A fluorescent probe for thiols based on aggregation-induced emission and its application in live-cell imaging

Article abstract of DOI:10.1016/j.dyepig.2014.04.020

A turn-on fluorescent probe DNBS-CSA is reported here with selective detection of thiols over other amino acids. Probe DNBS-CSA possesses the widely-used thiol-selective 2,4-dinitrobenzenesulfonyl (DNBS) group to react with thiols and release the fluoresc

Full text of DOI:10.1016/j.dyepig.2014.04.020

Dyes and Pigments 108 (2014) 24e31
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A fluorescent probe for thiols based on aggregation-induced emission
and its application in live-cell imaging
*
Lu Peng, Zhaojuan Zhou, Ruirui Wei, Kai Li, Panshu Song, Aijun Tong
Department of Chemistry, Beijing Key Laboratory for Analytical Methods and Instrumentation, Key Laboratory of Bioorganic Phosphorus Chemistry &
Chemical Biology, Tsinghua University, Beijing 100084, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A turn-on fluorescent probe DNBS-CSA is reported here with selective detection of thiols over other
amino acids. Probe DNBS-CSA possesses the widely-used thiol-selective 2,4-dinitrobenzenesulfonyl
(DNBS) group to react with thiols and release the fluorescent salicylaldehyde azine with aggregation-
induced emission (AIE) characteristics. The use of AIE dye prevented the sensor from aggregation-
induced fluorescence quenching which is challenging for other thiols sensors. Upon the addition of
cysteine (Cys) to DNBS-CSA in aqueous solution (10 mM PBS buffer at pH 7.4 containing 30% DMSO), an
intense fluorescence enhancement was observed at 558 nm with a large stokes shift of w170 nm. Under
the optimal condition, the fluorescence intensity linearly increased with the concentration of Cys in the
Received 17 October 2013
Received in revised form
20 February 2014
Accepted 14 April 2014
Available online 24 April 2014
Keywords:
Fluorescence
range of 8e18
m
M with a correlation coefficient of R2 ¼ 0.991. The fluorescence enhancement of DNBS-
Signal enhancement
Reaction-based probe
Thiols
Aggregation-induced emission
Live-cell imaging
CSA upon addition of Cys on test paper was also observed, enabling DNBS-CSA function as solid materials
(such as test papers) for thiols detection. The detection of thiols in serum samples was successfully
performed. With good cell permeability, the probe was applied to the imaging of thiols in HEK 293T cells.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
demonstrating the quantification of thiols [13e15]. However, most
of the reported thiols sensors are designed with fluorophores of
Biological thiols such as cysteine (Cys) and glutathione (GSH),
play crucial roles in biological and physiological processes [1]. Their
abnormal levels are linked to a number of diseases, such as liver
damage, skin lesions, slow growth, cancer, and AIDS [2,3]. There-
fore, the selective and sensitive quantification of thiols is of sig-
nificant importance. Compared with other chromatographical [4]
and electrochemical [5] detection techniques, the selective and
sensitive fluorescent probes for thiols have received increasingly
attention due to their simplicity and capability of imaging intra-
cellular thiols in vivo studies [6e8]. Various mechanisms have been
utilized to construct fluorescent chemosensors for thiols, such as
Michael addition [9e11], cyclization with aldehyde [12], cleavage of
sulfonate [13e15], and sulfonamide [16] by thiols, and others
[17,18]. As to the cleavage of sulfonate by thiols, 2,4-
dinitrobenzenesulfonyl (DNBS) has already been reported for pro-
tein labeling by G. Drewes et al. and other researchers [19e21].
Also, as to its application in detection of thiols, it was firstly re-
ported by H. Maeda et al. that the DNBS is a useful leaving group for
design selective thiol sensors and there have been several papers
aggregation caused quenching (ACQ) which would seriously
quench the fluorescence at high sensor concentration or in their
solid state [22e24]. It will become more severe especially when
imaging low-abundant molecular species in biological systems,
since the fluorescence signals could not be enhanced by increasing
the fluorophore concentration [22e24]. Also, it is difficult to apply
these sensors for thiol detection in solid state (such as on test pa-
pers) due to their ACQ characteristics. The paper-based sensors are
known to be more suitable for point-of-care testing alternative to
conventional analytical instrumentations, because they are simple,
portable, inexpensive, disposable, and use of low sample volume
[25e27]. In contrast, molecules of aggregation-induced emission
(AIE) would be more preferable, which would emit efficiently in the
aggregate state of a fluorophore [28e30].
Many AIE based probes have been utilized to sense metal ions
[31,32], anions [33], biomolecules such as heparin [34,35],
L-lactic
acid [36], -glucose [37] and others [38]. These detection methods
D
are mostly based on two ways to produce the fluorescent aggre-
gated molecule. One is the electrostatic attraction [33e36] between
the differently charged probe and analyte. The other is the coor-
dinative complexation processes [37]. However, electrostatic
attraction is easy to be affected by other charged species, thus
preventing the probes showing good selectivity. In addition, most
* Corresponding author. Tel./fax: þ86 10 62787682.
E-mail address: tongaj@mail.tsinghua.edu.cn (A. Tong).
http://dx.doi.org/10.1016/j.dyepig.2014.04.020
0143-7208/Ó 2014 Elsevier Ltd. All rights reserved.

L. Peng et al. / Dyes and Pigments 108 (2014) 24e31
25
buffer solutions used in the detection have to be acidic or alkaline
so that the probe could be charged, limiting the detection at neutral
pH. Therefore, AIE probes based on their selective reaction with
analytes would be more preferable. However, fewer reaction-based
AIE probes [22e24] have been reported. Herein, we designed a
novel AIE probe based on selective cleavage of the probe by thiols to
produce AIE fluorescence for fluorescence turn-on detection of
thiols both in solution and on test papers.
were purchased from commercial suppliers (Alfa Aesar Co., Tianjin,
China). Unless otherwise noted, all the other materials were pur-
chased from Sinopharm Chemical Reagent Beijing Co., Beijing,
China and used without further purification. Absolute DMSO of
analytical grade and deionized water (distilled) were used
throughout the experiment.
2.2. Apparatus
Previously, our group has reported a series of facilely synthe-
sized salicylaldehyde azine (SA) derivatives with AIE characteristics
[28] and investigated their applications in hydrazine [39], prot-
amine [40] and pH [41] detection. An adjacent hydroxyl group of
the rotatable NeN single bond in the probes plays a crucial role in
their AIE characteristics: intramolecular hydrogen bonds therein
ensure the free intramolecular rotation in their solution state with
fluorescence quenching while inhibition of the rotation occurred in
the aggregate state with fluorescence signal turned on.
Maeda et al. have demonstrated that the 2,4-
dinitrobenzenesulfonyl (DNBS) group is useful to design thiols
probes [14]. Based on this strategy, a fluorescence turn-on AIE probe
toward thiols, compound 2-(20,40-esulfonyl) 5-chlorosalicylaldehyde
azine (DNBS-CSA) (Scheme 1) was designed and synthesized. The
probe possesses 5-chlorosalicylaldehyde azine as a potential AIE
fluorophore and DNBS group, showing unique and high reactivity
towards thiols, as both a recognition moiety and an effective
quencher [13e15] of the AIE fluorophore. After thiols cleave the
DNBS group in aqueous solution at neutral pH (or on test papers)
to release free CSA with a hydroxyl group, the AIE fluorescence is
turned on.
All fluorescence and UV/vis absorption spectra were recorded
with JASCO FP-6500 and JASCO V-550 UVevis spectrophotometers
(Tokyo, Japan), respectively. All pH measurements were made with
a Model pHS-3C pH meter (Shanghai Precision & Scientific Instru-
ment Co. Ltd, Shanghai, China). All NMR spectra were recorded
using a JOEL JNM-ECA300 spectrometer (Tokyo, Japan) operated at
300 MHz. Electrospray ionization-mass spectra were obtained on a
high performance 1100 liquid chromatographyemass spectroscopy
spectrometer (Agilent Technologies, Kyoto, Japan) without using
the liquid chromatography part. High resolution mass spectra were
obtained on a Shimadzu LCMS-IT-TOF mass spectrometer (Kyoto,
Japan) equipped with an electrospray ionization (ESI) source. Cells
imaging was performed with a confocal microscope from Carl Zeiss
(LSM 710 META model) (Oberkochen, German).
2.3. Synthesis of 2-(20,40-dinitrobenzenesulfonyl)
5-chlorosalicylaldehyde (DNBS-CS) (Scheme 1(a))
To a solution of 5-Chloro-salicylaldhyde (626.2 mg, 4.0 mmol) in
dry CH2Cl2 (6 mL) was added Et3N (567 mL, 4.0 mmol). After stirring
at room temperature for 60 min, 2,4-Dinitrobenzenesufonyl chlo-
ride (1.0664 g, 4.0 mmol) in 12 mL of dry CH2Cl2 was added
dropwisely to the reaction mixture. After being stirred overnight at
room temperature, the reaction was stopped with 30 mL H2O. The
organic layer was separated and dried with Na2SO4 and then
filtered and evaporated in vacuo. 3 mL EtOH was then added and
the resulting precipitation was filtered and washed by 30 mL of
2. Experimental
2.1. Reagents
5-Chloro-salicylaldhyde, 2,4-dinitrobenzenesufonyl chloride,
2-hydroxybenzaldehyde hydrazone and N-ethyl maleimide (NEM)
Scheme 1. (a) Synthetic pathways for compounds DNBS-CS, DNBS-CSA and CSA. (b) Cleavage of sulfonyl bond of DNBS-CSA by thiols and production of AIE fluorophore CSA.

26
L. Peng et al. / Dyes and Pigments 108 (2014) 24e31
ethanol. After drying, DNBS-CS was obtained as a clear yellow solid
(316.5 mg, 21% yield). 1H NMR (300 MHz, DMSO-d6): 10.05 (s, 1H),
9.14 (d, 1H, J ¼ 2.4 Hz), 8.65 (dd, 1H, J1 ¼ 2.4 Hz, J2 ¼ 2.4 Hz), 8.35 (d,
1H, J ¼ 8.6 Hz), 7.95 (d, 1H, J ¼ 2.8 Hz), 7.82 (dd, 1H, J1 ¼ 2.8 Hz,
J2 ¼ 2.7 Hz), 7.32 (d, 1H, J ¼ 8.9 Hz). 13C NMR (DMSO-d6)
d (ppm):
187.51, 152.26, 148.66, 147.75, 136.17, 134.46, 134.01, 130.86, 130.80,
130.36, 128.23, 126.00, 121.77. ESI high resolution mass spectrom-
etry: calc. for
408.9514.
C13H7ClN2NaO8S [M
þ
Na]þ 408.9509, found
2.4. Synthesis of 2-(20,40-dinitrobenzenesulfonyl) 5-
chlorosalicylaldehyde azine (DNBS-CSA) (Scheme 1(a))
DNBS-CS (305.6 mg, 0.8 mmol) was dissolved in dry CH2Cl2
(10 mL). Then 2-Hydroxybenzaldehyde hydrazone (108.0 mg,
0.8 mmol) in dry CH2Cl2 (4 mL) was added. After being stirred
overnight at room temperature, the solvent was evaporated in
vacuo. 3 mL EtOH was then added and the resulting precipitation
was filtered and washed by 30 mL of ethanol. After drying, DNBS-
CSA was obtained as yellow solid (306.9 mg, 76% yield). 1H NMR
(300 MHz, DMSO-d6): 10.87 (s, 1H), 9.12 (d, 1H, J ¼ 2.4 Hz), 8.85
(s, 1H), 8.59 (m, 2H), 8.34 (d, 1H, J ¼ 8.6 Hz), 8.06 (d, 1H, J ¼ 2.8 Hz),
7.69 (m, 2H), 7.40 (m, 2H), 6.96 (m, 2H). 13C NMR (DMSO-d6)
Fig. 1. Fluorescence spectra of CSA (30 mM) in different DMSO-water solution (arrow
shows the increase of water fraction (v/v) from 10% to 90%) containing 10 mM PBS at
pH 7.4. Inset: Effect of water volume fraction on the AIE fluorescence intensity of CSA
(30 mM) measured at 558 nm (peaks in emission spectra). Excitation wavelength was
set at 400 nm.
d
(ppm): 164.28, 159.30, 155.00, 152.12, 148.65, 146.68, 134.44,
media were replaced with fresh media every two days. The cells
were seeded in a 6 cm dish at a density of 104 cells per dish in
culture media. After 24 h, the cells were treated without or with
134.26, 133.73, 133.26, 131.01 (2C), 129.44, 128.25, 128.13, 126.18,
121.55, 120.16, 118.59, 117.08. ESI mass spectrometry: calc. for
C
20H13ClN4O8S [M þ H]þ 505.0, found 505.2. ESI high resolution
100
washing with PBS buffer to remove the remaining NEM, the cells
were further incubated with 30 M of DNBS-CSA in PBS buffer for
m
M NEM in PBS buffer (10 mM, pH 7.4) for 30 min at 37 ꢂC. After
mass spectrometry: calc. for C20H13ClN4NaO8S [M þ Na]þ 527.0040,
found 527.0035.
m
40 min at 37 ꢂC. The cells were imaged by a confocal microscope.
The excitation source was a 405 nm argon laser and an emission
image was acquired using a long path filter (491e665 nm). All
images were taken under the same experimental parameters to
minimize possible variations in fluorescence intensity.
2.5. Synthesis of 5-Chlorosalicylaldehyde azine (CSA) (Scheme 1(a))
5-Chloro-salicylaldhyde (156.6 mg, 1.0 mmol) was dissolved in
EtOH (10 mL). Then 2-Hydroxybenzaldehyde hydrazone (136.2 mg,
1.0 mmol) in EtOH (4 mL) was added. After being stirred overnight
at room temperature, the resulting precipitation was filtered and
washed by 30 mL of ethanol. After drying, CSA was obtained as
yellow solid (139.7 mg, 51% yield). 1H NMR (300 MHz, DMSO-d6):
11.17 (s, 1H), 11.08 (s, 1H), 9.02 (s, 1H), 8.95 (s, 1H), 7.77 (d, 1H,
J ¼ 2.4 Hz), 7.71 (d,1H, J ¼ 7.6 Hz), 7.44 (d,1H, J ¼ 2.4 Hz), 7.40 (d,1H,
3. Results and discussion
3.1. The AIE characteristics of CSA
J ¼ 7.6 Hz), 7.00 (m, 3H). 13C NMR (DMSO-d6)
d
(ppm): 163.75,
Compound CSA was expected to be released from the reaction of
probe DNBS-CSA and thiols containing amino acid as shown in
Scheme 1. Aggregation-induced emission (AIE) behavior of CSA in
different volume fractions of DMSO/water mixed solvent was thus
studied. Absorption and fluorescence spectra of CSA in DMSO/wa-
ter (from 1:9 to 9:1, v/v) buffered by 10 mM PBS at pH 7.4 were
shown in Fig. 1, Fig. S1 and S2 in the supporting information. In a
good solvent of 9:1 DMSO/water, CSA was well dispersed in its
“solution” state with a structured absorption spectra, and displayed
weak fluorescence emission. However, when CSA was dispersed in
1:9 DMSO/water, a leveling-off in the visible region of its absorp-
tion spectra (commonly observed in nanoaggregate suspensions)
strongly suggested the formation of a poorly-soluble “aggregate”
state, and an intense fluorescence enhancement was observed. This
AIE effect was explained by the blocking of the nonradiative
intramolecular rotation decay of excited molecules through for-
mation of J- or H-aggregates [28]. As illustrated in Fig. 1, the AIE
effect of CSA occurred when the volume fraction of DMSO is less
than 70%. A lower DMSO volume fraction was found to enhance the
AIE fluorescence intensity, which correlates well with the fact that
more aggregates would be formed in poorer solvents. Also, The AIE
characteristics of CSA was investigated in ethanol/water and THF/
water (from 1:9 to 9:1, v/v) (see Fig. S3 and S4 in the Supporting
Information). Considering that the aggregate of CSA would
disperse better in DMSO/water than in EtOH/water and DMSO is
161.04, 159.23, 157.77, 133.92, 133.11, 131.26, 129.32, 123.65, 120.51,
120.15, 119.01, 118.72, 117.02. ESI mass spectrometry: calc. for
C
14H11ClN2O2S [M þ H]þ 275.1, found 275.1. ESI high resolution
mass spectrometry: calc. for C14H10ClN2O2S [MꢀH]þ 273.0431,
found 273.0434.
2.6. Procedure for fluorescence study
A 1.0 ꢁ10ꢀ2 M stock solution of probe DNBS-CSAwas prepared in
absolute DMSO. Stock solutions (100 mM) of amino acids (GSH, Cys,
Ala, Arg, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, Trp and Val),
MPA (3-Mercaptopropionic acid) and ABT (2-Aminobenzenethiol)
in deionized water were prepared. In a typical experiment, test
solutions were prepared by placing 6 mL of the probe stock solution
into a test tube, diluting the solution to 2 mL with buffered aqueous
DMSO (10 mM PBS, pH 7.4), and adding an appropriate amount of
each amino acid.
2.7. Imaging of HEK cells
HEK 293T (human embryonic kidney cells) were cultured in
culture media (Dulbecco’s modified Eagle’s medium supplemented
with 10% fetal bovine serum, 50 unit/mL of penicillin, and 50 unit/
mL of streptomycin) at 37 ꢂC in a humidified incubator, and culture

L. Peng et al. / Dyes and Pigments 108 (2014) 24e31
27
less volatile than THF, we chose DMSO/water for the following
detection procedures.
3.2. Fluorescence detection of Cys based on DNBS-CSA
The ability of probe DNBS-CSA as a sensor for thiols detection by
fluorescence enhancement was examined by using 30
mM of probe
DNBS-CSA and Cys in a concentration range from 0 M to 90
m
mM. As
shown in Fig. 2, upon the addition of Cys to DNBS-CSA in 10 mM
PBS buffer at pH 7.4 containing 30% DMSO, the absorption
maximum showed bathochromic shift from 305 nm to 361 nm,
resulting in a perceived color change from colorless to yellow, while
an enhancement of the fluorescence emission intensity at 558 nm
observed. As reported by Drewes et al., the cleavage of DNBS by
thiols would release colored 2,4-dinitrothiophenolate (DNTP) [19].
To verify the newly emerged absorption maximum at 361 nm was
due to the released DNTP, the absorbance change of 2,4-
dinitrobenzenesufonyl chloride (DNBSC) upon Cys was studied.
As can be seen in Fig. S5 there was also an increase in absorbance at
361 nm. Thus, the absorption at 361 nm could be ascribed to the
absorption of the product DNTP (see Scheme 1(b) and S1). Addi-
tionally, as indicated in Fig. S7 in the Supporting Information, the
fluorescence intensity of DNBS-CSA was linearly proportional to Cys
concentrations of 8e18
mM and the regression equation was
F ¼ 0.7212 [Cys] ( ꢁ 10ꢀ6 M) ꢀ0.213 with a linear coefficient of
R2 ¼ 0.991 (n ¼ 3).
3.3. Reaction mechanism
It has been reported by Maeda’s group [14] and others [13,15]
that thiols could selectively cleave sulfonate ester. To verify our
design principle of probe DNBS-CSA for thiols, the reaction mech-
anism was studied by 1H NMR and mass spectra. The reaction
product was isolated by filtration to confirm that CSA was formed
through the reaction of Cys with probe DNBS-CSA (see Scheme
1(b)). 1H NMR spectra of the isolated product, the probe DNBS-
CSA itself and the reference compound CSA are shown in Fig. 3.
Upon addition of Cys, three protons of DNBS-CSA corresponding to
the protons of 2,4-dinitrobenzenesulfonyl group around 8.59, 8.35
and 8.06 ppm dramatically disappeared with the concomitant
Fig. 2. (a) Absorption and (b) fluorescence spectra of DNBS-CSA (30
of various concentrations of Cys (0e90 M) in 30% DMSO-10 mM PBS buffer at 7.4.
Other conditions are the same as in Fig. 1. Inset: photographs of DNBS-CSA (30 M)
before and after addition of Cys (300 M) under a UV lamp (365 nm) and the fluo-
rescence intensity at 558 nm as a function of cysteine concentration.
mM) upon addition
m
m
m
Fig. 3. Partial 1H NMR spectra of DNBS-CSA (10 mM) upon addition of Cys (4.0 equiv.) in DMSO-d6. (A) Only DNBS-CSA, (B) the isolated aggregates after DNBS-CSA reacted with Cys
in DMSO for 40 min, (C) the reference compound CSA.

28
L. Peng et al. / Dyes and Pigments 108 (2014) 24e31
Fig. 4. Fluorescence spectra of compound CSA (30 mM) and DNBS-CSA (30 mM) before
and after addition of Cys (300
mM) in PBS buffer (10 mM, pH 7.4, 30% DMSO) for
40 min. Other conditions are the same as in Fig. 2.
appearance of a new peak at 11.16 ppm assigned to the released
hydroxyl group. Furthermore, the mass spectral analysis (Fig. S11,
S12 and S13 in the Supporting Information) and the same fluores-
cence spectrum (Fig. 4) of the reference compound CSA and that of
the probe reacted with Cys corroborated that the AIE fluorescence
was turned on in the reaction.
3.4. Selectivity
The selectivity of probe DNBS-CSA toward thiols was studied
(Fig. 5). Upon the addition of Cys, GSH, MPA or ABT, which are all
with a thiol group, the fluorescence intensity of DNBS-CSA was
greatly enhanced. However, no change was observed with other
amino acids such as Ala, Arg, Gln, Gluc, Gly, His, Lys, Met, Phe, Pro,
Fig. 6. Fluorescence intensity change of DNBS-CSA (30
mM) at 558 nm before and after
addition of Cys (300 M) (a) in PBS buffer (10 mM, 30% DMSO) with different pH and
m
(b) in PBS buffer (10 mM, pH ¼ 7.4) with different DMSO volume fraction. Other
conditions are the same as in Fig. 2.
Ser, Thr, Trp, Tyr and Val. Thus, probe DNBS-CSA showed good
selectivity to thiols.
3.5. Optimization of analytical pH and DMSO volume fraction
The pH value of solution was found to be essential to the
cleavage reaction. To investigate the pH effect, the fluorescence
intensities of 30
presence of 300
m
M DNBS-CSA at 558 nm in the absence and
mM Cys were examined at pH range from 4.0 to
10.0. As shown in Fig. 6(a), the probe DNBS-CSA itself does not show
fluorescence emission from pH 4.0 to 7.4 and its fluorescence in the
pH range of 7.4e10.0 is also negligible. Upon the addition of Cys,
there was significant fluorescence increase in pH range of 6.0e7.4.
At pH 4.0, no fluorescence enhancement could be observed, which
might be ascribed to the difficulty for thiols to cleave the sulfonyl
group under this condition. On the contrary, high pH environment
(>8.0) could cause the deprotonation of the hydroxyl group of CSA,
which would improve the solubility of CSA, thus decreasing the
fluorescence enhancement. Therefore, a pH range of 6.0e7.4 was
suggested for the detection of thiols using DNBS-CSA. Also, the
DMSO volume fraction of the solution showed great effect on the
Fig. 5. Fluorescence spectra of DNBS-CSA (30 mM) upon addition of 10 equiv. of various
amino acids, MPA (3-mercaptopropionic acid) and ABT (2-aminobenzenethiol) in 30%
DMSO-10 mM PBS buffer at 7.4. Inset: the corresponding fluorescence intensity of
DNBS-CSA upon addition of various amino acids and analytes (from left to right, 1:
blank 2: Ala, 3: Arg, 4: Gln, 5: Glu, 6: Gly, 7: His, 8: Lys, 9: Met, 10: Phe, 11: Pro, 12: Ser,
13: Thr, 14: Trp, 15: Val, 16: Cys, 17: GSH, 18: MPA and 19: ABT). Other conditions are
the same as in Fig. 2.

L. Peng et al. / Dyes and Pigments 108 (2014) 24e31
29
fluorescence enhancement as indicated in Fig. 6(b). High DMSO
volume fraction (40%) was benefit to the solubility of DNBS-CSA,
which could promote the cleavage reaction. However, the product
CSA is an AIE fluorophore that shows weak fluorescence intensity in
its “solution” state. Thus, 30% DMSO-10 mM PBS buffer at 7.4 was
chosen for thiols quantification using DNBS-CSA as a fluorescent
probe.
3.6. Detection of thiols in serum samples
According to the literature [18], fetal bovine serum (FBS) was
reported containing thiols. To further investigate the potential
utility of DNBS-CSA in biological samples, the content of thiols in a
serum sample was analyzed by the proposed method. As shown in
Fig. S8a, the fluorescence intensity of DNBS-CSA at 558 nm
increased with the addition of FBS. The fluorescence intensity
showed a linear relationship with the concentration of FBS (0e
13.3%, volume fraction) (see Fig. S8b). However, the fluorescence
intensity of DNBS-CSA at 558 nm was relatively low in the serum
sample pretreated with a thiol-blocking reagent NEM (see Fig. S9).
This demonstrated that the fluorescence enhancement was actually
attributable to the thiols in the serum. Also, the results showed
good recoveries and R.S.D. values (Table S1), which suggested the
accuracy and reliability of the present method for thiols detection
in practical samples analysis.
3.7. Detection of thiols on test papers
As DNBS-CSA showed aggregation-induced emission in the
presence of Cys in 10 mM PBS buffer at pH 7.4 containing 30%
DMSO, it inspired us to further investigate the possibility of
detection as solid materials for point-of-care detection application.
Test paper was selected and the fluorescence detection perfor-
mance was studied by an UV lamp with color photography as well
as solid fluorescence spectroscopy. In the detection process, the
probe spots were prepared by dropping 1 mL solutions of DNBS-CSA
in THF (10 mM) on the paper strips. After the evaporation of sol-
vent, PBS solutions (10 mM, pH ¼ 7.4) of Cys at various concen-
trations were dropped onto the probe spots. Due to its AIE
characteristics, the fluorescence enhancement of DNBS-CSA toward
Cys on papers was clearly observed by naked eyes. As shown in
Fig. 7, the probe spots emitted bright yellow fluorescence upon the
addition of Cys under UV illumination, while an enhancement of
the fluorescence intensity at 558 nm could be recorded. Addition-
ally, the probe spots emitted fluorescence only upon the addition of
thiols (Fig. S10 in the Supporting Information), demonstrating the
good selectivity of probe DNBS-CSA as solid materials toward thiols.
3.8. Live-cell imaging
Fig. 7. (a) Images of test papers for the detection of Cys at various concentrations (0e
5.0 mM, from left to right) in PBS solutions (10 mM, pH ¼ 7.4) under a UV lamp
(365 nm). (b) Fluorescence spectra and (c) fluorescence intensity at 558 nm of the
It is reported that there are lots of thiols in cells and the con-
centration of glutathione (GSH) in cells is as high as 1.0e15 mM,
which is the most abundant intracellular thiol [42e44]. Therefore,
probe DNBS-CSA was further applied for in vivo imaging of thiols in
probe spots of DNBS-CSA (30
mM) on test papers upon addition of various concen-
trations of Cys (0e9.0 mM). Other conditions are the same as in Fig. 2.
live-cells. After the HEK 293T cells incubated with 1 (30
mM in
3:1000 DMSO-PBS v/v, pH 7.4) for 40 min at 37 ꢂC, strong fluores-
cence was exhibited (Fig. 8(a)). Here, we chose the incubation
condition as 3:1000 DMSO-PBS v/v other than 3:7 DMSO-PBS v/v
for the following two consideration: first, as can be seen in Fig. 6(b),
even in pure PBS buffer without DMSO as co-solvent, DNBS-CSA
could show a fluorescence enhancement toward thiols; second,
lower DMSO-PBS v/v ratio would do less damage to the living cells.
To demonstrate the specific detection of thiols by DNBS-CSA in
Fig. 8(a), a control experiment was undertaken (Fig. 8(b)). Cells
were pretreated with excess amount of a thiol-reactive reagent
NEM (100 mM), which was expected to consume the free thiols in
cells [45], and then incubated with probe DNBS-CSA, a remarkable
decrease in fluorescence intensity was observed. This confirms the
specificity of probe DNBS-CSA for thiols over other analytes in living
cells.
4. Conclusion
In summary, we have developed a “turn-on” fluorescent probe
DNBS-CSA for thiols based on selective cleavage of DNBS-CSA by

30
L. Peng et al. / Dyes and Pigments 108 (2014) 24e31
Fig. 8. Confocal fluorescence images of living HEK 293T cells (left, fluorescence image; middle, phase contrast image; right, merged image): (a) HEK 293T cells incubated with DNBS-
CSA (30 M in 3:1000 DMSO-PBS v/v, pH 7.4) for 40 min; (b) HEK 293T cells were pre-incubated with 100 M NEM for 30 min and then treated with 30 M of DNBS-CSA for 40 min.
m
m
m
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thiols and the AIE characteristics of the product. Under optimized
condition (10 mM PBS buffer at pH 7.4 containing 30% DMSO),
DNBS-CSA exhibited sensitive fluorescence enhancement for thiols
and a good selectivity over other amino acids. Due to the AIE rather
than ACQ characteristics of CSA, the design takes advantage of as
solid materials for detection of thiols, enabling further fabrication
of test papers for in-field detection. Detection of thiols in serum
sample was successfully performed. The fluorescence turn-on
response of DNBS-CSA for imaging cellular thiols was also applied.
Acknowledgment
[15] Chen X, Zhou Y, Peng XJ, Yoon J. Fluorescent and colorimetric probes for
detection of thiols. Chem Soc Rev 2010;39:2120e35.
[16] Jiang W, Cao Y, Liu Y, Wang W. Rational design of a highly selective and
sensitive fluorescent PET probe for discrimination of thiophenols and aliphatic
thiols. Chem Commun 2010;46:1944e6.
We thank the National Natural Science Foundation of China (No.
21175079 and 21375074) for financial support.
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and specific organoselenium fluorescent probe for thiols and its application in
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probes for rapid, sensitive, and label-free detection of Hg2þ and biothiols in
complex matrices. Chem Commun 2012;48:1147e9.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2014.04.020.
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selective radioactive labeling of protein thiols under spectrophotometric
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