A highly sensitive fluorescent probe for selective detection of cysteine/homocysteine from glutathione and its application in living cells and tissues

Article abstract of DOI:10.1039/c8nj04183k

Biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play significant roles in maintaining biological homeostasis in organisms. Due to their particular significance in the physiology and pathology of humans, accurate determination and differentiation of Cys, Hcy, and GSH for understanding their roles in related diseases are in urgent demand. Herein, a new fluorescent probe NIPY-NBD was designed and synthesized for the detection of Cys/Hcy and GSH. The probe exhibits a variety of excellent properties including high sensitivity and low cytotoxicity as well as good cell membrane permeability. Furthermore, the probe showed a 100-fold (λ_ex/em = 310 nm/520 nm) and 210-fold (λ_ex/em = 340 nm/550 nm) fluorescence enhancement when reacted with Cys in buffer, and could swiftly respond to Cys and Hcy within 2.5 min. NIPY-NBD was further applied successfully in imaging in A549 cells and xenograft mouse tumor tissues. Taken together, NIPY-NBD may have great potential in detecting and distinguishing Cys/GSH in living animals and could be applied in tumor diagnosis.

Full text of DOI:10.1039/c8nj04183k

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A highly sensitive fluorescent probe for selective
detection of cysteine/homocysteine from
glutathione and its application in living cells
and tissues†
Cite this: New J. Chem., 2018,
42, 18172
a
b
a
a
a
Ziyan Zhou,‡ Guofeng Duan,‡ Yingying Wang, Shikui Yang, Xuyan Liu,
c
b
d
a
a
Liying Zhang, Runing Sun, Yungen Xu, Yueqing Gu and Xiaoming Zha
*
Biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play significant roles in
maintaining biological homeostasis in organisms. Due to their particular significance in the physiology and
pathology of humans, accurate determination and differentiation of Cys, Hcy, and GSH for understanding their
roles in related diseases are in urgent demand. Herein, a new fluorescent probe NIPY-NBD was designed and
synthesized for the detection of Cys/Hcy and GSH. The probe exhibits a variety of excellent properties
including high sensitivity and low cytotoxicity as well as good cell membrane permeability. Furthermore, the
probe showed a 100-fold (lex/em = 310 nm/520 nm) and 210-fold (lex/em = 340 nm/550 nm) fluorescence
enhancement when reacted with Cys in buffer, and could swiftly respond to Cys and Hcy within 2.5 min.
NIPY-NBD was further applied successfully in imaging in A549 cells and xenograft mouse tumor tissues.
Taken together, NIPY-NBD may have great potential in detecting and distinguishing Cys/GSH in living
animals and could be applied in tumor diagnosis.
Received 16th August 2018,
Accepted 24th September 2018
DOI: 10.1039/c8nj04183k
rsc.li/njc
for GSH, and they have homologous structures and similar
reactivity although playing different roles in the body. Accurate
Introduction
Biothiols including cysteine (Cys), homocysteine (Hcy) and determination of Cys, Hcy, and GSH is of great importance to
glutathione (GSH) play significant roles in maintaining understand their respective roles in related diseases including
1
,2
9,10
biological systems. Biothiols affect the redox environment cystinosis, liver damage, and cancer.
Therefore, numerous
to avoid or repair dysfunctional oxidative damage that may lead studies were focused on monitoring and differentiating Cys/Hcy
to cell death. Cys is an essential amino acid in the process of and GSH due to their particular roles in physiology and pathology
10
protein synthesis while an abnormal level of Cys leads to and their structural similarity.
edema, lethargy, cardiovascular diseases, and slowed growth in Currently, few methods have been applied for detecting
3
,4
children. An elevated level of Hcy in plasma is an important biothiols in vivo, so fluorescent probes are regarded as desir-
indicator of functional insufficiency and neurological and able tools owing to their non-invasiveness, sensitivity, real-time
5
,6
11–13
neuropsychiatric diseases. GSH could prevent cellular com- detection and simplicity.
Various fluorescent probes have
5
ponents from being damaged by toxins and reactive oxygen been developed to detect biothiols from other amino acids.
species (ROS). It should be noted that cysteine is a precursor However, the vast majority of the fluorescent probes are weak
7
,8
in some respects such as spectral overlap between multiple
a
Department of Pharmaceutical Engineering, Department of Biomedical
Engineering, School of Engineering, China Pharmaceutical University,
emitting channels, slow response, or relatively poor selectivity
in differentiating intracellular thiols. Thus, due to the similar
reactivity of the thiol groups in the three biothiols, developing
fluorescent probes for the discriminative detection of Cys/Hcy
6
39 Longmian Avenue, Nanjing 211198, China. E-mail: xmzha@cpu.edu.cn
b
c
Department of Traditional Chinese Medicine, Senior Vocational School, China
Pharmaceutical University, 639 Longmian Avenue, Nanjing 211198, China
Key Laboratory of Traditional Chinese Medicine Research and Development of
Hebei Province, Institute of Traditional Chinese Medicine, Chengde Medical
University, Chengde 067000, China
1
4–19
from GSH remains a challenge.
It is well known that 7-nitro-1,2,3-benzoxadiazole derivatives
can be used to distinguish Cys/Hcy from GSH (Table S1,
d
Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, China
3,14,20
ESI†).
The novel fluorophore NIPY-OH has a large Stokes
shift, good photostability, high fluorescent quantum yield and a
†
Electronic supplementary information (ESI) available: Supplementary data
2
1,22
simple synthesis route.
However, probes with the NIPY-OH
(
Fig. S1–S8 and Table S1). See DOI: 10.1039/c8nj04183k
‡
These authors contributed equally to this work.
moiety have rarely been reported and none for detecting Hcy/Cys
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2
1,22
13
against GSH.
What’s more, the existing probes for detecting (q, J = 7.1 Hz, 4H), 1.28 (t, J = 7.1 Hz, 6H). C NMR (126 MHz,
) d 154.58, 153.77, 153.14, 152.26, 150.40, 147.11, 144.64,
Based on this, we designed NIPY-NBD to detect 143.84, 133.54, 133.42, 128.86, 128.66, 126.40, 123.98, 122.46,
Cys/Hcy from GSH have long reaction times and small Stokes CDCl
3
3
,11,14,23,24
shifts.
Cys/Hcy and GSH discriminatively. The introduction of the 119.91, 118.51, 113.12, 110.67, 108.36, 103.63, 44.75, 12.51
+
NBD moiety as an electron acceptor can efficiently quench (Fig. S6, ESI†). HRMS (ESI): calcd for C H N O [M + H]
2
9
24 6 4
the fluorescence inherent to the NIPY-OH dye fragment m/z 521.1859, found 521.1932 (Fig. S7, ESI†).
through the photo-induced electron transfer (PET) process,
1
8,25,26
UV-vis and fluorescence response
thus the emission of the probe is switched off.
Conse-
quently, the significant fluorescent turn-on response of the The absorption and fluorescence spectra were measured in
probe can be obtained after removing the NBD moiety based PBS–DMSO buffer (v/v = 1/1). NIPY-OH has been reported previously
on Cys/Hcy/GSH-induced thiolysis of the NBD ether bond. to exhibit excellent optical properties, especially large Stokes
2
1,22
Herein, in the presence of Cys/Hcy/GSH, the probe could react shifts (210 nm), and thus could avoid self-quenching.
As
with these three biothiols and release NIPY-OH, which has a shown in Fig. 1(a), the probe NIPY-NBD only showed an absorp-
good fluorescence enhancement at lex/em = 310/520 nm. Mean- tion peak at 300 nm in PBS–DMSO buffer (v/v = 1/1). While in the
while, Cys/Hcy reacted with NBD-Cl leading to fluorescence presence of Cys, Hcy or GSH, a strong absorption peak at 310 nm
enhancement at lex/em = 470/550 nm, while the fluorescence of was observed, which is ascribed to the release of free NIPY-OH. It
the reaction product of GSH and NBD-Cl was weak at lex/em
=
should be noted that there was a distinct absorption peak at
NIPY-NBD showed high sensitivity and good 470 nm after adding Cys and Hcy, indicating the reactions
cell-membrane permeability as well as low cytotoxicity. It was between the probe and Cys/Hcy.
1
2,27
470/550 nm.
employed successfully to monitor Cys/GSH in living cells and
On the other hand, as shown in Fig. 1(b and c), almost no
xenograft mouse tumor tissues. Taken together, NIPY-NBD may fluorescence was observed in the NIPY-NBD solution (quantum
exhibit great potential in detecting and distinguishing Cys/GSH yield F = 0.014). Addition of Cys/Hcy to the solution of NIPY-NBD
and could be applied in tumor diagnosis.
in PBS–DMSO buffer (v/v = 1/1) led to a significant fluorescence
enhancement both at 520 nm (lex = 310 nm, quantum yield
2
8
F = 0.176 ) and at 550 nm (lex = 470 nm). As for GSH, only one
excitation maximum at 520 nm was observed (lex = 310 nm),
which is ascribed to the formation of the single fluorescent
product of NIPY-OH. Both the absorption and fluorescence
Results and discussion
Synthesis
The synthetic route to NIPY-NBD (N,N-diethyl-3-((7-nitrobenzo- spectra indicated the reaction between the probe and the
c][1,2,5]oxadiazol-4-yl)oxy)-4-(1-phenyl-3aH-isoindol-3-yl)aniline) biothiols. NIPY-NBD could detect Cys/Hcy and GSH through the
[
1
is shown in Scheme 1. NIPY-OH (200 mg, 560 mmol, H-NMR fluorescent properties at 520 nm (lex = 310 nm), and distinguish
of NIPY-OH is shown in Fig. S1, ESI†), potassium carbonate Cys/Hcy from GSH through the fluorescence intensity at 550 nm
(
115.99 mg, 839.26 mmol), and NBD-Cl (334.95 mg, 1.68 mmol) (lex = 470 nm).
were added into a round bottom flask containing 3 mL DMF The fluorescence spectra of NIPY-NBD upon reaction with
under nitrogen. The reaction mixture was stirred for 2 h at room various concentrations (0–50 mM: 0, 2, 4, 6, 8, 10, 15, 20, 30, 40,
21,22
temperature.
After extraction with dichloromethane and water, and 50 mM) of Cys, Hcy or GSH were recorded to study the
drying over anhydrous sodium sulfate, NIPY-NBD was obtained as linearity of fluorescence responses (Fig. 2). After adding Cys to
a black solid by flash chromatography on a silica gel (v : v, petro- the solution of NIPY-NBD in PBS–DMSO buffer (v/v = 1/1), a
leum ether : ethyl acetate = 1 : 1) after being dried under vacuum concentration-dependent fluorescence ‘‘turn-on’’ response
1
(
200 mg, 68.8%). H NMR (300 MHz, chloroform-d, Fig. S5, ESI†) d at 520 nm was observed as the concentration of Cys increases
8.25 (d, J = 7.5 Hz, 1H), 7.97 (d, J = 7.2 Hz, 1H), 7.72 (d, J = 7.8 Hz, (0–50 mM). The fluorescence intensity increased approximately
2
H), 7.63 (t, J = 7.8 Hz, 2H), 7.38 (t, J = 7.8 Hz, 2H), 7.24 (t, J = 8.4 Hz, 100-fold and exhibited a good linear relationship with the
2
1H), 6.84 (d, J = 7.2 Hz, 1H), 6.78 (t, J = 7.5 Hz, 1H), 6.68 concentration of Cys ranging from 0 to 10 mM (R = 0.9914).
(t, J = 7.5 Hz, 1H), 6.58 (s, 1H), 6.55 (d, J = 8.4 Hz, 1H), 3.48 The detection limit (LOD) of NIPY-NBD for Cys was further
Scheme 1 Synthetic route to the probe NIPY-NBD.
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The effects on pH and dynamic research for detection of Cys,
Hcy or GSH
For use in biological applications, the probe should be functional
at physiological pH. The pH effects on the fluorescence behavior of
NIPY-NBD were investigated in the pH range from 2 to 12. As
shown in Fig. 3, NIPY-NBD only indicates no significant fluores-
cence in the wide range of pH, which means NIPY-NBD itself could
be stable under different pH environments. However, strong
fluorescence intensity increase occurred within the pH range of
6–12 after adding 20 mM of Cys, Hcy (lex = 310 nm and 470 nm) or
GSH (lex = 310 nm). These results show that the probe NIPY-NBD
is suitable for use in a physiological environment.
The dynamic reactions of NIPY-NBD toward Cys, Hcy, and
GSH were evaluated in the PBS–DMSO buffer (v/v = 1/1) to
compare their reactivity. After mixing the probe with the biothiols,
the spectrum was scanned every 1 minute for 30 minutes to obtain
the response time between NIPY-NBD (10 mM) and Cys/Hcy/GSH
(20 mM). In the case of Cys/Hcy, the fluorescence intensity could
reach equilibrium states after a dramatic increase within
2.5 minutes at excitation wavelengths of both 520 nm (lex
=
310 nm) and 550 nm (lex = 470 nm). As for GSH, the reaction
completed within 3 min at 520 nm (lex = 310 nm) as well. Similar
to the results of the fluorescence spectrum of probe NIPY-NBD with
GSH, no fluorescence enhancement at 550 nm (lex = 310 nm) was
detected with the extended response time (Fig. 4 and Fig. S8, ESI†).
These results indicate that probe NIPY-NBD showed a significant
Fig. 1 Absorption spectra (a) and fluorescence spectra (b: lex/em = 310/ fluorescence enhancement for GSH at the excitation wavelength of
5
20 nm; c: lex/em = 470/550 nm) of NIPY-NBD (10 mM) and after reaction with 310 nm in a short time and could detect Cys/Hcy from GSH rapidly
Cys, Hcy or GSH (20 mM) in PBS–DMSO buffer (v/v = 1/1) for 30 min at 37 1C.
at the excitation wavelength of 550 nm (lex = 470 nm).
Selective and competitive experiments
calculated to be 21.4 nM (by S/N = 3). The results show that
NIPY-NBD has a high detection sensitivity for Cys. When the
excitation wavelength was 470 nm, the fluorescence curve
around 550 nm also showed a steady enhancement and an
about 210-fold fluorescence enhancement was observed.
The results between NIPY-NBD and Hcy are similar to those
between NIPY-NBD and Cys. For Hcy, a nearly 85-fold fluores-
cence enhancement at 520 nm and an approximately 205-fold
fluorescence enhancement at 550 nm were observed. The LOD
of probe NIPY-NBD to Hcy is 46.2 nM (S/N = 3). Furthermore, an
excellent linear relationship between the fluorescence intensity
With the aim to investigate the selectivity of NIPY-NBD toward Cys/
Hcy and GSH, the fluorescence responses of NIPY-NBD to various
metal ions, biothiols, amino acids, and reactive oxygen species were
tested in the PBS–DMSO buffer (v/v = 1/1). As shown in Fig. 5, Cys,
Hcy, and GSH with NIPY-NBD produced significant fluorescence
increase at 520 nm (lex = 310 nm), and Cys/Hcy with NIPY-NBD
showed a significant fluorescence enhancement at 550 nm (lex
=
470 nm). Furthermore, the coexistence of the competitive analytes
did not exert much influence on the reaction with Cys/Hcy or GSH.
Therefore, probe NIPY-NBD is proved to be highly selective to Cys/
Hcy and GSH assays compared to other competing species. These
results suggest that probe NIPY-NBD may be capable of selectively
detecting Cys/Hcy or GSH in clinical samples.
2
and the concentration of Hcy was recorded (R = 0.9919,
ranging from 0 to 10 mM).
As expected, in the presence of GSH, almost no fluorescence at
l
ex/em = 470/550 nm was recorded. However, significant fluorescence
Mechanism research
(up to about 110-fold) was observed at 520 nm with the increase of
GSH concentration (lex = 310 nm). Moreover, the LOD of probe The ether group in NIPY-NBD is the reaction site with Cys/Hcy/
NIPY-NBD to GSH was calculated to be 63.5 nM (S/N = 3), and there GSH (Fig. 6). It would generate thioether intermediates S-NBD-
was an excellent linear relationship between the concentration Cys, S-NBD-Hcy, and S-NBD-GSH after reaction with Cys, Hcy,
of GSH and the fluorescence intensity when l
= 310/520 nm and GSH. The thioether intermediates 7-S-cysteinyl-4-nitrobenz-2-
ex/em
2
(R = 0.9956 ranging from 0 to 10 mM). These results indicated that oxa-1,3-diazole (S-NBD-Cys) and 7-S-homocysteinyl-4-nitrobenz-2-
NIPY-NBD could well distinguish Cys/Hcy from GSH through the oxa-1,3-diazole (S-NBD-Hcy) could further convert to N-NBD-
fluorescent properties at lex/em = 470/550 nm, and quantitatively biothiol derivatives with high fluorescence (7-N-cysteinyl-4-
detect the concentration of Cys, Hcy or GSH through the fluores- nitrobenz-2-oxa-1,3-diazole (N-NBD-Cys) and 7-N-homocysteinyl-4-
cence intensity at lex/em = 310/520 nm.
nitrobenz-2-oxa-1,3-diazole (N-NBD-Hcy)) through the intramolecular
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Fig. 2 (a and b) Fluorescence responses of NIPY-NBD (10 mM) upon addition of Cys (0–50 mM). Inset: The linear correlation between the concentration
of Cys (0–10 mM) and the emission intensity (a: lex = 310 nm; b: lex = 470 nm). (c and d) Fluorescence responses of NIPY-NBD (10 mM) upon addition of
Hcy (0–50 mM). Inset: The linear correlation between the concentration of Hcy (0–10 mM) and the emission intensity (c: lex = 310 nm; d: lex = 470 nm).
(
e and f) Fluorescence responses of NIPY-NBD (10 mM) upon addition of GSH (0–50 mM). Inset: The linear correlation between the concentration of
GSH (0–10 mM) and the emission intensity (e: lex = 310 nm; f: lex = 470 nm).
+
cyclization, whereas S-NBD-GSH eventually exists in the form of and GSH were N-NBD-Cys (m/z = 285.0215 [M] ), N-NBD-Hcy
2
0
+
+
an S-derivative and shows no fluorescence. In a word, NIPY- (m/z = 299.0386 [M] ), and S-NBD-GSH (m/z = 471.0931 [M] ),
OH and N-NBD-Cys or N-NBD-Hcy were produced and showed respectively.
high fluorescence during the reaction, and S-NBD-GSH was
produced with no fluorescence. The results of electrospray
Cytotoxicity of NIPY-NBD
ionization mass spectrometry (ESI-MS) also confirmed the Human lung cancer cell line A549 was used to evaluate
mechanism (Fig. S2–S4, ESI†), and revealed that the corres- the cytotoxicity of NIPY-NBD. A549 cells were incubated with
ponding products by the reaction of NIPY-NBD with Cys, Hcy, different concentrations of NIPY-NBD (0–100 mM) for 24 h
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was observed (Fig. 8(m–p)), which indicated that the probe is
fluorescent due to the reaction with Cys/Hcy and GSH rather than
other substances in the cells. These results demonstrated that NIPY-
NBD is cell membrane permeable and could be used to detect Cys
and Hcy against GSH in living cells through different emission
channels (the DAPI channel for detecting Cys/Hcy and GSH, and the
FITC channel for identifying Cys/Hcy against GSH).
Tumor tissue imaging
We applied the probe NIPY-NBD for the visualization of exogenous
and endogenous Cys/GSH in A549 cancer tissues inoculated in nude
mice. As shown in Fig. 9(a–c), tumor tissues in xenograft nude mice
were injected and stained with 200 mM Cys for 1 h and then stained
with 100 mM NIPY-NBD for 1 h. Using a laser confocal fluorescence
microscope, a strong fluorescence was observed in tumor tissues in
both blue and green channels (blue channel – the excitation: BP 350/
50 & the emission: BP 460/50, and green channel – the excitation:
Fig. 3 Fluorescence intensity changes of the probe NIPY-NBD (10 mM)
at different pH values in the absence or presence of Cys, Hcy, and GSH BP 480/50 & the emission: BP 527/50). In addition, when tumor
tissues in xenograft nude mice were injected and stained with
00 mM NIPY-NBD for 1 h only, a weaker fluorescence was also
(
20 mM). (a: lex/em = 310/520 nm; b: lex/em = 470/550 nm).
1
observed in tumor tissues (Fig. 9(g–i)), which were known to express
at 37 1C. The cell viability was still over 85% even when the
concentration of NIPY-NBD was up to 100 mM (Fig. 7). The cell
viability implied that NIPY-NBD exhibits extremely low cytotoxicity
and encouraged us to explore its application both in vitro and in vivo.
excess amounts of Cys, while the concentration of endogenous Cys
29–31
could not reach 200 mM.
Furthermore, the xenograft nude
mouse tumors were treated with GSH and the probe (Fig. 9(d–f)).
Intense fluorescence was observed in tumor tissues only in the blue
channel, while a much weaker fluorescence was observed in the
green channel, which could be due to the reaction of the probe with
the endogenous Cys in the tumor tissues.
In conclusion, we can detect Cys from GSH in tumor tissues
through the strong fluorescence in the green channel, and
the probe NIPY-NBD was able to monitor exogenous and
endogenous Cys/GSH in tumor tissues. The results mentioned
above indicated that the probe had great potential of imaging
the endogenous and exogenous biothiols and could be useful in
various biological and clinical applications including cancer
diagnosis.
Cell imaging
The ability of NIPY-NBD to detect Cys/Hcy and GSH in living A549
cells was assessed. As shown in Fig. 8, weak fluorescence was
observed in the DAPI and FITC channels (blue channel –
the excitation: BP 350/50 & the emission: BP 460/50, and green
channel – the excitation: BP 480/50 & the emission: BP 527/50) when
the cells were cultured with 20 mM probe only (Fig. 8(q–t)), owing to
the endogenous biothiols, while the concentration could not reach
29–31
500 mM.
After treating the cells with Cys and the probe, an
apparent fluorescence enhancement was detected in the DAPI and
FITC channels, so that the cells were treated with Hcy and NIPY-NBD
(Fig. 8(a–h)). The strong fluorescence can also be observed in the
DAPI channel after being treated with GSH and the probe, while
there was almost no fluorescence observed in the FITC channel
under the same conditions (Fig. 8(i–l)). When the cells were incu-
Experimental section
Materials and equipment
bated with N-ethylmaleimide (NEM, 2 mM, as a thiol-blocking agent) All reagents and solvents were purchased from commercial
and then co-incubated with the probe, no significant fluorescence suppliers. Thin-layer chromatography (TLC) was performed
Fig. 4 Fluorescence intensity changes of NIPY-NBD (10 mM) with time after adding 20 mM of Cys/Hcy and GSH in PBS–DMSO buffer (v/v = 1/1).
(a: lex/em = 310/520 nm; b: lex/em = 470/550 nm).
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Fig. 5 Fluorescence responses of NIPY-NBD (10 mM) to various biological species (100 mM) in the absence (a and b) or presence (c and d) of Cys/Hcy and
GSH (20 mM). The bars show the fluorescence intensity after the addition of other analytes (a and c: lex/em = 310 nm/520 nm; b and d: lex/em = 470 nm/
5
50 nm). (1–23: KCl, ZnCl
2 2 3 2 2 2 2 3 2 2 3 2 2
, FeCl , FeCl , CaCl , MgCl , NaF, NaCl, NaBr, NaI, NaNO , NaClO, Na SO , Na S O , H O , Arg, Glu, Gly, His, Met, GSH,
Hcy, and Cys).
on silica gel GF254 plates (Qingdao Haiyang Chemical Plant, (Bruker Corp, Billerica, MA, USA) at 25 1C and referenced to
China) and visualized by ultraviolet (UV) lamps (l = 254 nm and tetramethylsilane (TMS). Chemical shifts were reported in ppm,
3
65 nm). Nuclear Magnetic Resonance Spectroscopy was per- and splitting patterns were designated as – s, singlet; d, doublet;
1
13
formed and H NMR spectra (300 MHz) and C NMR spectra t, triplet; and m, multiplet. The final compounds were identi-
75 MHz) were recorded on a Bruker ACF-300 spectrometer fied by acquiring mass spectra on an Agilent 1100 Series
(
Fig. 6 The proposed reaction mechanism of probe NIPY-NBD with Cys/Hcy and GSH.
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supplemented with 10% fetal bovine serum (FBS, Hyclone),
À1
À1
1
00 mg mL penicillin and 100 mg mL streptomycin in the
atmosphere containing 5% CO2 at 37 1C. A549 cells were
incubated with various concentrations of NIPY-NBD (0–100 mM)
after being cultured in 96-well plates for 24 h to evaluate the
cytotoxicity of NIPY-NBD. Then the cells were cultured for
another 24 h, and 10 mL of thiazolyl blue (MTT) solution
À1
(
5 mg mL , 0.5% MTT) was added for 4 h co-incubation. After
removing the supernatant, 200 mL dimethyl sulfoxide was
added to each well. Then the plates were shaken at low speed
for 15 minutes on the shaker to fully dissolve the crystals. The
absorbance of each well was measured at the OD = 490 nm by
Fig. 7 Cell viability of A549 cells treated with different concentrations of the enzyme-linked immunosorbent assay.
NIPY-NBD.
For confocal fluorescence imaging, the cells were firstly
seeded in the laser scanning confocal microscope (LSCM)
5
culture dishes with a density of 5 Â 10 cells per well for 24 h
(
Agilent Technologies, Santa Clara, CA, USA), an liquid
at 37 1C. Then the cells were incubated with Cys (500 mM),
Hcy (500 mM), GSH (500 mM), or NEM (2 mM) for 30 min.
Subsequently, 20 mM NIPY-NBD was added and incubated
at 37 1C for 30 min. The cells were washed three times with
PBS buffer. Fluorescent images were observed using a confocal
laser scanning microscope (CLSM, LSM700, Zeiss, Germany)
and processed using the ZEN imaging software.
chromatography/mass spectrometer detector ion trap (LC/MSD)
and a Mariner ESI-TOF spectrometer. UV-vis and fluorescence
spectra were recorded respectively on a Shimadzu UV-vis spectro-
photometer, UV-2550, and an Edinburgh Instruments Spectro-
fluorometer FS5. The pH values were determined using a PB 10
digital pH meter. Fluorescent images were observed using a con-
focal laser scanning microscope (CLSM, LSM700, Zeiss, Germany)
and processed using the ZEN imaging software.
To prepare the tumor tissues, fresh tumor tissues of the test
nude mice were taken and fixed in 10% neutral formalin
solution for more than 24 h. The tissue was cut into a suitable
tissue block (2–3 mm thick), placed in a tissue-embedded box
and immersed in the new fixing solution, and then placed in a
Leica ASP200S automatic vacuum dehydrator for dehydration.
Afterwards, the tissue blocks were dehydrated in the automatic
dewatering machine by 70%, 80%, 95%, 95%, 100%, and 100%
gradient alcohol (each gradient for 30 min). The tissue after
dehydration was transparent to xylene in a dehydrator for
General procedure for fluorometric analysis
All chemicals used were of analytical grade. A stock solution
of probe NIPY-NBD (1 mM) was prepared in PBS–DMSO buffer
(v/v = 1/1). Solutions of Cys, Hcy, GSH and other various
analytes were prepared in deionized water. Unless other noted,
all fluorescence responses were measured with excitation wave-
lengths of 310 nm and 470 nm (slit: 4 nm/4 nm) after incuba-
tion for 30 min at 37 1C in the testing solution.
The quantum yield of the compound was calculated accord-
ing to the equation:
15 min. After that the tissue was immersed in a liquid paraffin
at 60 1C for 90 min in a dehydrator. The wax-impregnated tissue
was taken out of the dehydrator and placed in the EG1150H
tissue embedding machine (Leica, Germany). Next, the tissue in
the embedding box is grasped with a pointed file, and the
tissue plane after trimming is embedded downward on the
work surface of the tissue embedding machine. The tissue-
coated wax blocks were sectioned on a Leica RM2245 hand
slicer with a section thickness of 30 mm and then the slices were
2
F = F Â (I /I ) Â (A /A ) Â (n /n )
u
s
u
s
s
u
u
s
where F denotes the quantum yield; I denotes the area under the
fluorescence curve; A denotes the absorbance at the excitation
wavelength; and n denotes the refractive index of the solvent.
The fluorescence quantum yields of NIPY-NBD were determined
3
2
2
8
compared to the reference compound – fluorescein (F = 0.79).
34,35
sealed with a neutral gum on the slide.
The limit of detection (LOD) was determined based on the
fluorescence titration and was calculated using the following
equation:
As for tumor imaging, the xenograft nude mouse A549
tumor was treated in the absence or presence of Cys/GSH
(
200 mM) for 1 h and then with the probe NIPY-NBD (100 mM)
1
4,35,36
for 1 h.
The fluorescence images of the tissue slices were
LOD = 3Sbi/a
acquired using fluorescence excitation and emission windows
Sbi is the standard deviation of the blank measurements of DAPI and FITC channels (blue and green channels, blue
(
measured ten times), and a is the slope between fluorescence channel – the excitation: BP 350/50 & the emission: BP 460/50,
3
3
intensity versus sample concentration.
and green channel – the excitation: BP 480/50 & the emission:
BP 527/50). All animal experiments were performed in strict
accordance with the Animal Management Rules of the Ministry
of Health of the People’s Republic of China (Document No. 55,
Cell culture, cytotoxicity assay, and confocal fluorescence
imaging
Human lung cancer cell line A549 (purchased from the American 2001) and approved by the Animal Care and Use Committee of
Type Culture Collection) was cultured in DMEM (Invitrogen) China Pharmaceutical University (Nanjing, China).
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Fig. 8 Bright-field and fluorescence images of A549 cells stained with the probe NIPY-NBD and Cys/Hcy/GSH: (a–d) bright-field and fluorescence
images of the cells incubated with Cys (500 mM, 30 min) and then treated with the probe (20 mM, 30 min); (e–h) bright-field and fluorescence images of
the cells incubated with Hcy (500 mM, 30 min) and then treated with the probe (20 mM, 30 min); (i–l) bright-field and fluorescence images of the cells
incubated with GSH (500 mM, 30 min) and then treated with the probe (20 mM, 30 min); (m–p) bright-field and fluorescence images of the cells incubated
with the probe only (20 mM, 30 min); (q–t) bright-field and fluorescence images of the cells incubated with NEM (2 mM, 30 min), and then co-incubated
with probe NIPY-NBD (20 mM, 30 min).
Conclusions
distinguish Cys/Hcy from GSH well through the fluorescent
In summary, we have designed and synthesized a new properties at 550 nm, and quantitatively detect the concen-
‘
‘turn-on’’ fluorescent probe NIPY-NBD for selectively detecting tration of Cys, Hcy or GSH through the fluorescence intensity at
Cys/Hcy and GSH. The probe shows a 100-fold (lex/em = 310 nm/ 520 nm. Due to the low toxicity and high sensitivity characteristics
20 nm) and 210-fold (lex/em = 340 nm/550 nm) fluorescence of NIPY-NBD, the probe was successfully applied in imaging for
5
enhancement within a response time of 2.5 minutes in Cys/Hcy/GSH in A549 cells and Cys/GSH in xenograft mouse
the presence of biothiols. The probe exhibits a large Stokes tumor tissues. In conclusion, the high selectivity of this probe
shift (210 nm), high sensitivity and a low detection limit for Cys/Hcy against GSH has been successfully demonstrated by
(
21.4 nM against Cys, 46.2 nM against Hcy and 63.5 nM against both UV-vis and fluorescence response and cell imaging. We
GSH), which was comparable with other reported thiol probes believe this probe would have a promising prospect in the future
Table S1, ESI†). More importantly, the probe NIPY-NBD could biological and clinical investigations.
(
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Jiangsu Province (2016-YY-042), the National Key Research
and Development Program (Grant No. 2017YFC0107700), the
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