A new colorimetric and near-infrared fluorescent probe for biothiols and its application to image GSH in living cells and zebrafish

Article abstract of DOI:10.1016/j.jphotochem.2019.111984

A new near-infrared (NIR) fluorescent probe based on 2-Methylchromone was exploited for relay recognition of Cu²⁺ and biothiols. The mechanism involved in complex between metal ion and probe, and subsequent displacement reaction, which make the probe can detect GSH by NIR fluorescence turn on emission. Probe showed a fluorescence band at 718 nm in PBS-EtOH (3:2, v/v, 10 mM PBS, pH 7.4) solution. Upon the addition of Cu²⁺, a large fluorescence quenching was observed due to the coordination between probe 1 and Cu²⁺. The 1-Cu²⁺ could be used to detect biothiols utilizing high affinity of biothiols with Cu²⁺. This “on-off-on” fluorescent probe was also successfully applied to image GSH in MCF-7 cells and zebrafish.

Full text of DOI:10.1016/j.jphotochem.2019.111984

Accepted Manuscript
Title: A new colorimetric and near-infrared fluorescent probe
for biothiols and its application to image GSH in living cells
and zebrafish
Authors: Yutao Yang, Xueli Yang, Keyan Zhou, Ming Jin, Wei
Li
PII:
DOI:
Article Number:
S1010-6030(19)30680-X
https://doi.org/10.1016/j.jphotochem.2019.111984
111984
Reference:
JPC 111984
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2 July 2019
20 July 2019
Please cite this article as: Yang Y, Yang X, Zhou K, Jin M, Li W, A new colorimetric and
near-infrared fluorescent probe for biothiols and its application to image GSH in living
cells and zebrafish, Journal of Photochemistry and amp; Photobiology, A: Chemistry
(2019), https://doi.org/10.1016/j.jphotochem.2019.111984
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A new colorimetric and near-infrared fluorescent probe for biothiols
and its application to image GSH in living cells and zebrafish
Yutao Yang, a, b*Xueli Yang, a Keyan Zhou,a Ming Jin, a Wei Li a, *
aKey Laboratory of Medicinal Chemistry, and Molecular Diagnosis of the Ministry of Education,
College of Chemistry & Environmental Science, Hebei University, Baoding 071002, P. R. China;
b
Key Laboratory of Chemical Biology of Hebei Province, Hebei University, Baoding 071002, P.
R. China;
E-mail:yutaoyang2016@163.com(Y.T. Yang);
liweihebeilab@163.com(W. Li);
Abstract Graphic
Highlights
 A new NIR fluorescent probe for biothiols was developed based on complex
and displacement.
 2. The probe displayed excellent sensitivity, high selectivity and low detection
limit.
 3. The probe was successfully applied to detect endogenous biothiols in
MCF-7 cells and zebrafish.
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Absract
A new near-infrared (NIR) fluorescent probe based on 2-Methylchromone was
exploited for relay recognition of Cu2+ and biothiols. The mechanism involved in
complex between metal ion and probe, and subsequent displacement reaction, which
make the probe can detect GSH by NIR fluorescence turn on emission. Probe showed
a fluorescence band at 718 nm in PBS-EtOH (3:2, v/v, 10 mM PBS, pH 7.4) solution.
Upon the addition of Cu2+, a large fluorescence quenching was observed due to the
coordination between probe 1 and Cu2+. The 1-Cu2+ could be used to detect biothiols
utilizing high affinity of biothiols with Cu2+. This “on-off-on” fluorescent probe was
also successfully applied to image GSH in MCF-7 cells and zebrafish.
Keywords: Fluorescent probe; Biothiols; Near-infrared; Complex and displacement;
Bioimaging
Introduction
In biological systems, a nuance sometimes leads to a distinctly different function.
Glutathione (GSH), cysteine (Cys) and homocysteine (Hcy) play very important roles
in biological processes [1]. Abnormal these species levels are reported to be closely
related to certain diseases [2]. As the most abundant intracellular thiol (1–10mM),
GSH maintains biological redox homeostasis with beneficial antioxidant activity [3].
The inappropriate levels of GSH are directly linked to cancer, macular degeneration,
HIV/AIDS and other diseases [4-6]. Cys deficiency is associated with the slow
2 / 25

growth, muscle and fat loss, hair depigmentation, liver damage and skin lesions [7].
Elevated levels of Hcy in plasma are involved in increased risk of cardiovascular
disease, neural tube defects, Alz-heimer’s disease, inflammatory bowel disease and
osteoporosis [8-11]. Hence, Monitoring the concentration of biological thiols is
helpful for early diagnosis and disease prevention.
In recent years, fluorescence imaging detection method has received extensive
attention due to its high selectivity and obvious change of signals. A variety of
reaction-based fluorescent probes for thiols has been developed. Among these probes,
cleavage reactions [12-14], metal complex displacement coordination [15-20] and
nucleophilic addition reactions [21-24] are the main mechanisms for designing thiols
fluorescent probes. However, the emission peaks of many fluorescent probes are in a
short wavelength region, and the photo bleaching or damaging to living cells or
tissues can be caused by bioimaging. Hence, near-infrared fluorescent probes have
attracted much attention due to the advantages of high sensitivity, minimum
autofluorescent interference and deep tissue penetration to biological samples.
Correspondingly, some near-infrared biothiols fluorescent probes have been designed
and developed [25-37]. Despite this, there are still very few NIR fluorescent probes
for detection of thiols. Based on our previous research experience [39-40], we
designed and synthesized a new NIR fluorescent probe 1 (Scheme 1). In particular,
the probe exhibited several meritorious features as follows: (i) the probe 1 could be
coordinated with Cu2+ through hydroxyl groups to form 1-Cu2+ complex; （ii）the
subsequent complex 1–Cu2+ could be developed as a platform to detect biothiols；（iii）
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this “on-off-on” NIR fluorescent probe can detect the endogenous biothiols in MCF-7
cells and zebrafish.
2. Experimental section
2.1. Materials and Instruments
2-Methylchromone, malononitrile, acetic anhydride, CuCl2 ，MgSO4, Na2CO3,
CaCl2·6H2O, AlCl3, MgCl2, ZnCl2, FeCl3, NaCl, AuCl3·3H2O, CdCl2, NiCl2, KCl,
MnCl2, PbNO3, Hg(NO3)2 were brought from Aladdin Reagents. Homocysteine,
Glutathione, Cysteine, amino acids, N-Ethylmaleimide (NEM) were obtained from
Sigma-Aldrich. The DMEM medium, E3 medium bovine serum albumin (BSA) were
purchased from Thermo Fisher Scientific. Solvents and reagents were of analytic
grade without further purification. Stock solution of probe 1 (2 mmol/L) was prepared
in DMSO. Stock solutions (20 mmol/L) of various analytes, amino acids and metal
ions were prepared in deionized water. The UV-visible and fluorescence spectra were
1
13
recorded on the Agilent Cary 60 and Agilent Cary Eclipse. H and C NMR spectra
were recorded on a Bruker AVIII spectrometer, using tetramethylsilane as the internal
standard. Mass spectrometry was obtained through Q-TOF 6520.
2.2 Synthesis of probe 1
As shown in Scheme 1, compound 2 (104 mg, 0.5 mmol), 4-dihydroxybenzaldehyde
(96 mg, 0.7 mmol) and piperidine (10 µL) were dissolved in 30 mL of acetonitrile
solution. The mixture was refluxed for 9 hours and evaporated in vacuum to remove
solvent. The resulting residue was dissolved in 30 mL of dichloromethane, then dried
with anhydrous MgSO4 and concentrated to obtain the crude product. It was purified
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by silica chromatography (DCM: MeOH=400:1), giving a red solid (1.44 g, 68%
1
yield). H NMR (600 MHz, DMSO-d6) δ (ppm): 9.74 (s, 1H), 9.19 (s, 1H), 8.71(d, J
= 8.4Hz, 2H), 7.90 (t, J = 7.8Hz, 3H), 7.78 (d, J = 7.8Hz, 2H), 7.59 (m, 4H), 7.16 (t,
13
J = 15.6Hz, 2H), 7.09 (t, J = 7.8Hz, 2H), 6.96 (s, 1H), 6.82 (t, J = 8.4Hz, 2H). C
NMR (150 MHz, DMSO-d6) δ:158.92, 152.84, 152.02, 148.71, 145.73, 139.75,
135.17, 126.64, 125.99, 124.55, 121. 62, 119.01, 117.43, 117.10, 116.04, 115.90,
+
115.77, 114.95, 105.64, 58.91. HRMS(ESI): [M+NH4] calcd for 346.1186, Found
346.1186. (Figure S1).
<Inserted Scheme 1>
2.3. Cell culture and imaging
MCF-7 cells were used to carry out the experiment. Cells were grown in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal bovine and 4.5 g/L
glucose at 37°C in an atmosphere of 5% CO2. The cells were firstly inoculated for 12
hours, and the cell density was 2×105 cells per dish. Then 1-Cu2+ was added (20 μM)
to further incubate the cell for 0.5h. Following by being washed with PBS and were
ultimately imaged. At the same time, two controlled experiments were completed.
For a control group, cells were treated with NEM (1 mM) for 0.5h, then incubated
with probe of 1 h. A comparison group of experiments, four concentrations of GSH (0,
10, 50, 100 µM)) were added to the incubation of cells with NEM and 1-Cu2+. Finally,
the cells were washed with PBS three times for imaging. Fluorescence images were
5 / 25

obtained with a Zeiss LSM880 confocal. The emission was collected at 591−742 nm
and the excitation wavelength is at 588 nm.
2.4. Zebrafish imaging
For further investigation of bioimaging in vivo, zebrafishes of 3 days old were
prepared in advance which were purchased from Nanjing Eze-Rinka Biotechnology
Co., Ltd. Zebrafishes were initially pretreated with 1-Cu2+ (20 μM) in E3 embryo
media (pH 7.5). For NEM treated control experiments, zebrafish were pretreated with
NEM (1 mM) at 28℃ for 0.5 h, the following incubated with GSH (1 mM) prior to
addition of 1-Cu2+ (20 μM) for another 0.5 h and then washed three times with E3
medium for imaging.
3. Results and Discussion
3.1. Spectral properties of probe 1 towards Cu2+
The spectral properties of the probe 1 was investigated in physiological condition
pH 7.4 PBS-EtOH (3:2, v/v, 10 mM PBS, pH 7.4) solution (Figure S2). As shown in
Figure 1a, the ultraviolet absorptions of probe 1 were mainly centered at 454 nm (ε =
1.4×104M-1 cm-1, Ф= 0.14). With the addition of Cu2+ (20 μM), the absorption
peak at 454nm decreased, accompanying the migration of absorption peak to 570 nm.
In the meanwhile, the color of solution changed from yellow to purple, which was
mainly attributed to the formation of 1-Cu2+ complex. Correspondingly, fluorescence
spectrum of probe 1 (20 μM) showed an emission at 718 nm with excitation at 570
nm, the emission peak at 718 nm decreased rapidly and the red fluorescence
quenching was observed when Cu2+ (20 μM) was added (Figure 1b). In addition,
6 / 25

photostability was an important criterion for measure the performance of a probe, and
the photostbility of probe 1 was also demonstrated. The results indicated that the
probe 1 possessed excellent photostability in vitro (Figure S3).
Furthermore, with addition of other metal ions (100 μM), such as Ca2+, Al3+, Mg2+,
Zn2+, Fe3+, Hg2+, Na+, Au3+, Cd2+, Ni2+, Mn2+, K+, Pb2+, Ba2+, there was no significant
change in UV-visible absorption and fluorescence spectra. In addition, the presence of
these other cations (100 μM) did not interfere with the determination of Cu2+ (Figure
S4). Figure 2 displayed the UV absorption and fluorescence titrations of probe 1 with
Cu2+. The ratio of A570/454 changed from 0.28 to 1.48, and tended to be stable when
Cu2+ (20 μM) was gradually added (Figure 2). It was linearly related to Cu2+
(R=0.9941) and detection limit was 6.97 μM at 3σ/k. Furthermore, the Job’s plot
proved stoichiometry between 1 and Cu2+ (Figure S5), and the binding constant was
found to be 4.11×104 M-1 based on Benesi–Hildebrand plot (Figure S6) The kinetic
studies have shown that the reaction was completed in 10 s (Figure S7).
<Inserted Figure 1>
<Inserted Figure 2>
3.2. Spectral properties of 1-Cu2+ towards biothiols
The pH range for determination of biothiol was studied and an optimal pH of 7.4
was selected for further studies (Figure S8) The selectivity of 1–Cu2+ (Ф= 0.03)
towards various amino acids (GSH, Hcy, Cys, Val, Ala, Gly, Phe, Leu, Trp, Met, Tyr,
Asp, Thr, Asn, Glu, Lys, Gln, Ser, Pro, His and Arg) and other analytes (HS-, PhSH)
was conducted. The UV absorption and fluorescence intensity were returned to
7 / 25

original status due to formation of free 1 when GSH, Hcy and Cys (20 μM) were
added to the solution of 1-Cu2+ (Figure 3) and there was no obvious change upon
addition of other analytes (200 μM). As shown in Figure 4, the titrations of 1-Cu2+
with GSH was carried out and the detection limit was 6.47 μM based on IUPAC
method. Hcy and Cys have the similar spectral properties and the detection limits
were 4.81 μM and 4.93 μM (FigureS9).
<Inserted Figure 3>
<Inserted Figure 4>
3.3. Proposed mechanism
As well demonstrated in the previous research [15], the displacement approaches were
used as a method of designing fluorescent probes for biothiols, utilizing high affinities
of biothiols with copper ions. The rationale design is depicted in Scheme 2, illustrated
as follows. The probe 1 has two -OH groups, which can be easily coordinated with
copper ions to form 1-Cu2+ complexes. Based on the nucleophilic displacement
strategy, 1-Cu2+ was used for detection of biothiol owing to the high affinities of
biothiols towards copper ions. The reversibility of 1 for relay recognition of Cu2+ and
GSH was carried out in fluorescence spectrum. The experiment has been repeated at
least 5 times, implying that probe 1 can be used as a reversible fluorescent probe for
detection of Cu2+ and GSH (Figure S10). 1-Cu2+ complex was also characterized by
mass spectrometry (Figure S11).
<Inserted Scheme 2>
3.4. Fluorescence Imaging in Living Cells
8 / 25

The application of 1-Cu2+ for intracellular biothiols in MCF-7 cells was
performed in our study. Cytotoxicity is an important indicator of the applicability of
probe in biological systems. The MTT experiment showed that the 1-Cu2+ at a
concentration of 20 μM has minimal cytotoxicity. These results implied that this probe
was suitable for biothiols detection in live cells (Figure S12). Hence, we investigated
the potential use of 1-Cu2+ in MCF-7 cells. Confocal laser scanning microscopic
fluorescence imaging was used to evaluate the capability of 1-Cu2+ for detection of
biothiols in MCF-7 cells. We firstly conducted time-dependent imaging of 1-Cu2+
toward biothiols in living cells. When MCF-7 cell was incubated with 1-Cu2+ (20
μM), fluorescence was produced in the cytoplasm and the fluorescence intensity
gradually increased over time, reaching the maximum during 1 h, indicating that
1-Cu2+ could be used to visualize endogenous biothiols in 1 h (Figure 5a).
Furthermore, we performed a series of control experiments to assess the responses
of 1-Cu2+ toward GSH. As shown in Figure 5, MCF-7 cells were pretreated with
N-ethylmaleimide (NEM) for 0.5h, followed by addition of 20 μM 1-Cu2+. A
negligible fluorescence emerged in red channel. Subsequently, when cells were
incubated with different concentration of GSH (10, 50, 100µM), strong fluorescence
were observed, and the results suggested that 1-Cu2+ is responsive to intracellular
biothiols in living cells.
3.5. Fluorescence Imaging in Zebrafish
Next, we investigated the utility of 1−Cu2+ in 3-day-old Zebrafish. As shown in
Figure 6, zebrafish themselves showed no fluorescence in the near-infrared region but
9 / 25

it was incubated with 1-Cu2+ (20 μM) displayed a strong fluorescence. When
zebrafish was treated with NEM (1 mM) for 30min, then incubated with 1-Cu2+ for
1h, showing very weak fluorescence; while the NEM-treated zebrafish was incubated
with GSH (1 mM) for 30min, following incubated with 1-Cu2+ for 1h, showed a
strong fluorescence. Hence, we think 1-Cu2+ is capable of detecting biothiols in
Zebrafish.
<Inserted Figure 5>
4. Conclusions
In summary, a new NIR fluorescent probe 1 was successfully developed and its
Cu2+ complex was applied to image biothiols in MCF-7 cells. With the addition of
Cu2+, the fluorescence quenching was observed in PBS-EtOH (3:2, v/v, 10 mM PBS,
pH 7.4) solution. Subsequently, the fluorescence was restored with the increase of
biothiols due to the high affinities of biothiols towards Cu2+. Consequently, a
“on-off-on” fluorescent probe was developed to detect biothiols in living cells and
Zebrafish.
Acknowledgments
We thank Hebei University Science Foundation for High-level personnel, Hebei
Province Nature Science Foundation for Youths (No. B2017201093) and Hebei
Province Higher School Science Research fund for Youths(No.QN2016112). We also
thank the National Natural Science Foundation of China (No.21807021). We also
thank Natural Science Foundation of China (No.21807021) and Key Laboratory of
10 / 25

Medicinal Chemistry and Molecular Diagnosis of the Ministry of Education
enhancement projects (Hebei University).
11 / 25

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Figure captions
Scheme 1 Synthesis of probe 1.
Scheme 2 Proposed sensing mechanism.
Figure 1 (a) Absorption and (b) fluorescence spectra of probe 1 (20 µM) with
addition of Cu2+ (20 µM) in PBS-EtOH (3:2, v/v, 10 mM PBS, pH 7.4) solution.
(insert: color and fluorescence changes of the solution of 1 with addition of Cu2+).
Figure 2 (a) Absorption spectra of probe 1 (20 µM) towards concentrations of Cu2+
(0-20 µM) in PBS-EtOH (3:2, v/v, 10 mM PBS, pH 7.4) solution (insert: plots of
Absorbance ratio of A570/454); (b) Fluorescence spectra of probe 1 (20 μM) towards
concentrations of Cu2+ (0-20 µM) in PBS-EtOH (3:2, v/v, 10 mM PBS, pH 7.4)
solution (λex = 570 nm, slits: 10/10 nm; insert: the linear relationship between the
fluorescence intensity and Cu2+ concentration (0-20 µM)).
Figure 3 Absorption spectra of 1−Cu2+ (20 µM) in the present of GSH, Hcy, Cys (20
μM) and other analytes (Gly, Ala, Val, Leu, Phe, Trp, Tyr, Asp, Asn, Glu, Lys, Gln,
Met, Ser, Thr, Pro, His and Arg, HS-, PhSH) (100µM) in PBS-EtOH (3:2, v/v, 10 mM
PBS, pH 7.4) solution (λex = 570 nm, slits: 10/10 nm).
Figure 4 (a) Absorption spectra of 1-Cu2+ (20 µM) towards concentrations of GSH
(0-20 µM) in PBS-EtOH (3:2, v/v, 10 mM PBS, pH 7.4) solution; (b) Fluorescence
spectra of 1-Cu2+ (20 μM) towards concentrations of GSH (0-20 µM) in PBS-EtOH
(3:2, v/v, 10 mM PBS, pH 7.4) solution (λex = 570 nm, slits: 10/10 nm; insert: the
linear relationship between the fluorescence intensity and GSH concentration (0-20
µM)).
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Figure 5 (a) Time-dependent imaging of 1-Cu2+ toward biothiols in MCF-7 cells. (A)
10min; (B) 30min; (C) 60min; (D, E, F) 2.5D images of were MCF-7 cells, λex = 588
nm, λem = 591-742 nm, Scale bar: 10µm. (b) Top: A, B, C, D: Cells were treated with
1 mM of NEM for 30min, then incubated with GSH (0, 10, 50, 100µM), Bottom:
Fluorescence intensity per cell.
Figure 6 Fluorescence Imaging in Zebrafish. λex = 588 nm, λem = 591-742 nm, Scale
bar: 500µm.
Scheme 1
Scheme 2
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Figure 1
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(b)
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Figure 2
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(b)
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Figure 3
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(b)
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Figure 4
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(b)
Figure 5
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(a)
(b)
Figure 6
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