A fluorescence and UV/vis absorption dual-signaling probe with aggregation-induced emission characteristics for specific detection of cysteine

Article abstract of DOI:10.1039/c8ra03756f

Biological thiols with similar structures, such as glutathione (GSH), N-acetyl-l-cysteine (NAC), homocysteine (Hcy) and cysteine (Cys), play important roles in human physiology and are associated with different diseases. Thus, the discrimination of these

Full text of DOI:10.1039/c8ra03756f

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A fluorescence and UV/vis absorption dual-
signaling probe with aggregation-induced emission
Cite this: RSC Adv., 2018, 8, 24346
characteristics for specific detection of cysteine
Ruru Li, Xiaoyu Huang, *ac Guolin Lu and Chun Feng *ab
ac
a
Biological thiols with similar structures, such as glutathione (GSH), N-acetyl-L-cysteine (NAC),
homocysteine (Hcy) and cysteine (Cys), play important roles in human physiology and are associated
with different diseases. Thus, the discrimination of these thiols is a great necessity for various
biochemical investigations and the diagnosis of related diseases. Herein, we present a new dual-signaling
probe consisting of a typical aggregation induced emission fluorogen of a tetraphenylethylene group
and 2,4-dinitrobenzenesulfonyl moiety. The probe can be used to selectively and quantitatively detect
Cys over a variety of bio-species, including GSH, NAC and Hcy, from both UV/vis absorption and
fluorescence channels. The mechanism study showed that the fluorescence and UV/vis absorption were
turned on as the probe undergoes displacement of the 2,4-dinitrobenzenesulfonyl group with Cys,
where the UV/vis and fluorescence signals originate from the dinitrophenyl-containing compounds and
aggregates of TPE-OH, respectively. In addition, the discrimination of Cys was achieved by more rapid
intramolecular displacement of sulfur with the amino group of Cys than NAC, Hcy and GSH. Moreover,
the probe shows ignorable cytotoxicity against HepG2 cells, which demonstrates the great potential of
the probe in selectively detecting Cys in vivo.
Received 2nd May 2018
Accepted 29th June 2018
DOI: 10.1039/c8ra03756f
rsc.li/rsc-advances
health, the selective detection of these thiols is of great
importance and a necessity for various biochemical investiga-
Introduction
Biological thiols such as glutathione (GSH), N-acetyl-L-cysteine tions and the diagnosis of related diseases. However, it is
NAC), homocysteine (Hcy) and cysteine (Cys) play essential challenging to selectively and quantitatively detect these thiols
(
1–6
roles in human physiology. Although these thiols have similar because of the structural similarity of Cys, Hcy, NAC and
structure, their roles in biological processes are diverse and GSH.
7
–10
associated with different diseases. For instance, GSH is a pivotal
In the past few years, some uorescent and colorimetric
indicator of the maintenance of xenobiotic metabolism, intra- probes have been developed to selectively detection Cys in terms
10,11
cellular signal transduction and gene regulation, which is of high sensitivity and simplicity of operation.
However,
highly involved in the aging and the pathogenesis of many most of these probes send out only one signal based on the
1,2
12–14
diseases. Since NAC was found to be a precursor of GSH in same chemo-sensor molecule,
which may be inuenced by
cells and a thiol antioxidant, it shows the potential to prevent possible variations in the sample environment, especially in
6
cancer and other mutation-related diseases. Hcy is a risk factor biological surroundings. In order to improve the accuracy of the
3,4
for Alzheimer's and cardiovascular diseases. Abnormal level results, the utilization of dual signaling probes is one of effec-
of Cys would lead to slow growth, skin lesions, liver damage and tive alternatives, which allows mutual correction on the same
5
15–20
edema etc. Due to the critical role of these thiols in human target analyte, yet through different transduction channels.
However, dual signaling probes for Cys are still very limited so
10,11,21
far.
a
Key Laboratory of Synthetic and Self-Assembly Chemistry for Organic Functional
In 2001, Ben Zhong Tang reported an abnormal phenom-
enon of aggregation induced emission (AIE) of silole, which
shows nonuorescence in solutions but strong emission once
Molecules, Center for Excellence in Molecular Synthesis, Shanghai Institute of
Organic Chemistry, University of Chinese Academy of Sciences, Chinese Academy of
Sciences, 345 Lingling Road, Shanghai 200032, People's Republic of China. E-mail:
22–24
cfeng@sioc.ac.cn; Fax: +86-21-64166128; Tel: +86-21-54925520
aggregated.
By taking advantage of attractive AIE phenom-
State Key Laboratory of Molecular Engineering of Polymers, Department of enon, a large number of intelligent AIE uorogens susceptible
b
Macromolecular Science, Fudan University, 220 Handan Road, Shanghai 200433,
to external stimuli, such as bio-species, mechanical force,
temperature change, pH variation, toxic vapor, and photonic
irradiation have been explored and developed. Tetraphenyl-
People's Republic of China
c
School of Physical Science & Technology, ShanghaiTech University, 100 Haike Road,
25
Shanghai 201210, People's Republic of China. E-mail: xyhuang@mail.sioc.ac.cn; Fax:
86-21-64166128; Tel: +86-21- 54925310
26–30
ethylene (TPE) is a typical AIE uorogen,
which has been
+
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widely used for the construction of various uorescent sensors approach was develop in the current work, which will allows to
due to its signicant increase in uorescence upon aggregation, mutual correction on the same target analyte to get more reli-
31–37
low cytotoxicity and ease in synthesis and modication.
Just able results. In addition, the reaction mechanism for selectively
recently, Wang and co-workers reported a new TPE-based AIE detecting Cys was systematically investigated in the current
probe bearing a 2,4-dinitrobenzenesulfonyl pendant, which is work, which is of great benet to design highly selective and
38
able to detect Cys selectively over Hcy and GSH in PBS. In this sensitive dual signaling probes for detecting biological thiols.
work, they found that uorescence would signicantly increase
upon the substitution reaction between the probe with Cys,
which would result in the leave of uorescent quencher of 2,4-
Experimental
Materials
dinitrobenzenesulfonyl group from TPE moiety. Although
previous works showed that some 2,4-dinitrobenzenesulfonyl-
based probes were not able to discriminate Cys from GSH or
Tetrahydrofuran (THF, Aldrich, 99%) was dried over CaH and
distilled from sodium and benzophenone under N prior to use.
2
2
39,40
Hcy,
this probe demonstrated a strong selectivity to Cys over
4
-Hydroxybenzophenone
trobenzenesulfonyl chloride (Alfa Aesar, 98%), benzophenone
Acros, 99%), L-cysteine (Cys, Acros, 99%), L-glutathione (GSH,
(Acros,
99%),
2,4-dini-
Hcy and GSH. All these results showed that the whole structure
of probes, besides 2,4-dinitrobenzenesulfonyl itself, also affect
the reactivity of substitution reaction toward thiol compounds,
(
Acros, 99%) and DL-homocysteine (Hcy, TCI, 90%) were used as
received without further purication. Cell Counting Kit-8 (CCK-
38–40
especially the reaction kinetics.
Herein, a TPE-based dual signaling probe was synthesized by
introducing a 2,4-dinitrobenzenesulfonyl group to a TPE moiety
8
, Dojindo, Japan) and DMEM medium (GIBCO/Invitrogen,
U.S.A). Other reagents not specially mentioned were
purchased from Aladdin and used as received without further
purication.
41
(Scheme 1). It is assumed that the 2,4-dinitrobenzenesulfonyl
group would not only make the TPE-based probe more soluble
in aqueous media, but also quench the possible uorescence of
TPE through photo-induced electron transfer (PET). Thus, the
replacement of 2,4-dinitrobenzenesulfonyl group by Cys would
not only signicantly increase the uorescent of TPE by the
combination of AIE and PET effect, but also enhance the UV/vis
absorption due to the producing of dinitrobenzene-based
compound. In addition, it is hypothesized that the discrimi-
nation of Cys could be achieved by more rapid intramolecular
displacement of sulfur with amino group of Cys than NAC, Hcy
and GSH. Therefore, the probe would be able to selectively
detect Cys over NAC, Hcy and GSH through the combination of
Instrumentation
1
13
All H and C NMR analyses were performed on a JEOL reso-
nance ECZ 400S (400 MHz) in DMSO-d , tetramethylsilane was
used as internal standard. Electron impact ionization mass
spectrometry (EI-MS) was performed by an Agilent 5937N
system. FT-IR spectra were recorded on a Nicolet AVATAR-360
spectrophotometer with a 4 cm resolution. UV/vis absorp-
tion spectra were recorded on a Hitachi U-2910 spectropho-
tometer. Fluorescence spectra were measured by a Hitachi F-
6
ꢀ1
2
2
700 uorescence spectrophotometer with a band width of
.5/5.0 nm. UV/vis and uorescence measurements were per-

uorescence and UV/vis absorption from two separated trans-
duction species. To test this hypothesis, the performance of
TPE-NP for selectively detecting Cys over other various bio-
species, including NAC, Hcy and GSH were examined by Fluo-
formed under room temperature.
rescent and UV/vis absorption spectroscopy and the mechanism Synthesis of 4-(1,2,2-triphenylvinyl)phenol (TPE-OH)
behind was also investigated by the combination of HPLC-MS
Benzophenone (7.26 g, 39.85 mmol), 4-hydroxybenzophenone
1
and H NMR. It should be pointed out that although the
strategy is similar to that of previous report, where only uo-
rescent channel was employed to detect Cys, a dual signaling
(
7.90 g, 39.85 mmol), zinc powder (11.46 g, 175.34 mmol) and
38
THF (200 mL) were added to a three-neck ask equipped with
a magnetic stirrer under N
ꢁ
2
. The mixture was cooled to 0 C
Scheme 1 Structures of TPE-NP sensor and its products upon treating with Cys.
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followed by adding TiCl
4
(16.63 g, 87.67 mmol) slowly via control group was only treated with physiological saline. The
a constant pressure drop funnel while the temperature was kept evaluation of cytotoxicity was determined by cell viability which
ꢁ
under 0 C. The suspending mixture was warmed to room was calculated on the basis of the absorbance of control group
temperature, stirred for 0.5 h and then heated at reux for and the absorbance of treated group.
another 15 h. The mixture was cooled to room temperature and
charged with HCl (320 mL, 1 M). The mixture was extracted by
2 2 4
CH Cl and the organic layer was dried over MySO and
Results and discussion
concentrated. The crude product was puried by silica column Synthesis of probe TPE-NP
chromatography (eluent: n-hexane/ethyl acetate, v/v ¼ 4/1) to
4
-(1,2,2-triphenylvinyl)phenol (TPE-OH) was prepared rstly
give 4-(1,2,2-triphenylvinyl)phenol (TPE-OH) (9.02 g, 65%) as
a white powder.
from benzophenone and 4-hydroxybenzophenone according to
previous report. Then, the sensor of 4-(1,2,2-triphenylvinyl)
phenyl 2,4-dinitrobenzenesulfonate (TPE-NP) was readily
synthesized by the reaction of TPE-OH with 2,4-dini-
trobenzenesulfonyl chloride. The structure of TPE-NP sensor
1
H NMR: d (ppm): 9.37 (s, 1H, OH), 7.10 (m, 9H, ArH), 6.97 (t,
6H, ArH), 6.75 (d, 2H, ArH), 6.48 (d, 2H, ArH).
Synthesis of 4-(1,2,2-triphenylvinyl)phenyl 2,4-
dinitrobenzenesulfonate (TPE-NP)
1
13
was well characterized by H NMR, C NMR, FT-IR and MS.
Fig. 1A shows H NMR spectrum of TPE-NP, which exhibits the
1
TPE-OH (4.50 g, 12.90 mmol), 2,4-dinitrobenzenesulfonyl resonance signals of 2,4-dinitrophenyl group (peak “a”, “b” and
chloride (6.88 g, 25.80 mmol), CH Cl (200 mL) and Et
N (2.61 g, “c”) at 9.08, 8.60 and 8.12 ppm and TPE moiety at 7.11 and
5.80 mmol) were added to a three-neck ask equipped with 6.94 ppm. The C NMR spectrum of TPE-NP demonstrates the
2
2
3
1
3
2
a magnetic stirrer under N
2
. The mixture was stirred at room expected resonance signals of TPE and 2,4-dinitrophenyl groups
ꢀ
ꢀ
1
temperature for 14 h. The solution was diluted with CH Cl (200 (Fig. 1B). In addition, the ESI-MS result (578.11 g mol ) was
2
2
1
mL), washed with 1 M HCl and brine (200 mL), and dried over also consistent with the theoretical value (578.11 g mol ). All
MySO . The solvent was removed by rotary evaporation, and the these results conrmed the successful synthesis of target sensor
4
crude product was puried by silica gel chromatography TPE-NP.
(
eluent: n-hexane/ethyl acetate, v/v ¼ 5/1) to give 4-(1,2,2-tri-
phenylvinyl)phenyl
2,4-dinitrobenzenesulfonate
(TPE-NP)
Selectively and quantitatively detection of Cys by TPE-NP
(
5.99 g, 80%) as a yellow powder.
1
Aqueous solution of TPE-NP (240 mM in PBS/DMSO (v/v ¼ 8/2),
pH ¼ 7.4) is colorless because of the absence of absorption
features in the visible region (Fig. 2A). The solution exhibits no
H NMR: d (ppm): 9.08 (s, 1H, ArH), 8.59 (d, 1H, ArH), 8.10 (d,
1
H, ArH), 7.11 (m, 9H, ArH), 6.94 (m, 10H, ArH).
1
3
C NMR: d (ppm): 121.16, 121.57, 126.85, 127.42, 128.03,

uorescence, probably due to relatively high solubility of TPE-
1
1
30.68, 132.72, 133.62, 139.09, 114.83, 142.56, 142.85, 143.47,
46.85, 148.28, 151.47.
NP in the media and photo-induced electron transfer between
+
tetraphenylethene
group
and
dinitrophenyl
moiety
MS (ESI): calc. for C32
01.11.
H
22
O
7
N
2
S ([M + 23] ): 601.11; found:
6
Synthesis of 4-phenyl 2,4-dinitrobenzenesulfonate (phenol-NP)
Phenol (1.00 g, 10.60 mmol), 2,4-dinitrobenzenesulfonyl chlo-
ride (5.66 g, 21.23 mmol), CH
2
Cl
2 3
(50 mL) and Et N (2.15 g,
21.25 mmol) were added to a three-neck ask equipped with
2
a magnetic stirrer under N . The mixture was stirred at room
temperature for 12 h. The solution was diluted with CH Cl (50
2
2
mL), washed with 1 M HCl and brine (50 mL), and dried over
MySO . The solvent was removed by rotary evaporation, and the
4
crude product was puried by silica gel chromatography
(
eluent: n-hexane/CH
2
Cl
2
, v/v ¼ 3/1) to give 4-phenyl 2,4-dini-
trobenzenesulfonate (phenol-NP) (2.30 g, 67%) as a white
powder.
1
H NMR: d (ppm): 9.11 (s, 1H, ArH), 8.60 (d, 1H, ArH), 8.21 (d,
1H, ArH), 7.12 (m, 3H, ArH), 6.93 (m, 2H, ArH).
Cytotoxicity assay in vitro against HepG2 cells
The cytotoxicity of the samples were assessed against HepG2
cells with the CCK-8 assay. In brief, the HepG2 cells were
respectively incubated with the TPE-NP with different concen-
trations for 24 h. Aer that, the cells were resuspended in fresh
1
13
medium and treated with DMEM solution. Furthermore, the Fig. 1 (A) H NMR and (B) C NMR spectra of TPE-NP in DMSO-d
6
.
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Fig. 2 (A) UV/vis spectra, (B) dependence of absorbance intensity at 350 nm with time, and (C) fluorescent spectra of TPE-NP solution (240 mM in
PBS/DMSO (v/v ¼ 8/2), pH ¼ 7.4) in the presence of Cys (200 mM). (D) The time course of the fluorescence intensity at 450 nm of TPE-NP solution
(
240 mM in PBS/DMSO (v/v ¼ 8/2), pH ¼ 7.4) in the presence of 200 mM Cys and 2000 mM of Hcy and GSH.
15,42,43
(Fig. 2C).
Upon the addition of Cys (200 mM), the colorless indicated that the displacement reaction occurred continually
solution became bright yellow. This observation showed that upon the addition Cys, the product of TPE-containing moiety
TPE-NP could be a “naked-eye” probe for Cys. Meanwhile, the did not aggregate until its concentration is higher than its
absorption band at around 350 nm increased steadily over time critical aggregation concentration. This might be the reason
and reached plateau aer about 2 h (Fig. 2B). Upon the addition why the uorescent intensity just increased obviously aer
of Cys, a new emission peak located at 450 nm emerged and around 50 min upon the treatment with Cys.
a dramatic uorescence enhancement was observed in Fig. 2C.
We then incubated the solution with various concentrations
The time-dependent uorescence response was also detected, of Cys (20–400 mM) for 2 h. One can notice that both UV/vis
which showed that the uorescence intensity at 450 nm almost absorption and uorescent intensity of TPE-NP solution
does not change before 50 min but then increased sharply to increase with the increasing in the concentration of Cys (Fig. 3A
about more than ten times of the weak background aer 80 min and B). We plotted the absorbance intensity at 350 nm and
(Fig. 2D). Although the steady increase of absorption at 350 nm uorescence intensity at 450 nm of TPE-NP solution as
Fig. 3 (A) UV/vis and (B) fluorescence spectra of TPE-NP solution (240 mM in PBS/DMSO (v/v ¼ 8/2), pH ¼ 7.4) as a function of Cys concentration.
The insets are the absorbance at 350 nm and the fluorescence intensity at 450 nm as a function of Cys concentration, respectively.
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a function of the concentration of Cys. Both absorbance and Especially, GSH, Hcy and NAC, three thiol-containing
uorescence intensities are linearly proportional to the compounds as Cys, did not cause obvious change in uores-

concentration of Cys (insets of Fig. 3A and B), resulting the cent emission, even the amount of Hcy and GSH are ten times of
detection limit of Cys in PBS low to 0.21 mM (S/N ¼ 3) from that of Cys (Fig. 2D and 4C). All these results show that TPE-NP

uorescent channel. In previous reports on the detection of Cys has a high selectivity toward Cys over Hcy, NAC, GSH and other
by uorescent sensors, the detection limit is in the range from related bio-species, even if the only difference of Hcy and NAC
44–46
0.121 mM to 1 mM.
Especially, the detection limit of Cys for from Cys is a methylene or an acetyl group at their residues.
a TPE-based probe reported by Wang and co-workers is about
38
0
.18 mM, close to the value of TPE-NP. All these results show
Mechanism of detecting Cys by TPE-NP
Since dinitrophenyl group is well-known uorescent
quencher, the uorescence of TPE-NP is probably quenched by
that Cys can be quantitatively detected on the basis of the
change in UV/vis absorption and uorescence intensities.
We then test selectivity of TPE-NP by examining UV/vis
absorption and uorescence responses of TPE-NP toward Cys
and a variety of bio-species, including amino acids (aspartic
acid, glutamic acid, phenylalanine, tyrosine, threonine, argi-
nine, histidine, asparagine, leucine, alanine, proline, valine,
glycine, lysine, glutamine, methionine, isoleucine, serine and
a
15,42,43
dinitrophenyl group due to PET effect.
On the basis of
15,42,43
previous reports,
we speculated that the displacement of
dinitrophenyl group of TPE-NP upon the treatment with Cys
gave TPE-OH and dinitrophenyl-containing compounds
(Scheme 1). Thus, uorescence emission of TPE-OH at 450 nm
+
+
tryptophan), as well as common biological metal ions (Na , K ,
was turned on and the produce of dinitrophenyl-based
compound resulted in the yellow color of solution (Scheme 1).
In order to testify the yellow color originated from
dinitrophenyl-containing compound, we synthesized a new
sensor of 4-phenyl 2,4-dinitrobenzenesulfonate (phenol-NP) by
2
+
2+
Ca and Mg ), reactive oxygen species (H O ), vitamin C, GSH,
2
2
NAC and Hcy. It was found that these common bio-species did
not cause signicant changes in color (Fig. 4A), UV/vis absorp-
tion (Fig. 4B) and uorescence (Fig. 4C) of solution except Cys.
Fig. 4 (A) Photographs, (B) UV/vis absorbance at 350 nm and (C) fluorescence intensity at 450 nm of TPE-NP solution (240 mM in PBS/DMSO (v/v ¼ 8/
), pH ¼ 7.4) upon incubation with 200 mM of various bio-species, including amino acids (aspartic acid, glutamic acid, phenylalanine, tyrosine, threonine,
arginine, histidine, asparagine, leucine, alanine, proline, valine, glycine, lysine, glutamine, methionine, isoleucine, serine, tryptophan, GSH and Hcy), metal
2
+
+
2+
2+
2 2
ions (Na , K , Ca and Mg ), H O , vitamin C and NAC for 2 h, respectively.
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Fig. 5 (A) UV/vis spectra of TPE-NP and phenol-NP solutions (240 mM in PBS/DMSO (v/v ¼ 8/2), pH ¼ 7.4) with and without the treatment with
Cys and phenol. (B) Photographs of TPE-NP and phenol-NP solutions (240 mM in PBS/DMSO (v/v ¼ 8/2), pH ¼ 7.4) with and without treatment
with Cys, TPE-OH and phenol. Inset of part (A) is the structure of phenol-NP.
the reaction between phenol, instead of TPE-OH, and 2,4-dini- resonance signals of TPE-OH at 6.47 and 6.70 ppm and three
trobenzenesulfonyl chloride (Fig. 5A). Aer the aqueous solu- peaks originated from the protons of dinitrophenyl group at
tion of phenol-NP (240 mM in PBS/DMSO (v/v ¼ 8/2), pH ¼ 7.4) 8.95, 8.86 and 7.23 ppm appear aer the incubation (Fig. 6B).
was treated with Cys (200 mM), the colorless solution turned These observations show that TPE-OH is one of the products of
yellow (Fig. 5B) and the absorption with a maximum wavelength the reaction and the up-shi of signals from dinitrophenyl
at about 350 nm obviously increased (Fig. 5A). Whereas, moiety suggests the leave of electron withdrawing group.
aqueous solutions (PBS/DMSO (v/v ¼ 8/2), pH ¼ 7.4) of TPE-OH
Since all of Cys, Hcy, NAC and GSH contain a thiol group,
and phenol is colorless (Fig. 5B). Thus, these results indicate one inevitably raises the question why Cys has much higher
that the uorescence and UV/vis absorption (yellow) are origi- reactivity toward TPE-NP than Hcy, NAC and GSH. On the basis
nated from the aggregates of TPE-OH and dinitrophenyl- of previous reports on the detection of thiols using nitrophenyl-
15,42,43
containing compounds, respectively. This means that the dual containing sensors
and the above observations, a possible
signals of UV/vis absorption and uorescence for detecting Cys mechanism is proposed as shown in Fig. 7A. At pH ¼ 7.4, the
quantitatively and simultaneously based on TPE-NP probe are deprotonated thiols of Cys, Hcy, NAC and GSH would serve as
originated from two separated signaling molecules. Thus, the active nucleophile to attack the dinitrophenyl of TPE-NP for
cross-referencing of the obtained results can be realized by this generating a sulfur-substituted intermediate (Fig. 7A). The
approach, which undoubtedly makes the results more reliable. primary amine of Cys, Hcy and GSH would further attack the
To verify the mechanism of the reaction of TPE-NP with Cys, dinitrophenyl to form thermodynamic ve-, six- or ten-
1
the products of the reaction were analyzed by HPLC and H membered cyclic transition state by intramolecular cycliza-
NMR (Fig. 6A and B). We can see that a peak at 20.1 min appears tion, which would assist the leave of TPE-containing moiety to
in HPLC curve (Fig. 6A) aer TPE-NP was incubated with Cys for give TPE-OH and SO . Since ve-membered ring is much more
2
2
h, where the retention time is the same as that of TPE-OH favored conformation than six- and ten-membered ring, the
(
20.1 min). Moreover, the molecular weight of this compound reaction rate for Cys would be much faster than those of Hcy
ꢀ
1
is 348.1 g mol , consistent with that of TPE-OH (348.1 g and GSH. To verify the importance of amino group in acceler-
mol ). One also can notice that two characteristic proton ating the displacement reaction, TPE-NP solution was
ꢀ
1
1
Fig. 6 (A) HPLC curves and (B) H NMR spectra in DMSO-d
6
of TPE-OH and TPE-NP before and after treating with Cys.
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Fig. 7 (A) Proposed mechanism for the reaction of TPE-NP with Cys and Hcy. (B) Fluorescence and (C) UV/vis spectra of TPE-NP solution (240
mM in PBS/DMSO (v/v ¼ 8/2), pH ¼ 7.4) in the presence of 3-mercaptoisobutyric acid, butanethiol and Cys (200 mM), lex ¼ 350 nm, respectively.
incubated with butanethiol and 3-mercaptoisobutyric acid for In vitro cytotoxicity of TPE-NP
2
h. In comparison to Cys, both butanethiol and 3-mercaptoi-
Finally, we performed proof-of-concept in vitro experiments on
the examination of the in vitro cytotoxicity of TPE-NP against the
HepG2 cells, which was evaluated by means of a standard CCK-8
cell assay. It could be seen that the cell viability of TPE-NP is
higher than 95% for HepG2 cells in a range of concentrations
from 5 to 50 mM, respectively. These results show that TPE-NP
have ignorable cytotoxic effect on the HepG2 cells in a range
of concentration (5–50 mM, 2.89–28.9 mg mL ), which demon-
strates the potential of TPN-NP in selectively detecting Cys in
vivo (Fig. 8).
sobutyric acid almost did not lead to obvious change in uo-
rescence and UV/vis spectra (Fig. 7B and C). The low reactivity of
TPE-NP toward NAC further shows the importance of amino
group in accelerating the displacement reaction. All these
results demonstrate that the proposed reaction process is
reasonable.
ꢀ1
Conclusions
In summary, a simple colorimetric and uorescent dual
signaling probe has been developed, and the probe can selec-
tively and quantitatively detect Cys from other common bio-
species including Hcy, NAC and GSH. The uorescence and
UV/vis absorption are originated from the aggregates of TPE-OH
and dinitrophenyl- containing compound, respectively. This
allows the mutual correction through different transduction
channels and signaling reporters, which will make the result
more reliable. It is proposed that the discrimination of Cys from
Hcy, NAC and GSH is achieved through the faster displacement
of sulfonate attaching to dinitrophenyl with thiolate for Cys,
followed by subsequent substitution of the thiolate by the
amino group of Cys. Given the low cytotoxicity of TPE-NP, the
Fig. 8 In vitro cytotoxicity of TPE-NP against HepG2 cells.
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interesting reaction mechanism provides
a
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Conflicts of interest
There are no conicts to declare.
Acknowledgements
The authors thank the nancial supports from National Key 22 J. D. Luo, Z. L. Xie, J. W. Y. Lam, L. Cheng, H. Y. Chen,
Research & Development Program of China (2016YFA0202900),
C. F. Qiu, H. S. Kwok, X. W. Zhan, Y. Q. Liu, D. B. Zhu and
Shanghai Scientic and Technological Innovation Project
B. Z. Tang, Chem. Commun., 2001, 1740–1741.
(16JC1402500 and 16520710300), the Youth Innovation 23 Y. N. Hong, J. W. Y. Lam and B. Z. Tang, Chem. Soc. Rev.,
Promotion Association of Chinese Academy of Sciences
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(2016233), Strategic Priority Research Program of Chinese 24 Y. N. Hong, J. W. Y. Lam and B. Z. Tang, Chem. Commun.,
Academy of Sciences (XDB20000000) and State Key Laboratory
of Molecular Engineering of Polymers (K2018-15).
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