A "turn-on" fluorescent probe used for the specific recognition of intracellular GSH and its application in bioimaging

Article abstract of DOI:10.1039/c5ra25204k

We have designed and synthesized a carbazole-based fluorescent probe (CZ-Nm) for the specific recognition of GSH over Cys and Hcy. The probe is almost non-fluorescent owing to the photoinduced electron transfer (PET) process from the carbazole fluorophore to the nitroolefin moiety. Upon treatment with GSH, owing to the Michael addition of GSH to the double bond of the nitroolefin moiety, the probe shows a fluorescence enhancement and absorption change. The CZ-Nm probe displays desirable properties such as acting as a "naked eye" probe, a wide linear range of 0-0.01 M, high sensitivity, and strong anti-jamming capability. More importantly, the probe can also be successfully applied to the detection of intracellular GSH with a bright fluorescence signal, good cell permeability and bio-compatibility.

Full text of DOI:10.1039/c5ra25204k

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A “turn-on” fluorescent probe for specially recognize on intracellular
GSH and its application in bioimaging
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Lanfang Pang, Yanmei Zhou*, Enze Wang, Fang Yu, Hua Zhou, Wenli Gao
Institute of Environmental and Analytical Sciences, College of Chemistry and
Chemical Engineering, Henan University, Kaifeng, Henan 475004, P.R. China
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Correspond author: Tel: +86-371-22868833-3422; Fax: +86-371-23881589
E-mail address: zhouyanmei@henu.edu.cn (Y.M. Zhou)
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Abstract: We designed and synthesized a carbazole-based fluorescent probe (CZ-Nm)
for the specific recognition of GSH over Cys and Hcy. The probe is almost
non-fluorescent owing to the photoinduced electron transfer (PET) process from the
carbazole fluorophore to the nitroolefin moiety. Upon treatment with GSH, owing to
the Michael addition of GSH to the double bond of nitroolefin moiety, the probe
showed a fluorescence enhancement and absorption change. The probe CZ-Nm
displayed high properties such as acting as a “naked eye” probe, a wide linear range
0-0.01 M, a high sensitivity, and strong antijamming capability. More importantly, the
probe can also be successfully applied to detect the intracellular GSH with bright
fluorescence signal, good cell permeability and bio-compatibility.
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Introduction
Cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are important
intracellular thiols that play vital roles in physiological and pathological events,1-3 and
the abnormal levels of these thiols are closely related to certain diseases.4,5 In
particular, GSH is the most abundant intracellular thiol (1-10 mM) in mammalian and
many prokaryotic cells, functioning as an essential endogenous antioxidant primarily
involved in regulating cellular redox activities and preventing cell damage through
scavenging free radicals and peroxides.6,7 The aberrant levels of GSH are directly
associated with numerous clinical diseases including cancer, human
immunodeficiency virus (HIV), Alzheimer's, liver damage, leukocyte loss,
cardiovascular disease and others.8,9 Accordingly, developing efficient techniques for
detection of GSH under physiological conditions is of considerable significance and
has attracted a great deal of attention.10,11 Among the various detection methods that
have been reported in the past decades,12 optical imaging through staining with a
fluorescent probe is considered to be the most convenient and efficient approach
owing to its high sensitivity, good flexibility as well as apparent simplicity.13,14 In
particular, the fluorescent probe could be successfully applied to intracellular
detection.15,16
Up to now, numerous thiol probes have been designed and synthesized utilizing the
strong nucleophilicity of the thiol group.17 Although these probes can selectively
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detect these biothiols from other amino acids, the discriminative detection of certain
kind of thiols still remains a significant challenge due to their similar structure and
reactivity.18 In recent years, pioneered by Strongin’s group, the selective detection of
Cys/Hcy over GSH by means of the cyclization of Cys/Hcy with aldehydes could be
realized.19-22 Since then, the discrimination detection of Cys from Hcy/GSH and Hcy
from GSH/Cys using the Michael addition combined with steric and electrostatic
interactions,23,24 Cys-induced substitution-rearrangement cascade reaction25,26 or the
cyclization of Cys with acrylates27,28 were also developed. By comparison, probes
capable of detecting GSH from Cys/Hcy have been addressed in relatively less
extent.29,30 To our knowledge, some fluorescent probes based on organic dyes for
highly selective detection of GSH have been designed.31 For example, the
Chmielewski's group designed a seminal GSH-specific fluorescent probe based on
breaking the disulfide bonds followed by intramolecular cyclization and cleavage of a
neighboring carbonate bond between a rhodamine 110 derivative and GSH.32 Yang's
group prepared a bis-spiropyran sensor based on supramolecular interaction for
detecting GSH selectively.33 Taking advantage of the thiols-induced addition reaction,
78 Wang34, Huo35 and Keillor36 designed innovated chemical sensors for selectively
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detect GSH. Also, Yang37, Guo38 and Lin39 also designed GSH-specific fluorescent
probes by means of the thiols-induced substitution reaction. Therefore, it is of high
interest to design GSH-specific fluorescent probes.
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Among the various organic fluorophores, the carbazole dye was used as the mother
molecule due to its widely accepted superiority40,41 (e.g., high fluorescence quantum
yield, good water-solubility, good absorption cross section, high photostability,
visible wavelength absorption and its modular nature for facile functionalization, etc.)
Focusing on the advancing new strategies in sensing and signaling, we now construct
a nitroethenyl-carbazole conjugate for both colorimetric and fluorescence “turn-on”
responses to GSH. In the absence of GSH, the nitroolefin moiety serves as an electron
acceptor for the photoinduced electron transfer (PET) process, quenching the
fluorescence of carbazole fluorophore (ΦF=0.006). Upon treatment with GSH, owing
to the Michael addition reaction, the double bond between the fluorophore and the
quencher is broken, blocking the PET process and leading to the fluorescence
recovery (ΦF=0.30).42,43 Moreover, We conclude that the reaction of GSH to
nitroolefin breaks the conjugated structure of the probe CZ-Nm, also inhibits the
intramolecular charge transfer (ICT) between the N-ethyl moiety and the nitroolefin
moiety, producing a blue shift in fluorescence spectra, and accompanied with an
obvious colour change in solution.44,45 (Scheme 1)
Experimental
Apparatus
1H and 13C NMR spectra were measured on a Bruker DMX-300 spectrometer
operating at 400 MHz in Chloroform-d. The MS spectra were performed on Agilent
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1100. The UV-Vis absorption spectra were recorded on a U-4100 spectrophotometer.
An Edinburgh FS5 spectrofluorometer was used for fluorescence measurements. An
Olympus Zeiss 710 laser scanning confocal microscopy was used for fluorescence
image of cells. The pH measurements were measured using Jingke PHS-3D digital
PH-meter.
Materials
Carbazole, sodium hydride, nitromethane and amino acids were purchased from
Aladdin. Iodoethane was purchased from Energy Chemical. Piperidine was purchased
from Sinopharm Chemical Reagent Plant. The solvents were used as received without
further purification. Distilled water was used throughout.
Synthesis
As shown in Scheme S1, the key intermediate N-Ethylcarbazole-3-carboxaldehyde
was synthesized according to the literature procedure. The probe CZ-Nm was further
prepared according to well known Knoevenagel Condensation.46 A solution of
compound N-Ethylcarbazole-3-carboxaldehyde (0.50 g, 2.24 mmol), MeNO2 (196 ꢀL,
6.72 mmol), and NH4OAc (0.52 g, 6.72 mmol) in 20 ml acetic acid was then heated at
reflux with magnetic stirring for 3 h. After cooling, the resulting solution was
concentrated by distillation under reduced pressure, and chromatography on silica gel
using DCM/PE (v/v, 1:2) as the eluent afforded an orangered solid (0.53 g, yield
90%). Mass spectrometry: m/z, calcd: 266.11, found: 267.03 ([M+H]+). 1H NMR (400
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MHz, CDCl3) δ (ppm): 8.27 (s, 1H), 8.23 (d, J = 13.5 Hz, 1H), 8.12 (d, J = 7.8, 0.9 Hz,
1H), 7.71 (d, J = 13.5 Hz, 1H), 7.65 (d, J = 8.6 Hz, 1H), 7.54( t, J = 8.3 Hz, 1H), 7.48
-7.42 (m, 2H), 7.32 (t, J = 8.0 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 1.47 (t, J = 7.3 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ (ppm): 142.21, 140.93, 140.52, 134.19, 126.87,
126.59, 123.73, 122.96, 122.55, 120.73, 120.67, 120.27, 109.19, 109.37, 37.92, 13.89.
General spectra measurements
A 1.0×10-3 M of stock solution of the probe CZ-Nm was prepared in DMF. Stock
solutions of the biologically relevant analytes (i.e. GSH, Cys, Hcy, Thr, Ser, Glu, Lys,
Phe, His, Gly, Ala, Val, Tyr) were prepared in distilled water with a concentration of
4.0×10-2 M for UV/Vis absorption and fluorescence spectra experiments. In a typical
experiment, test solutions were prepared by placing 40 ꢀL of the probe stock solution
into a solution of 4 mL buffered (0.1 M PBS, pH 7.4) aqueous DMF solution
(H2O/DMF=4:1, v/v). The fluorescence spectra were measured after the addition of
analytes for 30 min at room temperature (λex=360 nm).
Cells culture
PC12 cells were seeded in glass bottom culture dishes and grown in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 2.5 % fetal bovine serum
(FBS) and 15 % horse serum at 37 °C with 5 % CO2 atmosphere until harvesting for
the experiment. When harvesting, the DMEM was drawn out from the culture dishes,
and the dishes were rinsed three times with 10 mM phosphate buffer saline (PBS) and
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then treated with 4 mL trypsinase solution which contain 0.25 % EDTA for 3 min in
the incubator.
Results and discussion
Conditional experiments
During the initial attempt, the probe CZ-Nm exhibited the response to GSH. Then,
we firstly investigated the influence of fraction of water on the interaction of the
probe CZ-Nm with GSH. Among various fraction of water tests, a combination of 0.1
M PBS buffer-DMF (pH 7.4, v/v, 4:1) proved to be highly efficient for the sensing
process (Fig. S1). Therefore, we choose 0.1 M PBS buffer-DMF (pH 7.4, v/v, 4:1) as
our test system.
To test the effect of pH values on the fluorescence intensity changes of the probe
CZ-Nm in the absence and presence of GSH. The GSH was obtained at different pH
values (from 3.0 to 11.0, prepared with NaOH and HCl). Fig. S2 shows the
relationship between pH value and the fluorescence intensity at 420 nm. As it can be
seen, the probe CZ-Nm exhibited an obvious response in the range of 7.0-11.0. For
the detection of the GSH in vivo, the PBS buffer of pH 7.4 was selected as the general
measurement condition owing to the biologically relevant pH range is 5.8-8.0, and the
probe CZ-Nm could be used to detect intracellular GSH without interference.
The time dependent fluorescent responses of the probe CZ-Nm to thiols were
monitored at 420 nm under optimal conditions (0.1 M PBS buffer-DMF, pH 7.4, v/v,
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4:1). Fig. S3 shows the fluorescence intensity reached its maximum value at about 25
min. Therefore, a 25 min reaction time and a medium of 0.1 M PBS buffer-DMF (pH
7.4, v/v, 4:1) solution were selected in subsequent experiments in order to make the
reaction of the probe CZ-Nm with GSH sufficiently.
UV-Vis absorption and fluorescence spectra response of CZ-Nm with GSH
The absorption spectra and fluorescence spectra of the probe CZ-Nm (10 ꢀM) were
explored in PBS buffer-DMF (0.1 M, pH 7.4, v/v, 4:1) solution in the presence of
different amino acids and thiol compounds. As shown in Fig. 1, owing to the
influence of PET and ICT effect between the fluorophore and nitroolefin moiety, the
probe CZ-Nm has a very weak fluorescence (ΦF=0.006) under a 365 nm UV lamp
that can hardly be observed with the naked eye. The addition of GSH induced a
variation in the solution from no fluorescence to a strongly blue fluorescence
(ΦF=0.30), and the maximum fluorescence emission peak has a blue shift from 480
nm to 420 nm. However, relevant analytes such as Cys, Hcy, Thr, Ser, Glu, Lys, Phe,
His, Gly, Ala, Val and Tyr showed almost no changes in the fluorescence spectra,
which indicated that probe CZ-Nm selectively responded to GSH.
Similarly, the UV-Vis spectra also exhibited the response of the probe CZ-Nm to
GSH. Upon the addition of GSH, the absorption peak at 420 nm decreased and a new
peak at 267 nm appeared, accompanied with a colour change from yellow to
colourless (Fig. 2), implying the break of the intramolecular conjugated structure in
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CZ-Nm. However, the absorption spectra didn't change significantly in the presence
of different amino acids. As shown in Fig. 3, when the increasing concentration of
GSH was added, the absorption intensity at 420 nm was gradually decreased, and the
new absorption peak at 267 nm was gradually increased. Under the same conditions,
the probe CZ-Nm also showed similar responses to other thiol-containing compounds
Cys and Hcy. However, owing to the response of the probe CZ-Nm to GSH (3
seconds) is much faster than that to Cys and Hcy (5 min), exquisite selectivity for
GSH can be obtained by controlling the reaction time.
Linearity
Under the optimized condition, the responsive properties of the probe CZ-Nm to
GSH were further investigated by fluorescence titration. As shown in Fig. 4,
fluorescence intensity increased up to 7-fold upon increasing GSH concentrations
(0-500 equiv). As envisioned, a good linear increase of fluorescence intensity could
be observed with increasing concentration of GSH over a wide linear range of 0-0.01
M (Fig. 5). The regression equation is Y=123116 + 66972.9X (R=0.9987). Then, we
obtained a low detection limit of 6.4 ꢀM based on 3×δblank/k (where δblank is the
standard deviation of the blank solution and k is the slope of the calibration plot). The
relative fluorescence quantum yields were determined to be 0.30 with quinine sulfate
dehydrate in 0.1 M H2SO4 as a standard. The corrected emission spectra were
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measured for the quinine sulfate dehydrate standard (λex=360 nm; A (absorbance) <
0.01; ΦF = 0.56), and calculated using the following equation.47
ꢊ
ꢄ
ꢆꢂ ꢇꢈꢁꢂ ꢉꢁ
ꢀꢁ = ꢀꢂ ꢃ ꢅ ꢃ ꢅ ꢃ ꢅ ꢃ
ꢆꢁ ꢇꢈꢁꢁ ꢉꢂ
ꢁ
ꢅ
ꢄ
ꢂ
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Where subscripts x and s refer to the unknown and the standard, Φ stands for
quantum yield, F represents integrated area under the emission curve, A is the
absorbance intensity at the excitation wavelength, λex exhibits the excitation
wavelength, n is index of refraction of the solution.
Tolerance of CZ-Nm to GSH over other interferent
The competitive experiment was implemented to analyze the influence of other
amino acids and thiol compounds on the reaction of the probe CZ-Nm with GSH. As
shown in Fig. 6, the change of fluorescence emission intensity caused by GSH with
background species together such as Cys, Hcy, Thr, Ser, Glu, Lys, Phe, His, Gly, Ala,
Val and Tyr was similar to that caused by GSH alone. The results indicated that the
recognition of GSH using the probe CZ-Nm was hardly affected by other amino acids
and thiol compounds.
Proposed mechanism
According to the literature,48 we speculate the Michael addition reaction between
the probe CZ-Nm and GSH is likely to be responsible for the fluorescence
enhancement and UV-Vis spectral change. To investigate this mechanism, the
stoichiometry of a binding event between GSH and the probe CZ-Nm was first
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determined. The results obtaining from the Job’s plot showed a 1:1 stoichiometry
between GSH and probe CZ-Nm (Fig. S4). Mass spectrometry also provided evidence
for the reaction of GSH with nitroolefin. As shown in Fig. 7, the peak at m/z=267.03
corresponded to CZ-Nm. And the peak at m/z=572.16 corresponded to the reaction
product between GSH and the probe CZ-Nm. Overall, all the measurements proved
the occurrence of Michael addition between the probe CZ-Nm and GSH.
Laser scanning confocal microscopy experiments of PC12
To further demonstrate that the permeability and the monitoring of intracellular
GSH, the fluorescence microscopy experiments were carried out in PC12 cells. As
shown in Fig. 8b, the living PC12 cells exhibited almost no fluorescence signal
without the incubation of the probe CZ-Nm in control group with excitation at 405 nm.
However, when PC-12 cells were incubated with CZ-Nm (10 ꢀM) for 30 min at 37 °C,
the strong blue fluorescence was exhibited inside the cells (Fig. 8e). This indicated the
cell permeability of the probe (transduction the cellular membrane). In another control
group, the PC12 cells were pretreated with 0.5 mM thiol reactive N-ethylmaleimide
(NEM, a trapping reagent of thiol species) for 30 min to consume all free thiols in the
PC12 cells, followed by treatment with probe CZ-Nm for 30 min. The confocal
microscopic images did not show a remarkable fluorescence signal (Fig. 8h). In
addition, if the PC12 cells pretreated with 2 mM GSH for 30 min followed by
incubated with the probe CZ-Nm for another 30 min, a brighter fluorescence signal
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(Fig. 8k) can be observed than that shown in Fig. 8e. This demonstrated that the
fluorescence change is really dependent on the concentration of the intracellular GSH
and not other related species. Thus, the probe CZ-Nm might provide a simple way to
detect the intracellular GSH from Cys and Hcy, or other common amino acids.
Conclusion
In summary, we have developed a novel “turn-on” and carbazole-based fluorescent
probe (CZ-Nm) for the discriminative detection of GSH from Cys and Hcy, or other
natural amino acids in PBS buffer-DMF (pH 7.4, v/v, 4:1) solution. The above results
show that the probe CZ-Nm with GSH has a wide linear range and low detection limit
under physiological conditions. Furthermore, the confocal fluorescence microscopy
imaging of the PC12 cells was carried out successfully, which not only indicated the
cell permeability and bio-compatibility of the probe CZ-Nm, but also demonstrated
that this probe can be applied to monitor GSH in living cells.
Acknowledgments
The authors are grateful for the National Natural Science Foundation of China
(21576071), the international science and technology cooperation project of Henan
province (152102410023).
Notes and references
1. F. Wang, L. Zhou, C. Zhao, R. Wang, Q. Fei, S. Luo, Z. Guo, H. Tian and W. H.
Zhu, Chem. Sci., 2015, 6, 2584-2589.
13

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260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
2. H. M. Meng, Z. Jin, Y. Lv, C. Yang, X. B. Zhang, W. Tan and R. Q. Yu, Anal.
Chem., 2014, 86, 12321-12326.
3. C. Yin, F. Huo, J. Zhang, R. Martinez-Manez, Y. Yang, H. Lv and S. Li, Chem.
Soc. Rev., 2013, 42, 6032-6059
4. Y. H. Lee, W. X. Ren, J. Han, K. Sunwoo, J.-Y. Lim, J.-H. Kim and J. S. Kim,
Chem. Commun., 2015, 51, 14401-14404.
5. X. Xiong, L. Zheng, J. Yan, F. Ye, Y. Qian and F. Song, RSC Adv., 2015, 5,
53660-53664.
6. N. Fahimi-Kashani, P. Shadabipour and M. R. Hormozi-Nezhad, RSC Adv., 2015,
5, 82906-82915.
7. Q. Miao, Q. Li, Q. Yuan, L. Li, Z. Hai, S. Liu and G. Liang, Anal. Chem., 2015,
87, 3460-3466.
8. Y. Zhang, Y. Tang, X. Liu, L. Zhang and Y. Lv, Sens. Actuators, B, 2013, 185,
363-369.
9. Q. Y. Cai, J. Li, J. Ge, L. Zhang, Y. L. Hu, Z. H. Li and L. B. Qu, Biosens.
Bioelectron., 2015, 72, 31-36.
10. L. Y. Niu, Y. S. Guan, Y. Z. Chen, L. Z. Wu, C. H. Tung and Q. Z. Yang, J. Am.
Chem. Soc., 2012, 134, 18928-18931.
11. D. Su, C. L. Teoh, S. Sahu, R. K. Das, Y.-T. Chang, Biomaterials, 2014, 35,
6078-6085.
14

Page 15 of 24
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DOI: 10.1039/C5RA25204K
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
12. R. Peng, L. Lin, X. Wu, X. Liu and X. Feng, J. Org. Chem., 2013, 78,
11602-11605.
13. L. Song, H. Tian, X. Pei, Z. Zhang, W. Zhang and J. Qian, RSC Adv., 2015, 5,
59056-59061.
14. D. Zhang, Z. Yang, H. Li, Z. Pei, S. Sun and Y. Xu, Chem. Commun., 2016, 52,
749-752.
15. X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010, 39, 2120-2135.
16. C. Han, H. Yang, M. Chen, Q. Su, W. Feng, and F. Li, ACS Appl. Mater.
Interfaces, 2015, 7, 27968-27975.
17. Y. Yang, F. Huo, C. Yin, A. Zheng, J. Chao, Y. Li, Z. Nie, R. Martinez-Manez and
D. Liu, Biosens. Bioelectron., 2013, 47, 300-306.
18. J. Liu, Y. Q. Sun, Y. Huo, H. Zhang, L. Wang, P. Zhang, D. Song, Y. Shi and W.
Guo, J. Am. Chem. Soc., 2014, 136, 574-577.
19. O. Rusin, N. N. S. Luce, R. A. Agbaria, J. O. Escobedo, S. Jiang, I. M. Warner, F.
B. Dawan, K. Lian and R. M. Strongin, J. Am.Chem. Soc., 2004, 126, 438-439.
20. M. Zhang, M. X. Yu, F. Y. Li, M. W. Zhu, M. Y. Li, Y. H. Gao, L. Li, Z. Q. Liu, J.
P. Zhang, D. Q. Zhang, T. Yi and C. H. Huang, J. Am. Chem. Soc., 2007, 129,
10322-10323.
21. W. Lin, L. Long, L. Yuan, Z. Cao, B. Chen and W. Tan, Org. Lett., 2008, 10,
5577-5580.
15

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Page 16 of 24
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300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
22. S. Madhu, R. Gonnade and M. Ravikanth, J. Org. Chem., 2013, 78, 5056-5060.
23. S. Lim, J. O. Escobedo, M. Lowry, X. Xu and R. Strongin, Chem. Commun.,
2010, 46, 5707-5709.
24. H. S. Jung, T. Pradhan, J. H. Han, K. J. Heo, J. H. Lee, C. Kang and J. S. Kim,
Biomaterials, 2012, 33, 8495-8502.
25. L. Y. Niu, Y. S. Guan, Y. Z. Chen, L. Z. Wu, C. H. Tung and Q. Z. Yang, Chem.
Commun., 2013, 49, 1294-1296.
26. X. Gao, X. Li, L. Li, J. Zhou and H. Ma, Chem. Commun., 2015, 51, 9388-9390.
27. J. Zhang, J. Wang, J. Liu, L. Ning, X. Zhu, B. Yu, X. Liu, X. Yao and H. Zhang,
Anal. Chem., 2015, 87, 4856-4863.
28. B. Liu, J. Wang, G. Zhang, R. Bai and Y. Pang, ACS Appl. Mater. Interfaces, 2014,
6, 4402-4407.
29. X. Hou, X. Guo, B. Chen, C. Liu, F. Gao, J. Zhao and J. Wang, Sens. Actuators, B,
2015, 209, 838-845.
30. C. Xu, H. Li, B. Yin, Biosens. Bioelectron., 2015, 72, 275-281.
31. J. Yin, Y. Kwon, D. Kim, D. Lee, G. Kim, Y. Hu, J. H. Ryu and J. Yoon, J. Am.
Chem. Soc., 2014, 136, 5351-5358.
32. M. M. Pires and J. Chmielewski, Org. Lett., 2008, 10, 837-840.
33. N. Shao, J. Y. Jin, H. Wang, J. Zheng, R. H. Yang, W. H. Chan and Z. Abliz, J. Am.
Chem. Soc., 2010, 132, 725-736.
16

Page 17 of 24
RSC Advances
View Article Online
DOI: 10.1039/C5RA25204K
320 34. J. Chen, X. Jiang, S. L. Carroll, J. Huang, and J. Wang, Org. Lett., 2015, 17,
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
5978-5981.
35. F. Huo, J. Kang, C. Yin, Y. Zhang and J. Chao, Sens. Actuators, B, 2015, 207,
139-143.
36. Y. Chen, K. Tsao, É. D. Francesco, and J. W. Keillor, J. Org. Chem., 2015, 80,
12182-12192.
37. L. Y. Niu, Y. S. Guan, Y. Z. Chen, L. Z. Wu, C. H. Tung, and Q. Z. Yang, J. Am.
Chem. Soc., 2012, 134, 18928-18931.
38. J. Liu, Y. Q. Sun, Y. Huo, H. Zhang, L. Wang, P. Zhang, D. Song, Y. Shi, and W.
Guo, J. Am. Chem. Soc., 2014, 136, 574-577.
39. L. He, Q. Xu, Y. Liu, H. Wei, Y. Tang and W. Lin, ACS Appl. Mater. Interfaces,
2015, 7, 12809-12813.
40. L. Li, Y. Q Wu, Q. L. Zhou and C. Y. He, J. Phys. Org. Chem., 2012, 25, 362-372.
41. K. Wang, T. Zhang, Y. H. Hu, W. G. Yang, Y. Shi, Electrochim. Acta, 2014, 130,
46-51.
42. X. Guo, X. Zhang, S. Wang, S. Li, R. Hu, Y. Li, G. Yang, Anal. Chim. Acta, 2015,
869, 81-88.
43. M. Lan, J. Wu, W. Liu, H. Zhang, W. Zhang, X. Zhuang, P. Wang, Sens. Actuators,
B, 2011, 156 , 332-337.
17

RSC Advances
Page 18 of 24
View Article Online
DOI: 10.1039/C5RA25204K
339
340
341
342
343
344
345
346
347
348
44. M. Isik, R. Guliyev, S. Kolemen, Y. Altay, B. Senturk, T. Tekinay and E. U.
Akkaya, Org Lett., 2014, 16, 3260-3263.
45. Y. Liu, K. Li, M. Y. Wu, Y. H. Liu, Y. M. Xie and X. Q. Yu, Chem. Commun.,
2015, 51, 10236-10239.
46. L. G. Voskressensky, A. A. Festa, A. V. Varlamov, Tetrahedron, 2014, 70,
551-572.
47. R. R. Zhang, J. F. Zhang, S. Q. Wang, Y. L. Cheng, J. Y. Miao, B. X. Zhao,
Spectrochim. Acta A, 2015, 137, 450-455.
48. C. Chen, W. Liu, C. Xu, W. Liu, Biosens. Bioelectron., 2015, 71, 68-74.
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Scheme 1. The design concept of probe CZꢀNm for GSH.
Fig. 1. (a) Fluorescence spectra of CZꢀNm (10 ꢁM, λex=360 nm) in the presence of
thiols and other 10 amino acids (500 equiv) in PBS bufferꢀDMF (0.1 M, pH 7.4, v/v,
4:1) for 30 min. (b) The corresponding fluorescent photographs of CZꢀNm (10 ꢁM) in
a cuvette in the presence of different analytes (500 equiv) under a UV lamp.

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Fig. 2. (a) UV/Vis absorption spectral changes of probe CZꢀNm (10 ꢁM) at 420 nm
upon addition of various thiols and amino acids (100 equiv). (b) a color change
photograph for thiols and other amino acids.
Fig. 3. Absorbance spectra of reaction solution of CZꢀNm (10 ꢁM) in 0.1 M PBS
bufferꢀDMF (pH 7.20, v/v, 1:1) solution with different concentrations of GSH.

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Fig. 4. Fluorescence emission spectra of probe CZꢀNm (10 ꢁM, λex=360 nm) upon
increased GSH concentrations (0ꢀ1000 equiv) in PBS bufferꢀDMF (0.1 M, pH 7.4, v/v,
4:1).
Fig. 5. The change in the fluorescence intensity of probe CZꢀNm (10 ꢁM, λex=360 nm)
against varied concentrations of GSH from 0 to 0.01 M in PBS bufferꢀDMF (0.1 M,
pH 7.4, v/v, 4:1).

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Fig. 6. Fluorescence intensities of probe CZꢀNm (10 ꢁM, λex=360 nm) in PBS
bufferꢀDMF (0.1 M, pH 7.4, v/v, 4:1) solution upon addition of GSH (500 equiv) in
the presence of background species (500 equiv).
Fig. 7. Mass spectrometry monitoring Michael addition between probe CZꢀNm and
GSH. (a) Mass spectrometry of probe CZꢀNm, (b) Mass spectrometry of
CZꢀNm+GSH.

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Fig. 8. Confocal fluorescence images of PC12 cells. (a,b,c) PC12 cells without
incubation with probe CZꢀNm. (d,e,f) PC12 cells incubated with probe CZꢀNm (10
ꢁM) for 30 min (λex=405 nm). (g,h,i) PC12 cells incubated with NEM (0.5 mM) for
30 min. (j,k,l) PC12 cells incubated with GSH (2.0 mM) for 30 min, then incubated
with probe CZꢀNm (10 ꢁM) for another 30 min. (a),(d),(g) and (j) bright field image;
(b),(e),(h) and (k) fluorescence image in blue channel; (c),(f),(i) and (l) overlap image.

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A “turn-on” fluorescent probe for specially recognize on intracellular GSH and its
application in bioimaging.