A reversible coumarin-based sensor for intracellular monitoring cysteine level changes during Cu2+-induced redox imbalance

Article abstract of DOI:10.1016/j.saa.2021.120173

Biological thiols are crucial small molecule amino acids widely existing in cells, which play indispensable roles in maintaining redox homeostasis of living systems. Owing to their abnormal levels have close relation with many diseases, thus, developing more convenient, rapid and practical in-vivo detection tools is imminent. Herein, a reversible coumarin-based probe (HNA) was successfully constructed through a simple two-step synthesis. HNA can detect Cys/Hcy with high response speed and desirable selectivity based on Michael addition recognition mechanism. Free HNA has an orange emission at 580 nm, but after addition of Cys/Hcy, the conjugated structure of probe HNA was destroyed by the attack of sulfhydryl, resulting in a new green emission at 507 nm. Further, HNA has been applied to monitor Cys/Hcy in HeLa cells and zebrafish. Notably, HNA has also been successfully applied for real-time tracing Cys levels changes in living cells and zebrafish during the imbalance in redox status caused by copper (II). This provides a new strategy for studying the process of oxidative stress in cells.

Full text of DOI:10.1016/j.saa.2021.120173

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 263 (2021) 120173
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A reversible coumarin-based sensor for intracellular monitoring cysteine
level changes during Cu2+-induced redox imbalance
Jianbin Chao a, , Jiamin Zhao a,b, Jinping Jia a, Yongbin Zhang c, Fangjun Huo c, Caixia Yin d,
⇑
⇑
a Scientific Instrument Center, Shanxi University, Taiyuan 030006, China
b School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China
c Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China
d Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
A reversible probe (HNA) was
prepared based on coumarin.
HNA exhibited high response speed
and low detection limit for Cys/Hcy.
HNA can evaluate the effect of copper
(II)-induced oxidative stress on Cys
levels in living cells and zebrafish.
a r t i c l e i n f o
a b s t r a c t
Article history:
Biological thiols are crucial small molecule amino acids widely existing in cells, which play indispensable
roles in maintaining redox homeostasis of living systems. Owing to their abnormal levels have close rela-
tion with many diseases, thus, developing more convenient, rapid and practical in-vivo detection tools is
imminent. Herein, a reversible coumarin-based probe (HNA) was successfully constructed through a sim-
ple two-step synthesis. HNA can detect Cys/Hcy with high response speed and desirable selectivity based
on Michael addition recognition mechanism. Free HNA has an orange emission at 580 nm, but after addi-
tion of Cys/Hcy, the conjugated structure of probe HNA was destroyed by the attack of sulfhydryl, result-
ing in a new green emission at 507 nm. Further, HNA has been applied to monitor Cys/Hcy in HeLa cells
and zebrafish. Notably, HNA has also been successfully applied for real-time tracing Cys levels changes in
living cells and zebrafish during the imbalance in redox status caused by copper (II). This provides a new
strategy for studying the process of oxidative stress in cells.
Received 17 June 2021
Received in revised form 6 July 2021
Accepted 7 July 2021
Available online 14 July 2021
Keywords:
Coumarin
Cys/Hcy
Cu2+
Redox imbalance
Bioiaging
Ó 2021 Elsevier B.V. All rights reserved.
1. Introduction
functions in physiological activities [1–4]. Among them, Cys
possesses the simplest structure and mainly responsible for the
Biological thiols, which are widely present in organisms, are the
general term for sulfhydryl-containing compounds in organisms.
The most representative biological thiols are: cysteine (Cys),
homocysteine (Hcy) and glutathione (GSH), which have pivotal
regulation of the redox balance of organisms, with an intracellular
concentration of 30–200
mol.LÀ1 [5,6]. Hcy is mainly involved in
the regulation of cell homeostasis, and its intracellular concentra-
l
tion is as low as 5–15 l
mol.LÀ1 [7,8]. High intracellular concentra-
tion of GSH (1–10 mmol.LÀ1) is an important biochemical defense
system in organisms [9,10]. Aberrant levels of biological thiols in
cells may cause various health problems [11–15]. High
⇑
Corresponding authors.
E-mail addresses: chao@sxu.edu.cn (J. Chao), yincx@sxu.edu.cn (C. Yin).
https://doi.org/10.1016/j.saa.2021.120173
1386-1425/Ó 2021 Elsevier B.V. All rights reserved.

J. Chao, J. Zhao, J. Jia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 263 (2021) 120173
concentration of Cys can cause cardiovascular and neurotoxic dis-
eases, while low concentration is closely related to slow growth,
drowsiness, liver injury, skin lesions, fat loss and weakness [16–
20]. Studies have revealed that Hcy is a hazard element for angio-
cardiopathy, inflammation, osteoporosis, and mental diseases such
as Alzheimer’s disease and schizophrenia [21–25]. The abnormal
level of GSH is closely related to cancer, neurodegenerative disease
and cardiovascular disease [26–30]. Therefore, there are highly
required for more available tools to further understand the rele-
vant functions of biological thiols in human physiology and
pathology.
Compared with the previous detection methods for biological
thiols [31–33], non-invasive fluorescence probes have aroused
wide interest on account of their advantages of simple synthesis,
easy to operate, high sensitivity and real-time detection. For this,
many fluorescent probes with excellent performance have been
developed to identify thiols. However, since the three thiols pos-
sess similar molecular structures, it is still difficult to develop
effective probes to distinguish them at the same time. Neverthe-
less, some research groups have successfully developed a few
promising probes for discrimination of these three thiols. For
instance, Li [34] prepared a dual-site sensor for differentiation of
Cys, Hcy and GSH. Ren [35] developed a red-emitting probe to
detect Cys/Hcy and GSH. Fu [36] designed three sensors for identi-
fication of Cys and GSH.
J = 8.8 Hz, 1H), 7.16–7.12 (m, 2H), 6.82 (d, J = 8.1 Hz, 1H), 6.63
(s, 1H), 3.51 (d, J = 6.5 Hz, 4H), 1.16 (t, J = 6.4 Hz, 6H). 13C NMR
(150 MHz, DMSO d6): d 185.88, 160.46, 158.68, 157.37, 153.44,
148.79, 143.12, 136.25, 132.80, 131.01, 130.88, 129.85, 127.98,
127.39, 124.61, 124.09, 119.79, 116.19, 110.69, 109.54, 108.41,
96.40, 44.93, 12.85. HR-MS: m/z calculated for C26H23NNaO4
[M + Na]+: 436.15248, found: 436.15138.
2.2. Optical properties
Probe HNA (0.0016 g) was dissolved in DMSO to prepare a stock
solution (2 mM). Stock solutions of Cys, Hcy, other amino acids and
ions (0.01 M) were prepared in deionized water. All spectroscopy
experiments were tested in PBS/DMSO (v/v, 6/4, pH 7.4) system.
HeLa cells and zebrafish (4-day-old) were used for bio-imaging
studies and the specific culture methods are described in the sup-
porting information.
3. Results and discussion
3.1. Spectral properties
First, we studied the absorption spectrum of HNA to Cys in the
test system (Fig. 1a). After continuously adding Cys (0–400
lM),
the absorption at 480 nm synchronously weakened and blue
shifted to 460 nm, which corresponded to the destruction of con-
jugated structure. Subsequently, the fluorescence spectrum was
measured (Fig. 1b), the probe itself had an emission peak at
580 nm, but when Cys was added, a new emission peak appeared
at 507 nm and steadily enhanced. The fluorescence spectrum of
HNA titrated with Hcy was similar to that of Cys (Fig. S1). All these
changes were caused by the Michael addition reaction between the
sulfhydryl and the double bond, which destroyed the conjugation
structure of the probe HNA. In addition, we found that the fluores-
cence intensity was linear with Cys/Hcy concentrations at a wide
To our knowledge, coumarin dyes have the advantages of easy
modification, large stokes shift and stable optical properties [37–
39]. Given these, herein, we constructed a reversible coumarin-
based probe (HNA) through a simple two-step synthesis. HNA
exhibited high response speed, desirable selectivity and low detec-
tion limits for Cys/Hcy detection. After addition of Cys/Hcy, the
carbon-carbon double bond in the
a,b-unsaturated ketone struc-
ture was nucleophilically attacked by sulfhydryl, resulting in the
destruction of the conjugate structure of HNA, the emission peak
at 580 nm shifted to 507 nm, and green fluorescence was released.
Further, HNA has been used for imaging Cys and Hcy in HeLa cells
and in vivo. More interestingly, we further used HNA to monitor
Cys levels changes in HeLa cells and zebrafish during the imbalance
in redox status caused by copper (II).
range of 0–275
lM and 0–325 l
M, respectively (R2 = 0.9993 for
Cys and 0.9999 for Hcy) (Fig. 1c). The calculated detection limit
of Cys was 8.4 nM, and Hcy was 8.2 nM on the basis of IUPAC rec-
ommendation (CDL = 3Sb/m). These results suggested that HNA
was a reliable tool for distinguishing Cys/Hcy from GSH.
2. Experimental section
3.2. Kinetic study and reversibility of probe HNA
2.1. Synthesis of the probe HNA
Then, the kinetics of the HNA-Cys/Hcy system was investigated.
As illustrated in Fig. 2 and Fig. S2, the fluorescence of HNA at
507 nm remained almost unchanged during the whole test, sug-
gesting that the probe HNA was stable. However, after addition
Compound 1 (1 mmol, 0.259 g), 6-hydroxy-2-naphthaldehyde
(1 mmol, 0.172 g) were dissolved in anhydrous ethanol (10 mL),
then 150 lL piperidine was added. After refluxing for 36 h, the sol-
vent was evaporated and the crude product was further purified by
column chromatography (EtOAc/PE = 1/1, v/v) to get a red solid
(0.149 g, 36%) (Scheme 1). 1H NMR (600 MHz, DMSO d6) d 10.05
(s, 1H), 8.62 (s, 1H), 8.10 (s, 1H), 8.00 (d, J = 15.6 Hz, 1H), 7.82
(dd, J = 22.3, 12.1 Hz, 2H), 7.75 (t, J = 23.6 Hz, 2H), 7.71 (d,
of 400 lM Cys/Hcy, a significant enhancement in fluorescence
was observed at 507 nm, and reached a maximum in a short time.
This denoted that HNA is a rapid tool for detecting Cys/Hcy.
Reversibility is one of the vital parameters to evaluate the per-
formance of probes. For probe HNA, we used NEM (thiol blocking
Scheme 1. Synthetic route of probe HNA.
2

J. Chao, J. Zhao, J. Jia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 263 (2021) 120173
Fig. 2. Kinetics study of HNA (10 lM) and HNA-Cys system.
respectively, the absorption peak returned to 480 nm, and the
absorption intensity was slightly lower than that of adding HNA
alone. Additionally, we also studied this property through cyclic
addition of Cys and NEM/H2O2 in the probe solution. As shown in
Fig. 4, through five experimental cycles, the fluorescence intensity
has undergone a small and non-negligible change. All these hinted
that the probe HNA has good reversibility and can also be used to
evaluate the redox kinetics of Cys and H2O2.
3.3. Selectivity, interference and pH experiments
To evaluate the desirable selectivity of HNA to Cys/Hcy, we
investigated the changes in fluorescence intensity in the presence
of common amino acids (Ala, Asp, Arg, Thr, Leu, Tyr, Glu, Ser, His,
Trp, Val, Phe, Pro, Gly, Lys, Ile, Gln, Asn, Met) and ions (SO42À
,
SO23À, HSÀ, S2O32À, NO3À, CO23À, NO2À, ClÀ, BrÀ, IÀ, AcÀ, CrO42À, K+,
Fe2+, Mg2+, Na+, Cu2+, Mn2+, Pb2+, Co2+, Hg2+, Cr3+, NH4+). As depicted
in Fig. 5a, after addition of 400 lM interfering substances, the flu-
orescence intensity at 507 nm remained almost unchanged. Fur-
ther, interference experiments were also performed in the test
system, in the presence of these interfering substances (400
lM),
the fluorescence intensity of HNA-Cys/Hcy system did not change
significantly (Fig. 5b, c). All these confirmed that HNA can specifi-
cally identify Cys/Hcy in complex samples.
In addition, the pH-dependent experiments of HNA and HNA-
Cys/Hcy system were also performed (Fig. 6). The probe HNA itself
exhibited excellent stability at pH 2.0–10.0. After 400 lM Cys/Hcy
was added, obvious fluorescence enhancement was observed at pH
4.0–10.0, and the peak of fluorescence intensity appeared at
pH = 7.4. The above facts demonstrated that HNA was capable of
well applied to detect Cys/Hcy in physiological environment.
3.4. Response mechanism
Fig. 1. Spectral response of HNA to Cys (PBS/DMSO, v/v, 6/4, pH 7.4). (a) UV
absorption spectrum of HNA (10
Fluorescence spectrum of HNA (10
Linear fit within the Cys range of 0–275
l
l
M) within the Cys range of 0–400
M) within the Cys range of 0–400
M. kex = 460 nm, slit: 5/5 nm.
l
M; (b)
lM; (c)
We proposed that the response mechanism of HNA with Cys/
Hcy was attributed to Michael addition reaction. As described in
Scheme 3, upon addition of Cys/Hcy, the carbon–carbon double
l
bond in the
a,b-unsaturated ketone structure was nucleophilic
agent) and H2O2 (a representative ROS that can consume Cys) as
inducible factors to study its reversibility (Scheme 2). From the
UV absorption spectrum in Fig. 3, we found that free HNA had a
attacked by the sulfhydryl, so that the conjugate structure of the
probe HNA was broken, the ICT process was changed, and the
emission peak at 580 nm shifted to 507 nm, strong green fluores-
cence was released. Further, this assumption has been verified by
mass spectrometry and 1H NMR titration. The m/z peaks were
observed at 557.17087, 571.18633 (calculated: 557.17223,
strong absorption at 480 nm, after addition of 400
absorption decreased and shifted to 460 nm. However, when
NEM and H2O2 (400 M) were added to the above solution
lM Cys, the
l
3

J. Chao, J. Zhao, J. Jia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 263 (2021) 120173
Scheme 2. The possible reversible mechanism between NEM/H2O2 and Cys.
Fig. 4. Reversible study of HNA (10
(b) (400 M) cyclically.
lM) by adding Cys (400 lM) and NEM (a)/H2O2
Fig. 3. UV absorption spectra of HNA after adding Cys (400
H2O2(b) (400 M) in sequence.
lM) and NEM(a)/
l
l
571.18788). After 1H NMR titration, the characteristic signal of the
double bond in HNA at d 7.8 (a/c), d 8.0 (b/d) almost disappeared
and new peaks appeared at d 4.59, 4.03 and 3.76 for Cys, d 4.55
and 3.73 for Hcy, corresponding to the generated CH and CH2
protons.
NEM-treated cells were further incubated with Cys/Hcy (200
lM,
10 min) and HNA (10 M, 10 min), the green channel displayed
l
intense florescence. These results demonstrated the ability of
HNA for imaging Cys/Hcy in living cells.
Oxidative stress (OS) is a state of imbalance between oxidation
and antioxidation, that is, redox imbalance, which is considered to
be an important factor leading to aging and diseases [40,41]. Cys-
teine, as a potential endogenous antioxidant, plays a vital role in
retaining cell redox homeostasis. Moreover, related papers pointed
out that copper (II) stimulation can induce cells to produce ROS,
and the generated reactive oxygen species further undergo redox
reactions with Cys, resulting in a significant decrease in the intra-
cellular Cys content [42–44]. Since the intracellular content of Hcy
is much lower than that of Cys, under this premise, redox balance
was altered by H2O2/Cu2+, and the changes of Cys levels in HeLa
3.5. HeLa cells imaging
Encouraged by the superior optical properties of HNA, we fur-
ther applied it to cell imaging. As described in Fig. 7, after treat-
ment of HeLa cells with HNA (10
exhibited weak fluorescence. As a control, NEM-treated cells were
further incubated with HNA (10 M, 10 min), and weak orange flu-
lM, 10 min), the green channel
l
orescence was clearly observed, while no green fluorescence was
observed. Next, HNA was used for detecting exogenous Cys/Hcy.
4

J. Chao, J. Zhao, J. Jia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 263 (2021) 120173
Fig. 6. Fluorescence intensity of HNA and HNA-Cys system under different pH
conditions.
was almost unchanged within 20 min. When the incubation of
HeLa cells with HNA (10
lM, 10 min) and H2O2 (2 mmol/L)
successively, the green fluorescence was gradually reduced within
20 min. This implied that the addition of H2O2 induced oxidative
stress, which consumed Cys in cells. Next, we investigated the
effect of Cu2+ on the induction of oxidative stress in HeLa cells,
using Cu(NO3)2Á3H2O as the copper source. HeLa cells treated with
HNA (10 l
M, 10 min) and Cu2+ (2 mmol/L) sequentially, the inten-
sity of green fluorescence decreased significantly within 20 min.
All these results may be due to the fact that HeLa cells produced
ROS under the stimulation of copper (II), the generated ROS (sim-
ilar to H2O2) undergo a redox reaction with Cys, thereby reducing
the intracellular Cys levels. So the fluorescence intensity of the
green channel was gradually weakened.
3.6. Zebrafish imaging
To further evaluate the imaging performance of HNA in vivo, we
conducted laser confocal microscopy imaging experiments using
zebrafish as a model. As illustrated in Fig. 9, after the zebrafish
co-incubated with HNA (10
showed weak fluorescence. In contrast, NEM-treated zebrafish
were further incubated with HNA (10 M, 10 min), and no fluores-
lM, 10 min), the green channel
l
cence signal was observed in the green channel, but there was flu-
orescence emission in the orange channel. Then, the zebrafish
cleared by NEM (500
l
M, 30 min) were further treated with Cys/
M, 10 min), and the green
Hcy (200 M, 10 min) and HNA (10
l
l
channel showed intense fluorescence. These phenomena suggested
the excellent in vivo imaging performance of HNA.
In addition, the redox imbalance induced by Cu2+ in zebrafish
Fig. 5. The specific response of HNA to Cys. (a) Fluorescence spectrum of HNA
(10 mM) after adding various amino acids and ions (400
intensity of HNA (10 mM) for other amino acids in the presence and absence of
400 M Cys (1.blank, 2.Ala, 3.Asp, 4.Arg, 5.Thr, 6.Leu, 7.Tyr, 8.Glu, 9.Ser, 10.His, 11.
lM); (b) Fluorescence
was also evaluated. Zebrafish treated with HNA (10
lM, 10 min)
and Cu2+ (2 mmol/L) sequentially, and the changes in the fluores-
cence intensity of the green channel were collected over time.
From Fig. 10a, we observed that the green fluorescence gradually
weakened in 20 min. Similarly, we used H2O2 as the representative
of reactive oxygen species to study its effect on the redox process
in zebrafish (Fig. 10b). After the treatment of zebrafish with HNA
l
Trp, 12.Val, 13.Phe, 14.Pro, 15.Gly, 16.Lys, 17.Ile, 18.Gln, 19.Asn, 20.Met, 21.Hcy); (c)
Fluorescence intensity of HNA for various ions in the presence and absence of
400 l
M Cys (1.blank, 2.SO42À, 3.SO32À, 4.HSÀ, 5.S2O32À, 6.NO3À, 7.CO32À, 8.NOÀ2 , 9.ClÀ, 10.
BrÀ, 11.IÀ, 12.AcÀ, 13.CrO42À, 14.K+, 15.Fe2+, 16.Mg2+, 17.Na+, 18.Cu2+, 19.Mn2+, 20.
Pb2+, 21.Co2+, 22.Hg2+, 23.Cr2+, 24.NH+4). All data were recorded after mixing for
3 min.
(10 lM, 10 min) and H2O2 (2 mmol/L) separately, we observed that
the fluorescence signal of green channel gradually decreased
within 20 min. These phenomena indicated that Cu2+ can induce
the production of ROS in zebrafish, and excessive ROS consumed
Cys in zebrafish, resulting in the decrease of green fluorescence.
cells were investigated through the fluorescence response of HNA.
As illustrated in Fig. 8, HeLa cells on incubation with HNA (10
lM,
10 min) emitted green fluorescence, and the fluorescence intensity
5

J. Chao, J. Zhao, J. Jia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 263 (2021) 120173
Scheme 3. Proposed reaction process between HNA and Cys/Hcy.
Fig. 7. Imaging of Cys/Hcy in HeLa cells with HNA. Orange channel: kem = 580 30 nm (kex = 488 nm); Green channel: kem = 507 30 nm (kex = 458 nm).
6

J. Chao, J. Zhao, J. Jia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 263 (2021) 120173
Fig. 8. Time-dependent cell imaging with HNA (10
l
M) and PBS/H2O2/Cu2+. Green channel: kem = 507 30 nm (kex = 458 nm).
7

J. Chao, J. Zhao, J. Jia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 263 (2021) 120173
Fig. 9. Imaging of Cys/Hcy in zebrafish with HNA. Orange channel: kem = 580 30 nm (kex = 488 nm); Green channel: kem = 507 30 nm (kex = 458 nm).
Fig. 10. Time-dependent zebrafish imaging with HNA (10
l
M) and Cu2+ (2 mmol/L) (a)/H2O2 (2 mmol/L) (b). Green channel: kem = 507 30 nm (kex = 458 nm).
8

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 263 (2021) 120173
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Jianbin Chao: Conceptualization, Methodology, Supervision.
Jiamin Zhao: Formal analysis, Investigation, Resources, Data cura-
tion, Writing - original draft, Visualization. Jinping Jia: Conceptual-
ization,
Methodology,
Supervision.
Yongbin
Zhang:
Conceptualization, Methodology, Supervision. Fangjun Huo: Vali-
dation, Writing - review & editing. Caixia Yin: Validation, Writing
- review & editing.
Declaration of Competing Interest
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Acknowledgements
We thank the National Natural Science Foundation of China
(No. 21472118, 21672131), the Program for the Top Young and
Middle-aged Innovative Talents of Higher Learning Institutions of
Shanxi (No. 2013802), Talents Support Program of Shanxi Province
(No. 2014401), Shanxi Province Outstanding Youth Fund (No.
2014021002), Natural Science Foundation of Shanxi Province of
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