPY-S (10 μM) prior to and after addition of GSH (100
μM) at 37 C for 90 min in DMSO/PBS (v/v = 1:1).
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400
300
200
100
0
516nm
(a)
0.1 mM
0
566nm
450
500
550
600
Wavelength (nm)
80
(b)
70
60
50
40
30
R2=0.99597
0
2
4
6
8
10
concentration (uM)
Figure 4 (a) The emission spectra of QUPY-S (10 μM)) in DMSO/PBS (v/v = 1:1) with
increasing GSH (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μM) at 37 C for 90 min. (b)
A linear calibration curve between the fluorescent intensity at 516 nm of QUPY-S in
DMSO/PBS (v/v = 1:1) and the concentration of GSH in the range of 0-10 μM after
incubation for 90 min at 37 C.
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500
400
300
200
100
0
(a)
NaSH
Cys
Hcy
GSH
0
5
10
15
20
25
30
Time (min)
500
400
300
200
100
0
(b)
9 min
30 min
Blank
GSH
Cys
Hcy
NaHS
Figure 5 (a) Time-dependent changes of emission spectra of QUPY-S (10 μM)
in DMSO/PBS buffer (1:1, v/v, pH 7.4) at 37 C after incubation with GSH (red,

1 mM), Cys (blue, 80 μM), Hcy (black, 80 μM) as mimics of intercellular
concentration and NaSH (cyan, 50 μM, typical concentration in blood). (b)
Fluorescence intensity of QUPY-S (10 μM) in DMSO/PBS buffer (1:1, v/v, pH
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7.4) at 37 C with GSH (red, 1mM), Cys (blue, 80 μM), Hcy (black, 80 μM),
NaSH (cyan, 50 μM, typical concentration in blood) as mimics of intercellular
concentration and after 9 min (black column) and 30 min (red column).
29