A red-emitting fluorescent probe with large Stokes shift for real-time tracking of cysteine over glutathione and homocysteine in living cells

Article abstract of DOI:10.1016/j.saa.2019.02.050

Fluorescent probes with high quality for highly selective detection of cysteine (Cys) are still urgently in demand because of the indispensable roles Cys plays in the biological systems. Herein, a red-emitting fluorescent probe CP was developed for the highly selective detection of Cys over glutathione (GSH) and homocysteine (Hcy) by incorporating acryloyl group as the recognition unit into the 2-(2-(4-hydroxystyryl)-6-methyl-4H-pyran-4-ylidene) malononitrile (P-OH) fluorophore which is characterized by red emission, noteworthy Stokes shift, and appreciable photostability. Basically, CP demonstrated appreciable sensing performance toward Cys including short response time of 4 min, high sensitivity with approximately 147-fold emission enhancement, low detection limit of 41.696 nM, and good selectivity both in the solution and living cells, indicating its promising potential of visualizing Cys in biological systems.

Full text of DOI:10.1016/j.saa.2019.02.050

Accepted Manuscript
A red-emitting fluorescent probe with large Stokes shift for real-
time tracking of cysteine over glutathione and homocysteine in
living cells
Ming Qian, Liuwei Zhang, Jingyun Wang, Xiaojun Peng
PII:
S1386-1425(19)30169-6
https://doi.org/10.1016/j.saa.2019.02.050
SAA 16826
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 16 September 2018
Revised date:
Accepted
date:
16 January 2019
1
6 February 2019
Please cite this article as: M. Qian, L. Zhang, J. Wang, et al., A red-emitting fluorescent
probe with large Stokes shift for real-time tracking of cysteine over glutathione and
homocysteine in living cells, Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy, https://doi.org/10.1016/j.saa.2019.02.050
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ACCEPTED MANUSCRIPT
A red-emitting fluorescent probe with large Stokes shift for real-time tracking of
cysteine over glutathione and homocysteine in living cells
a,b
b
a,b,*
a
Ming Qian , Liuwei Zhang , Jingyun Wang , Xiaojun Peng
a
State Key Laboratory of Fine Chemicals, Dalian University of Technology, 116024,
Dalian, Liaoning, P. R. China
b
School of Life Science and Biotechnology, Dalian University of Technology, 116024,
Dalian, Liaoning, P.R. China
Abstract:
Fluorescent probes with high quality for highly selective detection of cysteine
(Cys) are still urgently in demand because of the indispensable roles Cys plays in the
biological systems. Herein, a red-emitting fluorescent probe CP was developed for
the highly selective detection of Cys over glutathione (GSH) and homocysteine (Hcy)
by incorporating acryloyl group as the recognition unit into the
2
-(2-(4-hydroxystyryl)-6-methyl-4H-pyran-4-ylidene)
malononitrile
(P-OH)
fluorophore which is characterized by red emission, noteworthy Stokes shift, and
appreciable photostability. Basically, CP demonstrated appreciable sensing
performance toward Cys including short response time of 4 min, high sensitivity with
approximately 147-fold emission enhancement, low detection limit of 41.696 nM, and
good selectivity both in the solution and living cells, indicating its promising potential
of visualizing Cys in biological systems.
Keywords: Fluorescent probe, Cysteine detection, Large Stokes shift, Fluorescence
imaging.
Introduction:
Cysteine (Cys), regarded as one of the significant biothiols in living systems,
play a crucial role in multiple physiological processes including the protein
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biosynthesis, mitochondrial protein turnover, metabolism regulation and
1
-3
detoxification administration . It was reported that the normal intracellular level of
4
Cys is in the range of 30-200 μM and an abnormal level of Cys is associated with
many syndromes such as Alzheimer's disease, liver damage, metabolic hair
5
-7
depigmentation and weakness . In view of the crucial physiological and pathological
roles Cys plays in biological systems, it is essential to develop an invasive analytical
tool for selective Cys detection so as to better elucidate the physiological processes of
Cys and understand the specific pathogenesis of Cys-related diseases. So far,
fluorescence analysis technique has been widely used in the investigation of
intermolecular interactions (such as between drug with human serum albumin,
8
-13
14-17
hemoglobin or DNA) , and detection of different analytes
. Fluorescence
detection based on small-molecular fluorescent probes that are capable of interacting
with the specific analyte to trigger fluorescent signal variation has attracted extensive
attention due to their inherent advantages including simplicity for implementation,
1
8-20
high sensitivity, good selectivity, and invasive in-situ detection in living systems
.
Among the reported fluorescent probes for the detection of various analytes, specific
reaction-based fluorescent probes that possess faster response, higher sensitivity
toward targeted analytes have attracted a great deal of attention. Over the past decade,
a large number of reaction-based fluorescent probes have been developed for biothiols
21-24
detection on the basis of serval sensing strategies including Michael addition
,
2
5-27
28
cyclization reaction with the aldehyde
, cleavage of disulfide and sulfonyl
2
9-31
ester
. Unfortunately, up to now, few reported fluorescent probes could distinguish
Cys from homocysteine (Hcy) and glutathione (GSH). Considering the fact that the
biothiols possess structural similarity, comparable reactivity, different amounts in
biological systems, distinct physiological and pathological functions, it is of great
challenges and significance to design brilliant fluorescent probe for discriminating
3
2
Cys from other two biothiols in living systems . Despite some advancement has been
obtained in terms of fluorescent probe design for selective detection of Cys, there
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remain some limitations waiting to be overcome to get the further application. Firstly,
some reported probes usually show relatively slow response rate or low
signal-to-noise ratio in the detection process, which is not favorable for real-time
3
3,34
tracking of Cys in living systems
. Besides, several fluorescent probes exploiting
fluorophores that emit fluorescence with a relatively short wavelength (< 600 nm)
generally suffer from the auto-fluorescence interference of intrinsic
3
5-42
biologically-related species
. Furthermore, certain reported long-wavelength
fluorescent probes that possess short Stokes shift (< 100nm) characterized by the
substantial overlap of excitation and emission bands are subjected to self-absorption
4
3-45
effect and unsatisfactory anti-interference capability
. Accordingly, developing
long-wavelength fluorescent probe with noteworthy Stokes shift for selective Cys
detection with rapid response and high sensitivity is still desirable for the practical
application.
Herein, in this study, a novel red-emitting fluorescent probe (named as CP)with
large Stokes shift was designed and synthesized for highly selective Cys detection in
living cells by introducing acryloyl group as the recogniztion group into the
2
-(2-(4-hydroxystyryl)-6-methyl-4H-pyran-4-ylidene) malononitrile fluorophore
4
6-48
(P-OH)
. Basically, the probe itself exhibits almost no fluorescence emission due
to the fluorescence masking effect of the acryloyl group. Upon recognizing with Cys,
CP will undergo the Michael addition between Cys and the acryloyl group to afford a
transient intermediate, followed by the intramolecular cyclization to release P-OH
fluorophore with strong red fluorescence. Accordingly, Cys was detected by
monitoring the changes of fluorescence intensity before and after the reaction. As
expected, this probe demonstrated excellent sensing properties toward Cys as
expected. Firstly, probe CP demonstrated good selectivity for Cys over various
analytes, especially, Hcy and GSH. Secondly, the probe exhibited short response time
of 4 min toward Cys in the solution. Thirdly, this probe had high signal-to-noise ratio
of 147-fold and high sensitivity with the ultralow detection limit of 41.696 nM. Lastly,
3

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CP was successfully applied to monitor the Cys level in living cells with low
cytotoxicity. To sum up, probe CP emerged as a promising reaction-based fluorescent
probe for the highly selective detection of Cys in living cells.
2
. Materials and methods
2
.1. Instruments and materials
All reagents used were obtained from commercial suppliers and used directly
without further purification unless otherwise stated. The synthesized compounds were
characterized by three different analysis method including high-resolution mass
1
13
spectra (HRMS), H NMR and C NMR. HRMS were obtained by the LTQ Orbitrap
1
13
XL mass spectrometer. H NMR and C NMR spectra were recorded in Deuterium
reagents by a Bruker Avance II 500 spectrometer with tetramethylsilane (TMS) as an
internal standard. All reactions were monitored using thin-layer chromatography
(TLC) which was performed on silica gel plates (60F-254). Column chromatography
was carried out on silica gel (200~300 mesh) obtained from Qingdao Ocean
Chemicals. The deionized water used for experiments was purified by a Millipore-Q
system (Millipore, USA). UV-vis absorption spectra and fluorescence emission
spectra were obtained employing an Agilent Cary 60 UV-vis spectrometer and
Agilent Cary Eclipse fluorescence spectrophotometer respectively using a 10 mm
standard quartz cuvette. Fluorescence images were obtained by confocal laser
scanning microscopy (Fluoview1200, Olympus, Japan) with a 100 × objective lens.
Scheme 1. The synthetic route of probe CP. Reaction conditions: (a) malononitrile,
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acetic anhydride, reflux; (b) p-hydroxybenzaldehyde, piperidine, acetonitrile, reflux;
(c) acryloyl chloride, DIEPA, 0 °C, anhydrous dichloromethane;
2
.2. Synthesis and Characterization
2
.2.1 The synthesis of 2-(2-(4-hydroxystyryl)-6-methyl-4H-pyran-4-ylidene)
malononitrile fluorophore (P-OH)
49
Compound 2 (516 mg, 3 mmol) and 4-hydroxybenzaldehyde (185 mg, 1.5
mmol), piperidine (0.3 ml) was added in 10 ml acetonitrile. The mixture was refluxed
for 6 h under N atmosphere. After the removal of the solvent, the residue was
2
dissolved with dichloromethane, washed with brine and deionized water three times
respectively, then the obtained organic phase was dried over Na SO . Subsequently,
2
4
the solvent was evaporated under reduced pressure to get the crude product, and the
crude product was purified by silica column chromatography (dichloromethane: ethyl
1
acetate = 100:5, v/v) to give an orange solid (42.7%). H NMR (500 MHz, DMSO-d )
6
δ 10.05 (s, 1H), 7.55 (d, J = 8.6 Hz, 2H), 7.46 (d, J = 16.1 Hz, 1H), 7.12 (d, J = 16.1
13
Hz, 1H), 6.86 – 6.79 (m, 3H), 6.66 (d, J = 1.3 Hz, 1H), 2.45 (s, 3H). C NMR
(
DMSO-d ): δ 163.89, 160.46, 159.72, 156.69, 137.84, 129.96, 125.94, 115.93,
6
−
1
2
15.54, 115.39, 105.88, 105.66, 55.00, 19.35. HRMS: calculated for [M-H] :
75.0821; found: 275.0827.
2
.2.2 The synthesis of the probe CP
P-OH (82.8 mg, 0.3 mmol), triethylamine (0.42 ml, 3 mmol) were dissolved in
1
0 ml anhydrous dichloromethane and stirred at 0 °C for 10 min, then acryloyl
chloride (54 mg, 0.6 mmol) was added dropwise. After reaction for 30 min, the
mixture was allowed to stir at room temperature for another 30 min. Afterwards, the
obtained solution was diluted with 50 ml dichloromethane and washed with brine and
distilled water for three times respectively, then dried over anhydrous Na SO . After
2
4
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the removal of the solvent, the crude product was purified by silica gel
chromatography using dichloromethane as the eluent to get the pure target product CP
1
as a yellow solid (62%). H NMR (500 MHz, DMSO-d ) δ 7.78 (d, J = 8.7 Hz, 1H),
6
7
2
1
.57 (d, J = 16.2 Hz, 1H), 7.43 – 7.32 (m, 1H), 7.29 (d, J = 8.6 Hz, 1H), 6.92 (d, J =
.2 Hz, 1H), 6.72 (d, J = 1.4 Hz, 0H), 6.56 (dd, J = 17.3, 1.3 Hz, 1H), 6.42 (dd, J =
13
7.2, 10.3 Hz, 1H), 6.18 (dd, J = 10.3, 1.3 Hz, 1H), 2.47 (s, 1H). C NMR
(
DMSO-d ): δ 164.67, 164.43, 160.08, 157.18, 151.92, 136.94, 134.39, 133.21,
6
1
29.64, 127.98, 122.92, 119.85, 115.84, 107.74, 106.41, 56.53, 19.89. HRMS:
−
calculated for [M-H] : 329.0926; found: 329.0940.
2
.3. General procedure for spectral measurements
The stock solution of probe CP (1 mM) was prepared by adding 1.65 mg pure
CP solid in 5 ml CH CN and maintained in the refrigerator at 4 °C for future use. For
3
the selectivity experiments, stock solutions of biologically relevant analytes were
prepared in distilled water. The fluorescence spectra of the resultant solution and the
P-OH fluorophore were measured using the excited wavelength of 500 nm. The
excitation slit width and emission slit width were fixed to 10 nm and 5 nm,
respectively. For the investigation of pH effects, the pH was accurately adjusted using
dilute hydrochloric acid solution and dilute sodium hydroxide solution and measured
with a Model PHS-3C meter.
2
.4. Cell culture and cytotoxicity assay
Cells were purchased from the Cell Bank of Shanghai Institute of Biochemistry
and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%
Fetal Bovine Serum (FBS) and 1% antibiotics. The culture was conducted in a cell
incubator under humidified environment of 5% CO and 37 °C. To conduct the
2
5
0
cytotoxicity assay , traditional MTT method was applied based on the mechanism
that succinate dehydrogenase in mitochondria of living cells can reduce exogenous
MTT to water-insoluble blue-purple crystalline methylene (Formazan) and deposit in
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cells, and the amount of formed formazan crystal was associated with the cell viability.
Briefly, HeLa cell lines were seeded into a 96-well plate with culture media (100 μl)
for overnight culture, then cells were incubated with different concentrations of CP (0,
2
, 4, 6, 8, 10 μM) for 24 h. Next, 10 μl MTT solution (5 mg/ml) was added to each
well. After incubation for 4 h, the culture media was discarded and 100 μl of dimethyl
sulfoxide (DMSO) was added to dissolve produced formazan. Untreated cells in
culture medium were used as the blank control. Finally, the OD value at 570 nm and
6
30 nm were measured with a microplate reader.
2
.5. Cellular imaging
5
1
For confocal luminescence imaging , HeLa cells in culture media (2 ml) were
5
seeded on 35 mm glass-bottomed dishes at a density of 1 × 10 cells per dish. After
the overnight culture, the cells were incubated with N-ethylmaleimide (NEM), probe
CP, biothiols and H O respectively according to the specific experiments.
2
2
Fluorescence images were obtained by confocal laser scanning microscopy
Fluoview1200, Olympus, Japan) with the excitation wavelength of 488 nm.
(
3
. Results and discussion
3
.1. Design and synthesis of probe CP
The probe CP was designed by employing P-OH as the fluorophore and acryloyl
moiety as the recognition unit. Fluorophore P-OH was picked up due to its red
emission properties, large Stokes shift of 141 nm (Figure S1) and excellent
photostability (Figure S2). On the other hand, the acryloyl group was selected because
it is not only an effective fluorescence quencher but also an excellent recognition
moiety that could react with Cys selectively through the Michael addition and the
intramolecular cyclization to release the fluorophore P-OH. As shown in Scheme 1,
CP can be readily synthesized from the reaction of P-OH with the commercially
available acryloyl chloride in the presence of triethylamine. Detailed synthetic
7

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procedures are provided in the experimental section. The structure of P-OH and CP
1
13
were successfully confirmed by H NMR, C NMR, and HRMS provided in the
supporting information.
3
.2. Time-dependent optical responses toward Cys
With probe CP in hand, its optical sensing performance toward Cys was firstly
investigated by measuring the time-dependent spectra changes in PBS-DMSO
pH=7.4, 1:1) solution. As demonstrated in Figure 1a, the probe itself exhibited a
(
maximum absorption peak at 390 nm. However, with the addition of 50 μM Cys, the
initial absorption peak of CP vanished gradually within 10 min, along with the
simultaneous emergence of two new intensified absorption peak at 425 nm and 500
nm which are consistent with the phenol form absorption peak and phenolate form
absorption peak of fluorophore P-OH, respectively (Figure S6a). In addition, as
shown in Figure S3, the color of the reaction mixture changed from pale yellow to
brown, demonstrating that this detection behavior of the probe for Cys could be
observed by the naked-eye. As for the time-dependent fluorescence spectra of CP
toward Cys, it could be observed in Figure 1b that the fluorescence intensity at 641
nm enhanced gradually as the reaction time increased and it reached the saturation
within 4 min. This quite short reaction time indicates a fast response behavior of CP
toward Cys and is much favorable for Cys detection. Additionally, this fluorescence
response behavior could also be observed under a 365 nm UV-lamp (Figure S3).
8

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Figure 1. The time-dependent response behavior of CP toward Cys in PBS-DMSO
solution (pH=7.4, 1:1). (a) The time-dependent absorption spectra change in 10 min
upon the addition of 50 μM Cys. (b) The time-dependent fluorescence spectra change
in 10 min upon the addition of 50 μM Cys.
3
.3. Fluorescence titration toward Cys
Subsequently, to evaluate the detection sensitivity of CP toward Cys, the
fluorescence titration experiment was conducted by measuring the fluorescence
change of CP toward different Cys concentrations in the range of 0-50 μM. It was
observed in Figure 2a that CP presented a notable fluorescence intensity enhancement
at 641 nm gradually toward the increasing amount of Cys and reached the plateau
with a 147-fold fluorescence enhancement upon the addition of 20 μM Cys.
Furthermore, it is worth noting that the fluorescence intensity at 641 nm was found to
2
be linearly proportional to the Cys concentration in the range of 0-6.0 μM (R =0.998)
with a low detection limit of 41.696 nM which was calculated according to the
5
2
formula of DL= 3σ/k , demonstrating that probe CP has high sensitivity for detecting
Cys quantitatively in the in vitro measurements (Figure 2b).
Figure 2. The fluorescence titration behavior of CP toward Cys in PBS-DMSO
solution (pH 7.4, 1:1). (a) The fluorescence spectra change of 10 µM CP with
addition of different Cys concentrations (0, 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 40, 45,
5
0 µM) for 10 min. (b) The fluorescence intensity at 641 nm changes with the
different Cys concentrations (0-50 µM), inset: the linear relationship between the
9

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fluorescent intensity at 641nm and the low concentration of Cys from 0 to 6 µM.
3
.4. Discriminating Cys over GSH and Hcy
Taking into consideration that Hcy and GSH are the most likely interferences for
Cys detection, the discrimination ability of CP for Cys over these two biothiols was
firstly investigated. As shown in Figure 3, with the addition of 50 μM Cys, the
emission intensity at 641 nm showed a rapid and great fluorescence enhancement at
first and reached the maximum within 4 min. By contrast, the addition of 50 μM Hcy
or 50 μM GSH induced slight fluorescence intensity change at 641 nm. Moreover,
within the same incubation time (4 min), the fluorescence intensity induced by Hcy or
GSH is 1:11 and 1:18 in ratio when compared with Cys, indicating the low reactivity
kinetic characteristic of CP toward GSH and Hcy (Figure S5). Noting that the
inherent amount of GSH in living systems is usually in the extremely high level of
millimole. Therefore, the response behavior of CP toward 1 mM GSH was
investigated. As shown in Figure S6, the treatment of 1 mM GSH showed tiny
fluorescence alternation with a more time-consuming manner. Based on these results,
probe CP exhibited higher reactivity and selectivity for Cys over Hcy and GSH.
Figure 3. The ability of CP for discriminating Cys from GSH and Hcy. (a) The
fluorescence spectra change of CP toward 50 μM GSH, 50 μM Cys, 50 μM Hcy in
PBS-DMSO buffer (pH = 7.4, 1: 1). (b) Time-dependent fluorescence intensity
change at 641 nm toward 50 μM GSH, 50 μM Cys, 50 μM Hcy in PBS-DMSO buffer
(pH = 7.4, 1: 1)
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3
.5. Selectivity over other biological analytes
To further evaluate the selectivity of CP toward Cys, the fluorescence response
behavior of CP toward other physiological amino acids was investigated. As shown in
Figure 4, only the addition of Cys (100 μM) could induce CP to display an obvious
response with enhanced fluorescence signal. By contrast, other 19 amino acids (100
μM) resulted in negligible fluorescence at 641 nm when compared with Cys. And the
specific intensity ratio between other amino acids and Cys (other amino acids :
Cys)was determined as follows: 1:49(Asp), 1:36(Gly), 1:53(Glu), 1:59(Ala),
1
1
:34(Trp), 1:33(Ser), 1:33(Val), 1:53(Ile), 1:32(Pro), 1:31(Phe), 1:31(Met), 1:42(Tyr),
:41(His), 1:46(Thr), 1:58(Leu), 1:40(Asn), 1:37(Gln), 1:32(Lys), 1:31(Arg).
Basically, the above fluorescence intensity ratios were all less than 1:30, which
indicated the good selectivity of CP toward Cys. Furthermore, the competitive
experiment suggested that the other amino acids exerted almost no fluorescence
intensity alternation, demonstrating that the co-existence of other amino acids didn’t
interfere with the recognition and detection of Cys by probe CP. In a word, CP
showed excellent selectivity toward Cys over other analytes and is feasible for the
selective detection and bioimaging of Cys in living systems.
Figure 4. The sensing performance of 10 μM CP toward other 19 amino acids (100
1
1

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μM) in PBS-DMSO buffer (pH = 7.4, 1:1) in the absence (black bars, the y-axis of
black bars is on the right) and presence (red bars, the y-axis of black bars is on the left)
of 50 μM Cys.
3
.6. Verifying the sensing mechanisms
Firstly, as observed in Figure S7, the fluorescence spectra of the reaction solution
were consistent with that of the fluorophore P-OH, which basically indicated that
P-OH was indeed released during the sensing process. To further explore the sensing
mechanism of CP toward Cys, the mass spectroscopy analysis was employed to
analyze the product released from the reaction process. As demonstrated in Figure S8,
−
predominantly, three [M-H] peak at m/z 119.98, 174.01, 275.15 were observed,
which were assigned to the Cys (Mr =121.02), the released seven-membered ring
cyclic amide by-product (Mr =175.03) and P-OH (Mr =276.09) respectively. On the
5
3,54
3
basis of the above results and the reported literatures
, a proposed sensing
mechanism of CP toward Cys was outlined in Scheme 2. Specifically, the reaction
started with a nucleophilic addition of Cys to the α, β-unsaturated carbonyl group of
probe CP to form the intermediate. Subsequently, this transient intermediate would
undergo the free amino-group mediated intramolecular cyclization process to produce
the fluorophore P-OH accompanied by the release of the seven-membered ring
cyclization product simultaneously. Based on the proposed sensing mechanism, the
different response behavior of CP toward biothiols could be reasonably explained.
Basically, the intramolecular cyclization process is no doubt the rate-limiting step
during the reaction process. Compared with Hcy, Cys could induce the more
kinetically favorable formation of a seven-membered ring product rather than the
kinetically disfavored eight-membered ring (the possible product for Hcy) and thus
resulted in the faster response behavior. On the other hand, GSH could only generate
1
2

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the conjugated thioether rather than conducting the intramolecular cyclization reaction
due to the hindrance effect of the bulk volume of GSH itself.
Scheme 2. Proposed sensing mechanism of CP toward Cys.
3
.7. The effect of pH
Afterwards, the pH effect on CP in the absence or presence of Cys was evaluated
to validate whether this probe is appropriate for the Cys detection in living systems.
As displayed in Figure 5, for the probe CP, the fluorescence intensity at 641 nm
exhibited almost no change over the pH range of 3.0−9.0, indicating that probe CP is
pH-unaffected and has good stability in a wide pH range. However, upon the addition
of Cys, an obvious enhancement of fluorescence intensity was observed in the pH
range of 6.0-9.0, which was attributed to the fact that the high pH environment would
−
boost the deprotonation of P-OH fluorophore to produce more deprotonated PM-O
which is the truly effective form responsible for the red fluorescence emission. In a
word, the results above indicated that CP can work smoothly over a wide pH range
and is suitable for biological applications.
1
3

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Figure 5. The fluorescence intensity of 10 μM CP at 641 nm under different pH
values (from 3.0 to 9.0) in the absence and presence of 50 μM Cys.
3
.8. Fluorescence imaging of Cys in living cells
On account of the excellent sensing performance in the solution, the practical
application of CP in living cells was evaluated by confocal fluorescence microscope.
Before the conduction of confocal fluorescence imaging, cytotoxicity test against
HeLa cells was carried out to investigate the biocompatibility of the CP. As shown in
the Figure S9, the viability of HeLa cells maintained to be more than 90% upon the
incubation with 0−10 μM of CP for 24 h, suggesting that CP possess low cytotoxicity
against the living cells and was safe enough to be used for the bioimaging of Cys.
Subsequently, the practical application of CP for Cys detection in confocal
fluorescence imaging was carried out in the HeLa cells. As demonstrated in Figure 6,
with merely the addition of probe CP, red fluorescence was observed in the cytoplasm
with the excitation wavelength of 488 nm. Nevertheless, when treated with a
5
5
recognized biothiols consumption agent (N-ethylmaleimide, NEM) prior to the
addition of CP, basically no fluorescence signal was observed due to the consumption
of intramolecular Cys. The results above indicated that CP could be internalized into
HeLa cells and response with the basal Cys. Furthermore, upon the exogenous
addition of 50 μM Cys, 50 μM Hcy, 1 mM GSH to the NEM-pretreated HeLa cells
1
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respectively, only the treatment of Cys induced distinct red fluorescence signal that is
about 7:1 in ratio compared to GSH and 20:1 in ratio for Hcy, furtherly indicating the
highly specific response behavior of CP toward Cys over GSH and Hcy in living cells.
Taking the above results together, we can draw the conclusion that CP could detect
and visualize both exogenous and endogenous Cys in living cells through a highly
specific manner. In addition, the treatment of 100 μM H O resulted in the attenuation
2
2
of the red fluorescence signal due to the decrease of the intracellular Cys
concentration induced by the oxidation of H O , suggesting that probe CP could be
2
2
exploited as an effective tool for monitoring the level of intracellular Cys. Finally, we
attempted to validate whether CP could achieve real-time detection of Cys in living
cells. As displayed in Figure S10, upon the exogenous addition of 50 μM Cys to the
NEM-pretreated HeLa cells, with the increase of incubation time at an interval of 5
minutes, the red fluorescence intensity enhanced increasingly and reached a steady
state after 20 min, implying that CP is capable of real-time tracking the Cys level in
living cells. To sum up, these findings above suggested that probe CP could be a
feasible tool to monitor the level of Cys via a rea-time and highly selective manner.
Figure 6. Confocal fluorescence images of Cys in living HeLa cells. (a) (2A): HeLa
cells incubated with 10 μM CP for 30 min. (2B): HeLa cells pretreated with 1.0 mM
NEM for 30 min and then incubated with 10 μM CP for 30 min. (2C, 2D, 2E) :HeLa
cells pretreated with 1.0 mM NEM for 30 min and then incubated with 50 μM Hcy,
1
.0 mM GSH, and 50 μM Cys for 1 h, respectively, and finally incubated with 10 μM
1
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CP for 30 min. (2F): HeLa cells pretreated with 100 μM H O for 30 min and then
2
2
incubated with 10 μM CP for 30 min. Scale bar: 20 μm. Channel 1A-1F and 3A-3F
are the corresponding bright-field and merge image of 2A-2F, respectively. (b) The
quantified mean intensity of channel 2A-2F obtained by image J software.
4
. Conclusion
In summary, a novel red-emitting fluorescent probe, namely, CP, was developed
for the highly selective detection of Cys in living cells. This probe was designed
based on the conjugated addition and intramolecular cyclization mechanism. By
exploiting the difference in reaction kinetics, CP could distinguish Cys from Hcy and
GSH directly. In addition, the rapid response (4 min), high sensitivity (147-fold
fluorescence enhancement), and noteworthy Stokes shift (141 nm) contributed to the
appreciable sensing properties of CP toward Cys. In view of the excellent sensing
performance in solution and the low cytotoxicity, CP was successfully applied in the
real-time monitoring of Cys level in living HeLa cells. Overall, this work provided a
potential red-emitting fluorescent probe for the real-time and highly selective
detection of Cys in biological systems.
ACKNOWLEDGMENT
This research was financially supported by the National Natural Science Foundation
of China (Grant Number. 21878041, U1608222).
AUTHOR INFORMATION
Corresponding Author
*
E-mail: wangjingyun67@dlut.edu.cn. Tel: +86-41184706356
Notes
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The authors declare no competing financial interest.
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exogenous and endogenous cysteine in biological samples. Anal. Chim. Acta 994(2017)
73-81.
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Graphic abstract
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Highlights
>
>
A red-emitting fluorescent probe CP with large Stokes shift was developed.
CP can discriminate cysteine from glutathione and homocysteine in the solution
and living cells.
>
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CP shows rapid response and high sensitivity toward cysteine.
The limit of detection was as low as 41.696 nM.
CP can monitor the exogenous and endogenous cysteine in living cells.
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