A NIR facile, cell-compatible fluorescent sensor for glutathione based on Michael addition induced cascade spirolactam opening and its application in hepatocellular carcinoma

Article abstract of DOI:10.1039/C8TB02309C

A NIR fluorescence probe with long emission wavelength at 746 nm and high quantum yield of 0.36 was designed and synthesized to selectively detect GSH over Hcy and Cys in living systems. 2-(1,3,3-Trimethylindolin-2-ylidene)acetaldehyde was attached to rhodamine by Knoevenagel condensation to shift the emission to near infrared region. Then, ethylenediamine was linked to M₂ get an important intermediate M₃. Finally, target molecule named RhNM was synthesized through combined N-[4-(carbonyl)phenyl] with M₃. RhNM shows excellent discrimination in detecting GSH from Hcy/Cys along with an obvious color change. The fluorescence band at 746 nm is rapidly enhanced 106-fold after adding GSH, and the quantum was calculated at 0.36. Titration experiments of GSH showed good linear relationship of intensity vs. GSG concentration, and the detection limit was calculated at 70 nM. The proposed mechanism for identifying GSH based on Michael addition involves reactions of the carbon-carbon double bond and subsequent spirolactam opening. Meanwhile, the probe RhNM can work in a wide range of pH of 4-10. The probe RhNM was used for endogenous and exogenous imaging in HepG2 cells for glutathione. There was an outstanding intracellular red fluorescence occurring upon the addition of GSH incubated with HepG2 cells. It has been successfully applied for the determination of GSH in diluted serum and for bio-imaging of GSH in living cells with low cell toxicity. All results indicated that the probe can be used as an ultra-sensitive, near-infrared fluorescent chemical sensor for selectively detecting GSH in living cell. It has great potential in imaging of live tissues and even, animal imaging.

Full text of DOI:10.1039/C8TB02309C

Journal of
Materials Chemistry B
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PAPER
A NIR facile, cell-compatible fluorescent sensor
for glutathione based on Michael addition
induced cascade spirolactam opening and its
application in hepatocellular carcinoma†
Cite this: J. Mater. Chem. B, 2018,
, 7486
6
Xin Yang
and Ying Qian
*
A NIR fluorescence probe with long emission wavelength at 746 nm and high quantum yield of 0.36
was designed and synthesized to selectively detect GSH over Hcy and Cys in living systems. 2-(1,3,3-
Trimethylindolin-2-ylidene)acetaldehyde was attached to rhodamine by Knoevenagel condensation to
shift the emission to near infrared region. Then, ethylenediamine was linked to M
intermediate M . Finally, target molecule named RhNM was synthesized through combined N-[4-
carbonyl)phenyl] with M . RhNM shows excellent discrimination in detecting GSH from Hcy/Cys along
2
get an important
3
(
3
with an obvious color change. The fluorescence band at 746 nm is rapidly enhanced 106-fold after
adding GSH, and the quantum was calculated at 0.36. Titration experiments of GSH showed good linear
relationship of intensity vs. GSG concentration, and the detection limit was calculated at 70 nM. The
proposed mechanism for identifying GSH based on Michael addition involves reactions of the carbon–
carbon double bond and subsequent spirolactam opening. Meanwhile, the probe RhNM can work in a
wide range of pH of 4–10. The probe RhNM was used for endogenous and exogenous imaging in
HepG2 cells for glutathione. There was an outstanding intracellular red fluorescence occurring upon the
addition of GSH incubated with HepG2 cells. It has been successfully applied for the determination of
GSH in diluted serum and for bio-imaging of GSH in living cells with low cell toxicity. All results indicated
that the probe can be used as an ultra-sensitive, near-infrared fluorescent chemical sensor for selectively
detecting GSH in living cell. It has great potential in imaging of live tissues and even, animal imaging.
Received 3rd September 2018,
Accepted 11th October 2018
DOI: 10.1039/c8tb02309c
rsc.li/materials-b
discriminate GSH, Hcy and Cys from each other due to the
similar structures and reactivities of these biothiols. Zhang
Glutathione (GSH), an essential and most abundant intra- et al. reported a turn ‘‘on’’ fluorescence probe based on
Introduction
3
7
cellular thiol, plays an important role in human physiological nucleophilic substitution reaction to detect GSH/Hcy/Cys over
1
–7
activities. It has been proved that the GSH is known to serve other relevant biological species within a few minutes. Further-
8
38
many cellular functions in the human body, including anti- more, Liu et al. developed a novel rhodamine probe with a
oxidation in cellular processes, maintenance of intracellular large stokes shift for biothiols detection. There are many other
9
–11
38–48
signal, and gene regulation.
It also has a close relationship fluorescence probes reported for detecting amino acid.
12,13
with some disease,
liver damage, Alzheimer’s, AIDS, and even cancers.
time imaging and quantification of intracellular GSH in living their application in biological system. By contrast, NIR fluores-
such as slowed growth, loss of leucocytes, However, the absorption and emission of these probes are all
14–20
Thus, real- in the visible range (300–650 nm) (Scheme 1), which can hinder
21
cells would provide important pathophysiological insights.
cence probes (650–900 nm) are highly needed for in vivo
Thus, it is of great value to develop effective methods for real- imaging studies due to their excellent properties, such as
22,23
time monitoring of intracellular GSH in biological systems.
reduced tissue attenuation, better tissue penetration, less
Various fluorescence probes have been designed and synthe- optical damage to living cells and minimum interference from
2
4–36
48–53
sized to sense amino acids.
However, most of them cannot background auto fluorescence.
However, most of the near-
infrared probes for biothiols have lower quantum yields, thus,
5
4–56
rendering them unsuitable for bio-imaging.
Our group has
School of Chemistry and Chemical Engineering, Southeast University, Nanjing,
5
7
reported a NIR probe with a quantum yield of 0.26. Moreover,
fluorescence probes with near-infrared emission upto 700 nm
and high quantum yield are rarely reported. As a result, it is
2
11189, China. E-mail: yingqian@seu.edu.cn
†
Electronic supplementary information (ESI) available: NMR, HRMS spectra and
other experimental data. See DOI: 10.1039/c8tb02309c
7
486 | J. Mater. Chem. B, 2018, 6, 7486--7494
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Journal of Materials Chemistry B
and thionyl chloride, were purchased from Macklin Biochemical
Co., Ltd (Shanghai, China), Aladdin or Maya reagent company.
(Benzotriazollyloxy)tris(dimethylamino)phosphonium hexafluo-
phosphate (BOP reagent) and indocyanine green (ICG) were
purchased from Sigma-Aldrich. All reagents and solvents were
used as received without further purification.
Stock solutions of the probe in ethanol and of GSH, His,
Phe, Lys, Asp, Hcy, Tye, Asn, Pro, Tyr, Gly, Glu, Arg, Ala, Cys,
À
2À
2À
2À
Thr, Cl , S , SO
3
, and CO
3
in distilled water were pre-
pared. All amino acids were purchased from Macklin Biochemical
Co., Ltd, Tokyo Chemical industry Co., Ltd and Sinopharm
Chemical Reagent Co., Ltd.
1
13
H NMR and C NMR spectra were recorded on a Bruker
DMX300 and Bruker ADVANCE 500 NMR spectrometers in
CDCl with tetramethylsilane (TMS) as internal standard. Mass
3
spectra were obtained on an Ultraflex II (MALDI-TOF) spectro-
meter. UV-visible absorption spectra were determined on a
Shimadu UV-3600 spectrophotometer. Fluorescence spectra were
measured on an HORIBA FL-4 Max spectrometer. pH adjust-
ments were made, when necessary, using a pHS-3C pH meter. All
reactions were monitored by thin layer chromatography using
Merck TLC Silica gel 60 F254.
Scheme 1 Recent reports for detecting GSH, Hcy and Cys, structure of
probe RhNM and specific recognition of GSH; on the left is the structure of
Fluorescence parameter
À4
probe and on the right is the probe-GSH complex, whereby after addition Stock solutions of the probe in ethanol (1.0 Â 10 M) and all
of GSH, strong red fluorescence in NIR region occurred.
À2
amino acids (1.0 Â 10 M) in distilled water were precisely
prepared. Test solutions of the probe and glutathione with final
À5
concentrations of 1 Â 10
M were prepared using stock
essential to explore a series of NIR fluorescence probes with
long emission wavelengths and high quantum yields to dis-
criminately detect GSH over Hcy and Cys in living systems.
A NIR fluorescence probe, with emission wavelength at
solutions by dilution with ethanol and PBS buffer solution
(V/V, 3 : 2, pH = 7.4).
The detection limit was then calculated by a fluorescence
titration, using the equation: detection limit = 3s/k.
5
8
59,60
Here,
7
46 nm and high quantum yield of 0.36, for selective detection of
GSH over Hcy/Cys in living systems was designed and synthesized.
-(1,3,3-Trimethylindolin-2-ylidene)acetaldehyde was attached
s is the standard deviation of emission intensity of probe
RhNM and k is the slope between emission intensity and
concentration. Cell viability was calculated according to the
equation: cell viability (%) = average value of sample OD570/
average value of blank OD570 Â 100%, where, OD570 is the
absorbance measured at 570 nm. The quantum yield was
calculated according to the equation:
(n /n ). Here, A is absorbance at the excitation wavelength, F is
area under the corrected emission curve, and n is refractive
index of the solvents used. Subscripts S and X refer to the
standard and the unknown, respectively. Indocyanine green
2
to rhodamine by Knoevenagel condensation in order to extend
the conjugate chain and shift the emission wavelength to NIR
region. GSH recognition sites lie in 4-maleimidobenzoic acid,
where an a,b-unsaturated bond is expected to react with –SH
group of GSH through Michael addition. Next, the intramolecular
amino induced spirolactam opening results in the transforma-
tion of spiro-cyclic form of the probe from close to open structure
and fluorescence is exhibited. Results demonstrated that the
probe showed highly sensitive and selective detection of GSH
with the emission wavelength at 746 nm. Furthermore, the probe
was successfully applied in imaging endogenous or exogenous
GSH with low toxicity in HepG2 cells. In summary, the designed
probe has great potential to sense biothiols in tissues and
animals, which will greatly promote real-time monitoring of
GSH in important physiological processes.
6
1
6
2
F
F(X) = FF(S) (A
S
F
X
/A
X S
F )
2
X S
(
ICG, F = 0.13 in DMSO) was used as the ref. 57.
f
MTT cytotoxicity assay
Cytotoxicity of the probe RhNM was evaluated by vitro MTT
assay. The logarithmic phase of HepG2 cells were inoculated
4
in a 96-well cell culture plate (1 Â 10 cells per well), and
2
incubated at 37 1C and 5% CO for 24 hours. Next, the cells
were incubated with different concentrations (0–20 mM) of the
probe RhNM for another 24 h. Then, the cells were incubated
Experimental section
Materials and methods
À1
with 5 mg mL MTT for 4 h at 37 1C, and 5% CO . The super-
2
natant was then discarded, 100 mL DMSO was added to each
All chemical reagents and organic solvents used, including well and oscillation was conducted for 5 minutes. The absor-
acetic anhydride, cyclohexanone perchloric acid, fisher aldehyde bance of each well of the 96-well plate was then assessed at
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5
70 nm on a Benchmark Plus plate reader. Cell viability was give the yellow solid (0.21 g) of probe RhNM at a yield of 28.4%.
1
calculated according to the formula mentioned above.
3
H NMR (300 MHz, CDCl ): d 8.50 (s, 1 H), 8.06 (d, J = 4.2 Hz, 2 H),
7.91 (d, J = 3.6 Hz 1 H), 7.52–7.46 (m, 5 H), 7.21–7.14 (m, 3 H),
6.88–6.84 (m, 3 H), 6.63 (d, J = 3.9 Hz, 1 H), 6.35 (d, J = 4.5 Hz,
2 H), 6.27 (d, J = 4.2 Hz, 1 H), 5.39 (d, J = 6.3 Hz, 1 H), 3.62–3.58
Cell culture and NIR imaging
HepG2 cells were cultured in cell culture dishes with 10% fetal
(
1
6
m, 1 H), 3.52–3.49 (m, 2 H), 3.38–3.33 (m, 4 H), 3.27–3.25 (m,
bovine serum and 90% DMEM medium under 5% CO at 37 1C
2
6
H), 3.16 (s, 3 H), 1.75–1.73 (d, J = 6.9 Hz, 6 H), 1.69–1.57 (m,
until the density of cells reached 2 Â 10 cells per mL. Then, the
1
3
H), 1.20 (t, J
, ppm): d: 170.67, 169.11, 166.08, 157.98, 152.82, 151.86,
48.89, 148.30, 145.35, 138.88, 134.32, 133.79, 133.40, 132.66,
1 2
= 3.3 Hz, J = 3.6 Hz, 6 H). C NMR (300 MHz,
cells were planted in a cover glass bottom confocal dish and
CDCl
3
continuously incubated at 37 1C in 5% CO
2
atmosphere for
1
1
1
4
24 h. The probe RhNM was dissolved in DMSO, added to the
31.18, 128.33, 128.16, 127.69, 125.54, 123.66, 121.52, 119.95,
19.32, 108.71, 105.71, 104.21,102.74, 97.70, 92.00, 67.93, 53.38,
cover glass bottom confocal dish, and incubated for 0.5 h. Finally,
the cells were washed three times with PBS buffer solution
5.47, 44.34, 42.36, 39.23, 29.67, 29.12, 28.47, 28.30, 25.27,
(pH = 7.4), and fluorescence imaging was performed on a confocal
+
2
3.03, 22.15, 12.54. HRMS calcd, for [M + CH
3
OH + H] : 832.4074,
laser scanning microscope (FLUOVIEW FV3000. OLYMPUS).
found: 832.4066.
Synthesis
The synthesis of 4-maleimidobenzoic acid. A mixture of para-
aminobenzoic acid (1.00 g, 7.3 mmol) and maleic anhydride
The synthetic route adopted for preparation of probe RhNM
was illustrated in Scheme 2. Four steps synthesis protocol was
(0.75 g, 7.8 mmol) was dissolved in dry acetone (6.00 mL).
Then, the reaction mixture was stirred at room temperature for
2 hours, allowing sufficient time for complete precipitation. After
the reaction was completed, the precipitate was filtered and
washed with acetone to give an intermediate product as a white
solid (1.66 g). Yield: 96%.
adopted: The intermediate M
reported literature. For M
1
was synthesized according to
1
synthesis, 2-(4-diethylamino-2-
6
3
hydroxybenzoyl)benzoic acid was reacted with cyclohexanone
at 90 1C for 1.5 h, perchloric acid was then added, and the
product was finally extracted to obtain a dark red solid.
The synthesis of probe RhNM. A mixture of 4-(2,5-dioxo-2H-
pyrrol-1(5H)-yl)benzoic acid (0.20 g, 0.92 mmol) and thionyl
chloride (0.13 mL, 1.84 mmol) was refluxed for at least 10 hours.
Unreacted thionyl chloride was evaporated to yield the inter-
mediate complex which was used without further purification.
The compound obtained above was suspended in dry dichlor-
A mixture of crude (Z)-4-(4-carboxy-2-oxobut-3-en-1-yl)benzoic
acid (1.60 g, 6.75 mmol) and acetic anhydride (3.20 mL,
55.90 mmol) was stirred for 30 min at 80 1C. After the solid
was dissolved, sodium acetate (0.28 g, 3.39 mmol) was added to
the solution and stirred was continued for 6 h. After the
reaction was completed, the mixture was poured into ice water
and stirred for another 2 h. The precipitate was filtered and
washed with cold water until the pH is neutral and dried over-
night to yield a pale yellow solid. The crude product was purified
omethane (10.0 mL). Thereafter, M (0.83 g, 1.38 mmol) and
3
triethylamine (0.21 mL, 1.54 mmol) were added to the solution
and stirred at room temperature for 4 hours. After the reaction
was finished, the solvent was removed under vacuum, dichloro-
methane was added to the residue and the organic phase was
extracted three times with saturated sodium sulfate solution.
The extracted organic phases were combined and treated with
anhydrous sodium sulfate. Remaining solvent was removed by
rotary evaporation to yield the crude product as a pale green solid.
The crude product was purified by column chromatography to
3 2 2 2
by column chromatography, using CH CH OH/CH Cl , as a pale
1
3
yellow solid (0.69 g). Yield: 47%. H NMR (300 MHz, CD OD):
d 8.1 (m, 2 H), 7.52 (m, 2 H), 6.99 (s, 2 H).
The synthesis of M . A mixture of M (2.37 g, 6.30 mmol)
2
1
and 2-(1,3,3-trimethylindolin-2-ylidene)acetaldehyde (1.32 g,
.56 mmol) was dissolved in acetic anhydride (40.0 mL), and
6
then the reaction mixture was heated to 50 1C and further
stirred for 2.5 h. Then, water was added to the reaction mixture
2 2
to quench the reaction. The solvent was extracted with CH Cl ,
the organic layer was separated and dried over anhydrous
Na SO and solvent was removed under reduced pressure to
2
4
give the crude product as a green solid. The crude product was
purified by silica gel flash chromatography using CH Cl /
2
2
1
CH
CDCl
t, J = 7.5 Hz, J
3
CH
): d 8.60 (s, 1 H), 8.28 (d, J = 7.8 Hz, 1 H), 8.02 (s, 1 H), 7.75
=7.2 Hz, 1 H), 7.64 (t, J = 7.5 Hz, J = 7.5 Hz,
2
OH (10 : 1). Yield: 3.15 g (89.4%). H NMR (300 MHz,
3
(
1
2
1
2
1
6
H), 7.45–7.37 (m, 2 H), 7.28–7.16 (m, 3 H), 6.72–6.55 (m, 3 H),
.10 (s, 1 H), 3.69 (s, 2 H), 3.53 (s, 4 H), 2.97 (s, 2 H), 2.89
(
s, 2 H), 1.80 (s, 8 H), 1.30 (t, J = 6.6 Hz, J = 6.9 Hz, 7 H).
1 2
1
3
C NMR (300 MHz, CDCl , ppm): d: 172.37, 166.63, 163.16,
3
155.72, 154.21, 151.80, 142.60, 141.13, 140.14, 135.25, 132.43,
131.14, 130.13, 129.09, 128.81, 128.49, 128.27, 124.71, 121.92,
120.72, 115.40, 113.53, 112.25, 110.09, 98.39, 95.35, 48.68, 44.90,
Scheme 2 The synthesis route of probe RhNM.
30.93, 28.26, 26.26, 23.96, 20.16, 12.23.
7
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The synthesis of M
Journal of Materials Chemistry B
3
. A mixture of M
2
(2.75 g, 4.92 mmol), naked eye. However, there was no color change when His, Phe,
À
2À
ethylenediamine (0.34 mL, 5.00 mmol) and BOP reagent (2.3 g, Lys, Asp, Tye, Asn, Pro, Tyr, Gly, Glu, Arg, Ala, Thr, Cl , S
,
2
À
2À
5.20 mmol) was dissolved in dry dichloromethane. The mixture SO3 , and CO3 were added. This was also observed when Cys
was stirred at room temperature for two hours and the solvent and Hcy were added to the blank probe solution: it remained
was removed under vacuum. The crude product was purified by colorless, as is clearly seen in Fig. 1.
silica gel column chromatography using CH
25 : 1) to afford the compound M as a yellow solid (1.98 g). RhNM in ethanol/PBS = 3 : 2 (V/V) were investigated. Fig. 2(a)
Yield: 67.0%. H NMR (300 MHz, DMSO-d ): d 7.45 (d, J = indicated that a blank probe RhNM solution exhibited no
.2 Hz, 1 H), 7.57–7.46 (m, 2 H), 7.28 (d, J = 1.2 Hz, 1 H), 7.16 (t, characteristic absorption at 721 nm. However, in the presence
= 6.9 Hz, J = 7.2 Hz, J = 7.5 Hz, 2 H), 6.83–6.77 (m, 2 H), 6.38– of GSH (10 mM), an absorption band at 721 nm, accompanied
.34 (m, 1 H), 6.26–6.21 (m, 2 H), 5.74 (s, 3 H), 5.42 (d, J = 12.6, by color change from colorless to green, was observed. Molar
2
Cl
2
/CH
3
CH
2
OH
Besides, absorbance and fluorescence spectra of the probe
(
3
1
6
7
J
1
2
6
1
3
1
4
À1
À1
H), 3.15–2.99 (m, 6 H), 2.89 (s, 1 H), 2.73 (s, 1 H), 2.44–2.37 (m, extinction coefficient was calculated to be 3.81 Â 10 M cm
H), 1.64 (m, J = 1.8 Hz, 6 H), 1.56 (s, 2 H), 1.32–1.23 (m, 4 H), and the quantum yield reached 0.36. Compared with the blank
.12 (t, J = 6.9 Hz, J = 6.9 Hz, 6 H). HRMS calcd for probe RhNM solution, the absorption intensity was enhanced
1
2
+
+
C
39
H
45
N
4
O
2
[M + H] : 601.3543, found: 601.3532.
at more than 56-fold upon addition of GSH. Moreover, there
was a weak absorption peak at 721 nm when His, Phe, Lys, Asp,
Tye, Asn, Pro, Tyr, Gly, Glu, Arg, Ala, Thr, Cl , S , SO
À
2À
2À
3
,
2
À
Results and discussion
CO
3
, and especially Cys and Hcy were added to the probe
solution. However, the peak intensity was similar to the
blank probe solution. The fluorescence intensity change was
shown in Fig. 2(b). A remarkable fluorescence enhancement
was observed upon addition of GSH (10 mM) with an emission
peak located in the NIR region at 746 nm. Significantly, the
fluorescence turn on showed 106-fold enhancement, as expected.
Design and synthesis of probe with emission wavelength
located in near-infrared region
A unique NIR fluorescence probe RhNM was designed for
selectively detecting GSH from Hcy/Cys from conjugation of
2,2-(1,3,3-trimethylindolin-2-ylidene)acetaldehyde and 4-male-
imidobenzoic acid with a rhodamine derivative. The probe
RhNM was synthesized in four steps as shown in Scheme 2.
As depicted in Scheme 2, 2-(1,3,3-trimethylindolin-2-ylidene)-
acetaldehyde was conjugated with rhodamine by Knoevenagel
condensation to shift the wavelength to NIR region. Ethylene-
diamine was added to M
. Finally, 4-maleimidobenzoic acid was linked to M
3
2
to obtain an important intermediate
M
3
structure
as the GSH reaction site. The a,b-unsaturated bond was expected
to react with –SH group in GSH through Michael addition.
Finally, a new NIR fluorescence probe RhNM with high quantum
yield was constructed. The addition of GSH could significantly
change fluorescence measured at 746 nm. However, there is no
similar phenomenon observed upon addition of Hcy and Cys.
RhNM was successfully applied in imaging of endogenous or
exogenous GSH with low toxicity in HepG2 cells.
1
All intermediate structures were confirmed by H NMR,
1
1
3
C NMR or HRMS. Target molecule RhNM was confirmed by
13
H NMR, C NMR and HRMS. (Fig. S1–S9, ESI†).
Colorimetric and discrimination of probe RhNM for GSH from
Hcy/Cys
As can be visually observed from Fig. 1, the control solution of
probe is colorless under sunlight. Color of the blank RhNM
solution changed from colorless to green within a few minutes
when GSH was added to the solution, which was visible to the
Fig. 2 (a) Absorbance responses of probe (10 mM) in the presence of
various amino acids, include GSH, His, Phe, Lys, Asp, Hcy, Tye, Asn, Pro,
À
2À
2À
2À
Tyr, Gly, Glu, Arg, Ala, Cys, Thr, Cl , S , SO
3
, and CO
3
(10 mM) in
EtOH/PBS buffer solution (V/V, 3 : 2, pH = 7.4) and (b) fluorescence
responses of probe (10 mM) in the presence of various amino acids and
2
À
2À 2À
À
Fig. 1 Photograph of the probe solutions under sunlight before and after
SO
3
, CO
3
, S , and Cl (10 mM) in EtOH/PBS buffer solution (V/V, 3 : 2,
2
À
2À 2À À
adding different amino acids and SO
3
, CO
3
, S , Cl ions.
pH = 7.4).
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Fig. 3 Blue bars represent the histogram of Fig. 2(b). Red bars show the
fluorescence intensity of probe (10 mM) upon addition of other amino acids
Fig. 4 Fluorescence titration spectra of probe (10 mM) in EtOH/PBS buffer
solution (V/V, 3 : 2, pH = 7.4) upon addition of varying concentrations of
GSH (0–10 mM) (lex = 600 nm, lem = 746 nm).
(
(
10 mM) in the presence of GSH (10 mM) in EtOH/PBS buffer solution
V/V 3 : 2, pH = 7.4).
However, there was no obviously fluorescence enhancement Table 1 Detailed photophysical properties of RhNM in the absence and
presence of GSH and other amino acids
for upon addition of Hcy, Cys and other amino acids to the
blank probe sample under identical conditions. The absorption
and fluorescence spectra of probe RhNM show excellent per-
formance toward GSH. The results illustrated that only GSH
could induce the change in rhodamine spirolactam from RhNM + GSH 721 3.81
close form to open form, affording the probe RhNM with the
l
abs
e
max
l
em Quantum The color
4
À1
À1
(nm) (10 M cm ) (nm) yield (F) under sunlight
RhNM
721 0.067
—
—
Colorless
Green
746 0.36
735
735
RhNM + Hcy 721 0.068
RhNM + Cys 721 0.067
—
—
Colorless
Colorless
ability to discriminate GSH from Cys/Hcy with high quantum
l
abs: UV absorption maximum; emax: maximum molar extinction coeffi-
yield.
Moreover, the interference from other amino acids should
cient; lem: maximum emission wavelength FF(X) was calculated accord-
ing to the equation: FF(X) = FF(S) (A F /A F )(n /n ) .
S X X S X S
2
be taken into consideration. As shown in Fig. 3, no obvious
variation in fluorescence intensity of the RhNM–GSH complex
was observed with or without the presence of other competitive
species, indicating that the probe RhNM is barely influenced by
The proposed mechanism of Michael addition induced cascade
spirolactam opening toward GSH
other co-existing amino acids and can be utilized for selective The proposed mechanism of probe RhNM identifying GSH based
detection of GSH. These results further demonstrated that on Michael addition induced cascade spirolactam opening is as
the probe RhNM could be used as a selective fluorescent GSH shown in Fig. 5. The proposed mechanism of GSH identification
chemosensor.
involves reactions of the carbon–carbon double bond and sub-
To evaluate sensitivity of the probe, UV-vis and fluorescence sequent spirolactam opening, as follows: the probe RhNM exhibited
titration of probe RhNM with GSH was carried out. As shown in no fluorescence due to the closed spirolactam form of rhodamine
Fig. S10 (ESI†), there was an obvious absorption band located at moiety. Once GSH was added to the blank probe RhNM solution,
721 nm when GSH was added to the blank probe solution. As hydrogen ions interacted with the carboxyl groups of GSH following
the concentration of GSH increased, absorbance of the probe the formation of an unstable intermediate. Spontaneously, due
increased simultaneously within 5 mM of the GSH. The results to the decreased distance between the rhodamine moiety and
showed that the probe is sensitive under a small change in negatively charged carboxylate group, a highly acid microenviron-
concentration. Moreover, the change in fluorescence titration ment might be generated. Eventually, the spirocyclic moiety of
spectrum was investigated with the concentration of added rhodamine is proposed to be transformed into the fluorescent
GSH at a maximum of 10 mM. As Fig. 4 vividly depicts, with the open-ring form. As a result, a strong fluorescence band located at
addition of GSH to the blank probe solution, a fluorescence 746 nm was observed. The proposed sensing mechanism was
band with a maximum at 746 nm was observed in the NIR red confirmed by mass spectra analysis. Fig. 5 showed the probe in
region, which suggests a great potential for applications in the presence and absence of GSH. When 10 equiv. of GSH was
imaging of organisms or cells. In addition, a good linearity was added the blank probe solution, a new mass peak appeared at
+
obtained over the concentration range of 1–3 mM of GSH. The m/z = 1107.4630, which was attributed to [RhNM + GSH] . The
detection limit was calculated at 70 nM according to the equa- exact mass was calculated to be 1107.4644, which confirmed
tion of 3s/k. The quantum yield was calculated as 0.36 by using the proposed sensing mechanism of probe toward GSH.
IGG as the standard. Compared photophysical data was shown
Time/pH-dependence in the detection process of GSH
in Table 1. The results clearly illustrated that the probe could
sensitively detect GSH levels in EtOH/PBS buffer (V/V = 3 : 2) The response time of probe was studied at room temperature to
solution.
evaluate the sensitivity of probe toward GSH and Cys, and Hcy.
7490 | J. Mater. Chem. B, 2018, 6, 7486--7494
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Fig. 5 (a) Proposed mechanism for the response of probe RhNM toward GSH based on Michael addition induced cascade spirolactam opening,
b) HRMS spectrum of RhNM–GSH complex.
(
There was an obvious color change within a few minutes upon
the addition of GSH (10 mM), as shown in Fig. 6, which
gradually stabilized thereafter. Moreover, there was no signifi-
cant change in fluorescence intensity when Hcy and Cys were
added during the same time period. Results indicated that the
probe could be used as a sensitive sensor for the detection of
GSH from Cys and Hcy. To illustrate that the probe RhNM
could sense GSH under physiological condition, the influence
of pH on GSH detection was investigated. As Fig. 6(b) shows,
the probe with no GSH exhibited strong fluorescence only when
pH o 4, this phenomenon could be illustrated by the spiro-
lactam opening of rhodamine under acidic conditions. At
pH 4 4, there was no obvious fluorescence over a wide range
of pH 4–10. Whereas, the probe exhibited strong fluorescence in
the presence of GSH, which can be contributed to the opening of
spirolactam of rhodamine that turns ‘‘ON’’ the fluorescence.
RhNM was stable after reacting with GSH and possessed excellent
fluorescence response in pH range of 4–10, which was suitable for
detecting GSH under physiological pH conditions.
MTT assay and red fluorescence imaging of probe RhNM in
HepG2 cells
Conventional MTT assay was conducted to investigate cytotoxi-
city of the probe and verify its sensitivity for detecting bio-
molecules in living cells. As shown in Fig. 7, upon exposure to
the probe in concentrations of 0–20 mM for 24 h, there was no Fig. 6 (a) Time-dependent fluorescence intensity of probe (10 mM) in
EtOH/PBS buffer solution (V/V, 3 : 2, pH = 7.4) with 10 mM of GSH and Hcy,
Cys. (b) Fluorescence intensity of the free probe (10 mM) and probe in
the presence of GSH (10 mM) at different pH in EtOH/PBS buffer solution
apparent loss of cell viability under the experimental condi-
tions. This result showed that the probe has low cytotoxicity to
cultured cells and has a great potential in cell study.
(
V/V, 3 : 2, pH = 7.4).
Based on above results, confocal fluorescence microscopic
imaging, including endogenous and exogenous fluorescence
imaging of the probe, was also investigated. Fig. 8 shows that after penetrate cells. In contrast, when the cell were treated with
HepG2 cells were incubated with the probe (10 mM) for 30 min, 0.5 mM N-ethylmaleimide (NEM, a known thiol blocking
there was remarkable intracellular red fluorescence observed. reagent) and then incubated with the probe at 37 1C for 30 min,
Results also indicated that the probe has good ability to the cells exhibited no fluorescence (Fig. 8(d–f)). To further explain
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2-(1,3,3-Trimethylindolin-2-ylidene)acetaldehyde was conjugated
to rhodamine by Knoevenagel condensation to elongate the
probe’s emission to near infrared region. Then, it was reacted
with ethylenediamine to synthesize an important intermediate.
Finally, the target molecule, named RhNM, was synthesized
through conjugated N-[4-(carbonyl)phenyl] as the GSH reaction
site. The a,b-unsaturated bond was expected to react with SH
group in GSH through Michael addition, due to which the new
NIR fluorescence probe RhNM with high quantum yield was
constructed. RhNM shows excellent discrimination of GSH from
Hcy/Cys, along with an obvious color change from colorless to
green. The fluorescence is rapidly enhanced by 106-fold at
746 nm after adding GSH, and the quantum was calculated to
Fig. 7 Cytotoxicity of probe in HepG2 cells. Cells were treated with be 0.36. Titration experiments of GSH showed a good linear
different concentrations of probe for 24 h, and cell viabilities were deter-
mined by the MTT assay. Data are expressed as mean Æ SD.
relationship between intensity and GSH concentration, and the
detection limit was calculated at 70 nM. The proposed mecha-
nism for identifying GSH based on Michael addition induced
cascade spirolactam opening. Meanwhile, the probe RhNM can
detect GSH in a wide range of pH 4–10. The probe RhNM was
used for endogenous and exogenous imaging in HepG2 cells for
glutathione. There was an outstanding intracellular red fluores-
cence observed upon the addition of GSH when incubated with
HepG2 cells. Results show that it has been successfully applied
for the determination of GSH in diluted serum and bio-imaging
of GSH in living cells with low cell toxicity. All results indicated
that the probe can be used as an ultra-sensitive, near-infrared
fluorescent chemical sensor for selective detection of GSH in
living cell. It has great potential in imaging live tissues and even
animal. We hope that the novel strategy can provide a template
for researchers to explore new NIR fluorescence probes with
improved sensitivity, greater fluorescence turn ‘‘ON’’ and fast
response time for probing bio-molecules in biological systems.
Conflicts of interest
Fig. 8 Top: Confocal microscopy images of HepG2 cells incubated with the
probe (10 mM) for 30 min; middle: confocal microscopy images of HepG2
cells preincubated with NEM (0.5 mM) for 30 min and then treated with the
There are no conflicts to declare.
probe (10 mM) for 30 min; bottom: confocal microscopy images of HepG2
cells incubated with NEM (0.5 mM) for 30 min, then incubated with the probe
Acknowledgements
(10 mM) for 30 min, and finally, after addition of GSH (5 mM). lex = 600 nm.
We thank the financial support from the Fundamental Research
Funds for the National Natural Science Foundation of China
the high sensitivity of probe RhNM toward GSH in HepG2 cells,
.5 mM GSH was added to NEM pretreated HepG2 cells. These
(No. 61178057).
0
cells were then incubated with the probe for almost 30 min, upon
which a strong fluorescence emerged (Fig. 8(g–i)). These results
implied that the probe was capable of detecting endogenous levels
of GSH and of imaging GSH in living cells. This suggests that
the NIR probe could perhaps be further applied in tracing and
detecting abnormal GSH content in biological systems, while
not affecting normal human activities.
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