A highly sensitive two-photon fluorescent probe for glutathione with near-infrared emission at 719?nm and intracellular glutathione imaging

Article abstract of DOI:10.1016/j.saa.2019.03.042

A near-infrared turn-on two-photon fluorescent probe ST-BODIPY for glutathione-specific detection was designed and synthesized by attaching triphenylamine to BODIPY skeleton through the Knoevenagel condensation to prolong the maximum emission wavelength t

Full text of DOI:10.1016/j.saa.2019.03.042

Accepted Manuscript
A highly sensitive two-photon fluorescent probe for glutathione
with near-infrared emission at 719nm and intracellular
glutathione imaging
Chunmei Huang, Ying Qian
PII:
S1386-1425(19)30280-X
https://doi.org/10.1016/j.saa.2019.03.042
SAA 16932
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 29 August 2018
Revised date:
Accepted
date:
22 February 2019
1
7 March 2019
Please cite this article as: C. Huang and Y. Qian, A highly sensitive two-photon fluorescent
probe for glutathione with near-infrared emission at 719nm and intracellular glutathione
imaging, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy,
https://doi.org/10.1016/j.saa.2019.03.042
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ACCEPTED MANUSCRIPT
A highly sensitive two-photon fluorescent probe for glutathione with
near-infrared emission at 719 nm and intracellular glutathione imaging
Chunmei Huang, Ying Qian*
School of Chemistry and Chemical Engineering, Southeast University, Nanjing
2
11189, China
Abstract
A near-infrared turn-on two-photon fluorescent probe ST-BODIPY for
glutathione-specific detection was designed and synthesized by attaching
triphenylamine to BODIPY skeleton through the Knoevenagel condensation to
prolong the maximum emission wavelength to the NIR region. And
2
,4-dinitrobenzenesulfonyl group (DNBS), as the fluorescence quencher and thiol
recognition moiety, was modified in 8 position of BODIPY. In the presence of GSH,
the probe afforded an “off-on” signal response with a significant NIR fluorescence
enhancement centered at 719 nm accompanying by quantum yield increased to 0.44,
which was ascribed to the glutathione-induced S Ar (aromatic substitution) reaction.
N
Surprisingly, we found that the probe could discriminate GSH from other biothiols
including Cys and Hcy upon the addition of intracellular concentrations of them.
Time-dependence also demonstrated that the probe could distinguish GSH from Cys
and Hcy under physiological environment. The limit of detection (LOD) for GSH was
calculated as 25.46 nM from the titration experiments, which is lower than most
previously reported GSH-selective probes. Under the Ti:sapphire pulsed laser’s 800
nm irradiation, ST-BODIPY toward GSH generated an “off-on” signal response with
a significant enhancement of fluorescence emission at 719 nm after treatment with

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GSH. Besides, the 2PA cross section value (σ
2
) was calculated to be 410 GM,
suggesting that it could not only function well as an excellent two-photon fluorescent
probe for the detection of intracellular GSH, but also be applied for two-photon
imaging with high sensitivity in living cells. Moreover, ST-BODIPY probe has been
successfully employed for monitoring exogenous and endogenous GSH in MCF-7
cells with satisfying results, perhaps it was feasible for detecting abnormal contents of
GSH in a biological system and accomplishing the goal of maintaining normal human
activities.
Key
words
：
two-photon;
near-infrared;
Glutathione
sensor;
bistriphenylamine-BODIPY; endogeous cell imaging.
1
. Introduction
Intracellular biothiol, including cysteine (Cys), glutathione (GSH), and
homocysteine (Hcy), in biological systems have drawed considerable interest in
recent years due to the essential influence on involving in various physiological and
pathological processes[1-3]. Among these intracellular biothiol, GSH, the most
abundant intracellular non-proteinogenic biothiol, whose normal intracellular level is
in the range of 1.0 to 15.0 mM[4-6], is regarded as an important biomolecule in
participating in the process of the intracellular redox activities to regulate the
metabolism and cellular homeostasis[7-11]. However, variations in the levels of GSH
directly implicated many diseases, such as liver damage, leucocyte loss, psoriasis,
cancer, and HIV infection[12-18]. Accordingly, selective and sensitive detection of GSH

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is of great benefit to monitoring these biothiols under complicated biological
conditions[19-22]
.
In the past few years, thiol-based fluorescent probes have attracted great
attention for the purpose of detecting biothiols. However, on account of the fact that
Cys/Hcy have similar reactivity of the thiol groups in the thiol-containing amino
acids, there were scarcely sensors that could detect GSH from Cys and Hcy[23-30]
Though a few probes provided selective fluorescent detection for GSH (Scheme 1),
the absorption and emission of them were in the visible range (300-650 nm)[31-33]
.
.
Accordingly, Zhao et al[34] developed BODIPY-based fluorescent probe used to detect
mitochondrial GSH in living cells with detection limit of 109 nM and its fluorescence
emission was located at 599 nm. Kim et al[35] has explored GSH-selective one-photon
fluorescent probe based on rhodamine using for HeLa cells and zebrafish imaging,
whose fluorescence emission wavelength was 579 nm and its detection limit was 0.17
μM. Superior to one-photon, two-photon probes, especially taking with a large
two-photon excitation cross-section, provided lots of advantages in cell imaging[36,37]
.
Considering the fact that near-infrared (NIR, 650-900 nm) two-photon fluorescent
dyes would display extraordinary advantages by virtue of the less damage to living
cells and better tissue penetration in the living systems, the exploring of two-photon
near-infrared (650-900 nm) GSH-selective probes were in urgent need, especially
NIR fluorescence emission wavelength reached 700 nm [38, 39]
.
Herein, A near-infrared two-photon fluorescent probe ST-BODIPY was designed
for the selective detection of GSH by introducing triphenylamine into 3 and 5 position

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of BODIPY. As a result of extended conjugation, the ST-BODIPY revealed emission
well-into the near-infrared region. 2,4-dinitrobenzenesulfonyl group(DNBS) was
incorporated at the 8-position of BODIPY as the fluorescence quencher and thiol
recognition moiety (Scheme 1). In the presence of GSH, the probe displayed an
“
off-on” signal response accompanying with an obvious NIR fluorescence
enhancement centered at 719 nm with lower limit of detection, which attributed to the
glutathione-induced S Ar reaction resulting in the cleavage of the DNBS group from
N
the probe molecule. Importantly, experimental results demonstrated the probe could
discriminate GSH from Cys and Hcy under intracellular concentrations. What’s more,
ST-BODIPY could be served as a two-photon fluorescent turn-on probe for detection
of GSH and its 2PA cross section value (σ ) was 410 GM at 719 nm excited by the
2
Ti:sapphire pulsed laser’s 800 nm irradiation, indicating that it was suitable for the
two-photon fluorescence bioimaging. At last, the probe was also used to detect
endogenous and exogenous GSH in living cells, which further displayed its valuable
applications in biological system.

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Scheme. 1. Recently reported GSH-selective chemosensors and design of specific
GSH-selective NIR fluorescent probe ST-BODIPY in this work
2
2
. Experimental section
.1 Materials and general methods
Reagents

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2
,4-dinitrobenzenesulfonyl group was purchased from Aladdin. Triphenylamine,
piperidine and toluene were purchased from Macklin Biochemical Co.,Ltd (Shanghai,
China). Triethylamine was purchased from Adamas. acetic acid and ethyl acetoacetate
were purchased from Maya reagent company. Indocyanine green (ICG) was
purchased from Sigma-Aldrich. All solvents used in spectroscopic measurements
were of analytical grade.
The used amino acides were obtained by commercial purchase. Glutathione
(GSH), cysteine (Cys), and homocysteine (Hcy) were purchased from Macklin
Biochemical Co., Ltd (Shanghai, China), Shanghai HuiXing Biochemical Reagent Co.,
Ltd, Tokyo Chemical industry Co., Ltd (Shanghai, China) respectively. Other amino
acids were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
Apparatus
1
Chemical structures were verified by NMR analysis and mass spectrometry. H
NMR and 13C NMR spectra were recorded on Bruker ADVANCE 500 NMR
spectrometer and Bruker AVANCE III HD 600 NMR spectrometer in DMSO-d with
6
tetramethylsilane (TMS) as internal standard. Chemical shifts were given in parts per
million (ppm). The mass spectra data were obtained on a Broker Autoflex TOF/TOF
mass spectrometry. UV-visible absorption spectra were determined on a Shimadzu
UV-3600 Spectrophotometer. Fluorescence spectra were measured on a HORIBA
FL-4 Max spectrometer. 1×1×3 cm quartz cuvettes were used for absorption and
emission spectral titration. The two-photon fluorescence emission spectra were
acquired by recording with Exemplar Plus modular spectrophotometer (MODEL NO.

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Mira 900) in the following standard configuration: wavelength range of 200-1080 nm,
2
5 μm slit, a LVF filter, a ruled grating (800 mm/250 nm), and a spectral resolution of
1
.5 nm, using a mode-locked Ti:sapphire pulsed laser with the two-photon excitation
condition( λex = 800 nm, 76 MHz, 100 fs).
Calculation of Detection limit
The detection limit, indicating the sensitivity of the probe, was found from 10
blank solutions based on the definition by IUPAC (CDL=3 Sb/m). Detection limit for
GSH was calculated by Eq.
Detection limit =3δ/k
Where δ standard deviation of 10 blank measurements and k=slope obtained
from the graph of fluorescence intensity ratio vs concentration of GSH[40]
Determination of the Fluorescence Quantum
.
The quantum yield was calculated according to the equation:
A
As
r
F
Fr
s
n
nr
s
2
φs
 φ   ( )
r
Where A is the absorbance at the excitation wavelength, F is the area under the
corrected emission curve, and n is the refractive index of the solvents used. Subscripts
s and r refer to the unknown and to the standard, respectively, indocyanine green (ICG,
Φ .
= 0.13 in DMSO)[41] was used as reference[42]
f
MTT cytotoxicity assay

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Cytotoxicity of the probe ST-BODIPY was examined by vitro MTT assay. The
logarithmic phase of MCF-7 cells were inoculated in a 96-well cell culture plate
4
(
1×10 cells per well), and incubated at 37 ℃ under an atmosphere of 5% CO
2
for 24
hours. Next, the cells were incubated with different concentrations (0-20 μM) of
ST-BODIPY for another 24 h. Then, the cells were incubated with 5 mg/mLMTT.
After that the supernatant was removed following by the addition of 100 mL DMSO
to each well and oscillation for 5 minutes. The absorbance of each well of the 96-well
plate was then obtained at 570 nm on a Benchmark Plus plate reader. Cell viability
was calculated according to the formula:
Cell viability (%) = A/A × 100%
0
where A is average value of sample OD570, A is average value of blank
0
OD570 and OD570 is the absorbance measured at 570 nm[43]
.
Cell culture
MCF-7 cells, human breast cancer cells, were firstly cultured in 1640 medium
supplemented with 10 % fetal bovine serum (FBS) in a humidified incubator at 37 ℃
under an atmosphere of 5% CO , Then were seeding on confocal dishes and allowed
2
to cultivate for 24 h. Before fluorescence imaging, all the cells were washed with PBS
thrice and then incubated with PBS free fresh media for subsequent cell imaging.
When starting the imaging of endogenous GSH detection, the cells were pretreated
with 100 μM N-Ethylmaleimide (NEM), a well-known thiol-blocking agent, for 90
min before incubation with ST-BODIPY (10.0 μM), then incubated with GSH (160

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μM) for 30 min. Confocal fluorescence microscopy (FLUOVIEW FV3000.
OLYMPUS) was used for all the imaging.[44]
2
.2 Synthesis
The synthetic route of ST-BODIPY is presented in scheme 2 and compound
[
45,46]
and 4-(diphenylamino)benzaldehyde[47] was obtained according to literature
M
1
procedures.
The synthesis of
-(diphenylamino)benzaldehyde (109 mg, 4.0 mmol) and piperidine (250 μL) were
M
2
:
Compound
M
1
(340 mg, 1.0 mmol),
4
dissolved in dry toluene (20.0 mL) in a Dean-Stark apparatus, followed by the
addition of three drops of AcOH. And then stirred at 100 ℃ for 12 h under a nitrogen
atmosphere. The solvent was concentrated in vacuo and flash column chromatography
1
(
petroleum ether/dichloromethane, 1:2) afforded the product (162 mg, 19 %). H
NMR (600 MHz, DMSO-d
6
) δ: 9.83 (s, 1 H), 7.48-7.45 (m, 6 H), 7.39-7.34 (m, 10 H),
= 7.1 Hz, J =7.1 Hz, 4 H), 7.09 (d, J = 8.2 Hz, 8
7
.17 (d, J = 8.2 Hz, 2 H), 7.14 (t, J
1
2
H), 6.96-6.93 (m, 6 H), 6.92 (s, 2 H), 1.49 (s, 6 H).
The synthesis of ST-BODIPY: Compound M
2
(190 mg, 0.2 mmol), excess Et N
3
(
156 μL) and 2,4-dinitrobenzenesulfonyl chloride (125 mg, 0.4 mmol) were dissolved
in CH Cl . After being stirred at room temperature for 3 h, The reaction mixture was
washed with water, and the organic layers were combined and dried over anhydrous
Na SO . The solvent was concentrated at reduced pressure, and the residue was
2
2
2
4
purified by column chromatography (petroleum ether/dichloromethane, 1:1) afforded
1
the product (130 mg, 54 %). H NMR (600 MHz, DMSO-d
6
) δ: 9.14 (s, 1 H), 8.60 (d,

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J = 8.7 Hz,1 H), 8.19 (d, J = 8.6 Hz, 1 H), 7.53-7.47 (m, 8 H), 7.41-7.38 (m, 4 H),
7
.37 (t, J
1
= 7.9 Hz, J
2
= 7.7 Hz, 8 H), 7.14 (t, J
1
= 7.4 Hz, J
2
= 7.4 Hz, 4 H), 7.10 (d,
) δ:
52.20, 151.51, 148.93, 148.36, 148.13, 146.35, 145.32, 140.39, 137.59, 137.41,
1
3
J = 7.7 Hz, 8 H), 6.96-6.94 (m, 6 H), 1.34 (s, 6 H). C NMR (300 MHz, DMSO-d
6
1
1
1
1
36.55, 135.37, 134.40, 133.79, 132.29, 130.99, 130.11, 129.64, 129.44, 129.30,
28.44, 127.18, 126.24, 124.89, 123.99, 122.77, 121.57, 121.07, 119.45, 118.54,
-
18.34, 115.97, 14.32. HRMS m/z calcd for C63
H47BF
2
N
6
O
7
S: [M] = 1080.3288,
found: 1080.3253.
Scheme 2 Synthetic route of ST-BODIPY
. Results and discussion
.1 Design and synthesis of the conjugated bistriphenylamine-BODIPY NIR
3
3
fluorescent probe
In the design of NIR fluorescent probe ST-BODIPY depicted in Scheme 2, to
afford the NIR dye Compound M , the 3 and 5 position of Compound M were
2
1
conjugated with 4-(diphenylamino)benzaldehyde through Knoevenagel condensation
in toluene under a nitrogen atmosphere with catalysis of piperidine and AcOH. The
2
,4-dinitrobenzenesulfonyl (DNBS) unit, as the fluorescence quencher and thiol

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recognition moiety, linked to the 8 position of BODIPY. In the presence of GSH,
glutathione-induced S Ar reaction led to cleavage of the DNBS to generate free
N
fluorophore in concert with turn-on fluorescence. The data of the structure
characterization are described in the experimental section and supporting information
(Fig. S1-S4, Supporting information). The target product ST-BODIPY were
1
13
confirmed by H NMR, C NMR and mass pectrometry.
.2 Two-photon turn-on NIR fluorescent probe ST-BODIPY for glutathione
based on GSH-induced S Ar reaction
3
N
Under the two-photon excitation condition, the probe alone displayed
non-fluorescent emission on account of DNBS as fluorescence quencher moiety that
switched off the TPEF(two-photon excited fluorescence) signal. However, in the
presence of GSH, an apparent emission band centered at 719 nm appeared which
attributed to GSH-induced S Ar reaction ( Fig. 1). The remarkable variation was
N
accompanied by a dramatic color change from colorless to brightly pink under
Ti:sapphire pulsed laser’s irradiation, which was easily observed by naked eyes
(
Scheme 3). What’s more, the 2PA cross section value (σ
2
) of ST-BODIPY for GSH
was examined to be 410 GM with rhodamine B[48] used as calibration standard (both
at the concentration of 10 M) according to the equation[49]:
-4
A
As
r
F
Fr
s
n
nr
s
2
φs
 φ
r

 ( )
Here, Subscripts s and r stand for the unknown and to the standard, σ is the TPA
cross-section value, c is the concentration of solution, n is the refractive index of the
solution, F is the TPEF integral intensities of the solution emitted at the exciting

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wavelength, and Φ is the fluorescence quantum yield. All the Two-photon
experimental results demonstrated that ST-BODIPY was favorable for the detection
of GSH as a Two-photon fluorescent turn-on NIR fluorescent probe. Maybe it was
expected to have great potential applications in two-photon fluorescence bioimaging.
3
2
2
1
1
5
.5x10
.0x10
.5x10
.0x10
.0x10
Probe
Probe+GSH
λem=719 nm
3
3
3
2
In THF/PBS buffer,
-4
0
1
M
Input laser: 800 nm,
1
00 fs, 76MHz
625
650
675
700
725
750
Wavelength (nm)
Fig. 1 Two-photon excitation fluorescence spectra of ST-BODIPY (100 μΜ) in the
absence or presence of 1600 μΜ of GSH. Conditions: PBS buffer (pH= 7.4) and THF
(1/1, v/v). λex = 800 nm.
the proposed mechanism of NIR fluorescent probe ST-BODIPY for GSH was
that the fluorescence enhancement response of ST-BODIPY was via thiol group of
GSH induced S Ar reaction which led to the cleavage of the DNBS group from the
N
probe molecule (Scheme 3). To verify the mechanism, ST-BODIPY was treated with
1
GSH in DMSO-d
6
at room temperature and then identified by H NMR. As depicted
in Fig. 2, the three protons on 2,4-dinitrobenzenesulfonyl group upfield shifted from
.14, 8.60, 8.19 ppm (H , H , H ) to 8.91, 8.52, 8.02 ppm (Ha’, Hb’, Hc’) respectively,
which could be ascribed to the fact that the electron withdrawing O=S=O on
9
a
b
c

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2
,4-dinitrobenzenesulfonyl group was replaced by electron donating −S group after
the nucleophilic aromatic substitution reaction.
Scheme 3. Proposed reaction mechanism for GSH-selective detection and its turn-on
fluorescence response toward GSH under 800 nm two-photon excitation light
1
Fig. 2 Partial H NMR spectra of ST-BODIPY, ST-BODIPY + GSH in DMSO-d
6
3
.3 Glutathione-specific detection of NIR fluorescent probe
As displayed in Fig. 3a, ST-BODIPY alone exhibited non-fluorescence.
However, the addition of GSH to the solution of ST-BODIPY generated a significant
fluorescence enhancement at 719 nm indicating an “off-on” signal response. Besides,

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the probe ST-BODIPY had no influence on the thiol-lacking amino acid, such as Gly,
Pro, Tyr, Ala, Arg, His, Thr, Asp, Glu, Ser, leu, Val, Trp. As is known to us, GSH is
the most abundant intracellular non-proteinogenic biothiol with intracellular
concentration of 1-15 mM compared with Cys (30-200 μM) and Hcy (12.4 μM).
Intracellular concentrations of Cys, Hcy, and GSH were respectively added to the
solution of ST-BODIPY, the probe showed remarkably selective response toward
GSH with a high quantum yield of 0.44. In contrast, the probe exhibited weak
fluorescence towards Cys (Φ
F
= 0.23) and Hcy (Φ = 0.12) ( Table 1). These results
F
indicated that ST-BODIPY can discriminate GSH from Cys and Hcy. Furthermore,
due to the co-existence of various amino acids in the physiological environment, the
interference from other amino acids should be taken into account. As shown in Fig. 3b,
competitive experiment indicated that the probe can be utilized for the selective
detection of GSH and not be interfered by other amino acids.
6
1
1
1
1
8
6
4
2
.6x10
.4x10
.2x10
.0x10
.0x10
.0x10
.0x10
.0x10
Probe+GSH
6
6
6
5
5
5
5
Probe , Probe + Gly, Pro,
Tyr, Ala, Arg, His, Thr, Asp,
Glu, Ser, leu, Val, Trp,
Probe+Cys
Probe+Hcy
0
.0
7
00
720
740
760
780
800
820
840
Wavelength (nm)
Fig. 3 (a) The fluorescence intensity of probe ST-BODIPY (10 μΜ, λex = 670 nm) in
the absence and presence of thiol amino acids including Cys (30 μM), Hcy (12.5 μM)
and GSH (1 mM) in intracellular concentrations and other thiol-lacking amino acids
(200 μM). (b) Fluorescence responses of ST-BODIPY in the presence of different

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amino acids. The blue bars showed the intensities of ST-BODIPY + GSH (160 μΜ) +
2
00 μΜ amino acids. The red bars displayed the intensities probe ST-BODIPY in the
absence and presence of thiol amino acids including Cys (30 μM), Hcy (12.5 μM) and
GSH (1 mM) in intracellular concentrations and other thiol-lacking amino acids (200
μM). Conditions: PBS buffer (pH= 7.4) and THF (1/1, v/v).
The absorption spectral properties of NIR fluorescent probe ST-BODIPY were
examined in a PBS buffer (pH 7.4) and THF (1/1, v/v) mixed solvent system.
According to Fig. S5, the probe ST-BODIPY showed two absorption peaks at 435 nm
4
−1
−1
4
−1
−1
(
ε = 3.14x10 cm ·M ) and 691 nm (ε = 6.91x10 cm ·M ).
Table.1 The detailed photophysical properties of ST-BODIPY in the absence and
presence of GSH and other amino acids
compounds
λ
abs
ε
max
λ
em
Fluorescence
quantum yield
4
−1 −1
(
nm)
(10 cm M )
(nm)
(
F
Φ )
probe
691
679
679
679
693
6.91
7.86
7.89
7.70
6.88
----
719
726
720
----
----
0.44
0.12
0.23
----
Probe+GSH
Probe+Hcy
Probe+Cys
Probe+His
λ
abs: UV absorption maximum; εmax: molar extinction coefficient; λem: fluorescence
maximum; Φ : fluorescence quantum yield. Conditions: PBS buffer (pH= 7.4) and
THF (1/1, v/v).
F
3
.4 Glutathione-sensitive NIR fluorescent probe ST-BODIPY with low detection
limit

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The fluorescence titration experiment was carried out to evaluate the ability of
NIR fluorescent probe ST-BODIPY to detect GSH (Fig. 4a). In the free probe, there
was almost no emission peak, while the emission band centered at 719 nm was
observed and enhanced up to a saturation gradually after adding GSH with the
increase of the concentrations. The emission spectra of ST-BODIPY to GSH had a
good liner relationship and the detection limit for GSH was obtained to be 25.46 nM
(3σ/k) ( Fig. 4b), which was lower than most probes’ in previous work.
6
1
1
1
1
8
6
4
2
.6x10
.4x10
.2x10
.0x10
.0x10
.0x10
.0x10
.0x10
6
6
6
5
5
5
5
6
5
5
5
5
1.0x10
Equation
y = a + b
Adj. R-Squ 0.99308
Value
Intercept 27146.139 12181.9329
Slope 1.70261E1 4.10115E8
Standard Er
8
.0x10
B
B
6.0x10
4
2
.0x10
.0x10
0
.0
0.0
7
00
720
740
760
780
800
820
840
0.0
-5
-5
-5
-5
-5
-5
1
.0x10 2.0x10 3.0x10 4.0x10 5.0x10 6.0x10
Wavelength (nm)
GSH (M)
Fig.4 (a) Fluorescence spectra of probe ST-BODIPY (10 μΜ, λex = 670 nm) with
increasing amount of GSH. (b) Calibration curve of the emission response as a
function of GSH concentration. Conditions: PBS buffer (pH= 7.4) and THF (1/1, v/v).
To further details of the applicability of the probe to detecting GSH in biological
environment, the influence of the pH on GSH before and after adding GSH has been
examined shown in Fig. 5. Emission intensity of the probe toward GSH between pH
6
.0-9.0 maintained almost constant values, demonstrating that the NIR probe
ST-BODIPY could work under physiological pH conditions, such as in living cells
and tissues. Moreover, Time dependence on the recognition behavior of probe
ST-BODIPY toward GSH, Hcy, and Cys testified that reaction rate to GSH was much

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faster than Cys and Hcy upon the addition of intracellular concentrations of them (Fig.
6
), which reconfirmed that probe ST-BODIPY showed remarkable selective detection
towards GSH under physiological condition.
6
1.4x10
1.2x10
1.0x10
8.0x10
6.0x10
4.0x10
2.0x10
probe
probe +16eq. GSH
6
6
5
5
5
5
0.0
0
2
4
6
8
10
12
14
pH
Fig. 5 Fluorescence intensity of ST-BODIPY (10 μΜ, λex = 670 nm) in the absence (●) or
presence (■) of GSH (160 μΜ) as a function of pH. Conditions: PBS buffer (pH= 7.4) and
THF (1/1, v/v).
6
6
5
5
5
5
GSH
Cys
Hcy
1.2x10
1.0x10
8.0x10
6.0x10
4.0x10
2.0x10
0.0
0
20
40
60
80
100
Time (min)
Fig. 6 Time-dependent changes of fluorescence spectra of probe ST-BODIPY (10 μM)
with GSH ( 1 mM), Cys ( 30 μM) and Hcy (12 μM) in THF/PBS buffer ( pH=7.4, 1: 1,
v/v) (λex=670 nm)

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3
.5 Endogenous and exogenous cell imaging of NIR fluorescent probe
ST-BODIPY
Conventional MTT assay was investigated to certify cytotoxicity of the probe.
As displayed in Fig. 7, it showed no apparent loss of cell viability when the probe was
exposed in concentrations of 0-20 μM for 24 h under the experimental conditions,.
This result demonstrated that the probe has low cytotoxicity to cultured cells.
1.0
0.8
0.6
0.4
0.2
0.0
0
2
4
6
8
10
12
14
16
18
20
22
Concentration (μM)
Fig. 7 Cytotoxicity of probe in MCF-7 cells treated with different concentrations of
ST-BODIPY for 24 h, and cell viabilities were determined by the MTT assay. Datas
were expressed as mean ± SD.
Based on above results, the practical utility of probe ST-BODIPY for selective
fluorescent imaging of GSH in living cells was studied (Fig. 8). A significant bright
NIR fluorescence was found when MCF-7 cells were incubated with probe
ST-BODIPY (Fig. 8 (a - c)), indicating that probe ST-BODIPY was capable of
permeating into cells and reacting with endogenous GSH to generate NIR
fluorescence. When the MCF-7 cells were preincubated with 0.5 mM
N-ethylmaleimide (NEM), a known thiol-blocking reagent, before incubating with

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ST-BODIPY(10 uM), no emission was detected (Fig. 8 (d - f)), which demonstrated
that thiol species were completely reacted by NEM. Upon the addition of GSH (1
mM) to the NEM pretreated MCF-7 cells followed by incubating with ST-BODIPY
(10 μΜ), an outstanding NIR red fluorescence emerged (Fig. 8 (g - i)). All these
results showed that probe ST-BODIPY not only could be used to detect the
endogenous GSH, but also could be applied for cell imaging upon addition of GSH in
living cells. Perhaps it could be further applied for detecting abnormal contents of
GSH in a biological system and maintaining normal human activities.
Fig. 8 The upside (a-c): confocal microscopy images of MCF-7 cells incubated with
probe ST-BODIPY (10 μΜ) for 90 min; the middle (d-f): confocal microscopy
images of MCF-7 cells preincubated with NEM (0.5 mM) for 30 min and then treated
with probe ST-BODIPY (10 μΜ) for 90 min; the downside (g-i): confocal microscopy
images of MCF-7 cells incubated with NEM (0.5 mM) for 30 min, then incubated

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with ST-BODIPY (10 μΜ) for 90 min, and finally added GSH (1 mM). Excitation at
6
00 nm.
4
. Conclusion
To sum up, we have developed a turn-on near-infrared two-photon fluorescent
probe ST-BODIPY for the selectively and sensitively detection of GSH from other
amino acids. Briefly, 4-(diphenylamino)benzaldehyde was conjugated at 3-position
and 5-position of BODIPY with longer wavelength as NIR-fluorophore by
Knoevenagel condensation. 2,4-dinitrobenzenesulfonyl (DNBS) was introduced at
8
-position of BODIPY playing a role of fluorescence quencher and thiol recognition
moiety. Under the Ti:sapphire pulsed laser’s excitation at 800 nm, ST-BODIPY could
be regarded as a two-photon fluorescent turn-on probe for GSH with the appearance
and remarked enhancement of fluorescence emission peak at 719 nm accompanied by
a dramatic color change from colorless to brightly pink. Due to its effective TPA
action cross section value (σ =410 GM), it could be used for two-photon imaging for
2
GSH with high sensitivity in living cells. In THF/PBS mixture solution, ST-BODIPY
had a special ability to recognize GSH over Cys and Hcy when intracellular
concentrations of them were added which was also testified from time-dependence. In
the addition of GSH, the enhancement of fluorescence emission was observed at 719
nm on account of the GSH-induced S Ar reaction that led to the cleavage of the
N
DNBS group and the release of the fluorophore. Furthermore, this probe displayed
high sensitivity for GSH and its detection limit was as low as 25.46 nM. The quantum
yield was worked out to be 0.44. Furthermore, ST-BODIPY has been successfully

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used for detection of endogenous and exogenous GSH. cell imaging experiment
certified that the probe ST-BODIPY could infiltrate into MCF-7 cells and react with
endogenous GSH to generate NIR fluorescence, which further displays its valuable
applications in biological system.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We are very grateful to prof. Y. P. Cui, prof. C. G. Lu and Dr. Z. Q. Qi advanced
Photonics Center of Southeast University for their assistance with the measurement of
two-photon fluorescence. We thank the financial support from the Fundamental
Research Funds for the National Natural Science Foundation of China (No.
6
1178057).
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Graphical abstracts: