Ultrasensitive and selective fluorescent sensor for cysteine and application to drug analysis and bioimaging

Article abstract of DOI:10.1016/j.ab.2021.114138

A fluorescent sensor based on coumarin-maleimide conjugate was developed for efficient discrimination of Cys from Hcy and GSH in both organic and aqueous solution. Addition of Cys to the non-fluorescent sensor solution in DMF induced bright blue fluorescence and enhanced the fluorescence intensity by 320-fold while other amino acids and biothiols (Gly, Hcy, GSH, Glu, Val, Tyr, Arg, Trp, Lys, His, Leu, Phe, Asp and Met) did not bring about remarked change. The sensor responds to Cys extremely rapidly. If Cys was added to the sensor solution, the fluorescence intensity increased by 170-fold immediately and attained the maximum value in 5 min. A linear relationship was observed between Cys concentration within 2–20 μM and the fluorescence intensity of the sensor solution. The detection limit of the sensor toward Cys is as low as 4.7 nM. The sensor is also effective for specific detection of Cys in aqueous (DMF/H₂O = 9:1, v/v) solution. Practical application of the sensor to drug analysis and bioimaging of living Hela cells has been verified. Possible sensing mechanism of the sensor toward Cys has been proposed.

Full text of DOI:10.1016/j.ab.2021.114138

Analytical Biochemistry 620 (2021) 114138
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Ultrasensitive and selective fluorescent sensor for cysteine and application
to drug analysis and bioimaging
,
,
Luping Hua, Tao Zhengb **, Yanxi Songc, Ji Fana, Hongqi Lia *, Ruiqing Zhanga, Yi Sunb
a Key Laboratory of Science and Technology of Eco-Textiles, Ministry of Education, College of Chemistry, Chemical Engineering & Biotechnology, Donghua University,
2999 North Renmin Road, Shanghai, 201620, PR China
b Department of Health Technology, Technical University of Denmark, Kgs, Lyngby, 2800, Denmark
c College of Environmental Science and Engineering, Donghua University, 2999 North Renmin Road, Shanghai, 201620, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A fluorescent sensor based on coumarin-maleimide conjugate was developed for efficient discrimination of Cys
from Hcy and GSH in both organic and aqueous solution. Addition of Cys to the non-fluorescent sensor solution
in DMF induced bright blue fluorescence and enhanced the fluorescence intensity by 320-fold while other amino
acids and biothiols (Gly, Hcy, GSH, Glu, Val, Tyr, Arg, Trp, Lys, His, Leu, Phe, Asp and Met) did not bring about
remarked change. The sensor responds to Cys extremely rapidly. If Cys was added to the sensor solution, the
fluorescence intensity increased by 170-fold immediately and attained the maximum value in 5 min. A linear
Fluorescent sensor
Coumarin
Cysteine
Maleimide
Bioimaging
Drug analysis
relationship was observed between Cys concentration within 2–20 μM and the fluorescence intensity of the
sensor solution. The detection limit of the sensor toward Cys is as low as 4.7 nM. The sensor is also effective for
specific detection of Cys in aqueous (DMF/H2O = 9:1, v/v) solution. Practical application of the sensor to drug
analysis and bioimaging of living Hela cells has been verified. Possible sensing mechanism of the sensor toward
Cys has been proposed.
1. Introduction
subsequent cyclization reaction [25–33], sulfonamide [34,35] or sulfo-
nate esters cleavage [36–38], S–S bond [39,40] or Se–N bond cleavage
As the main representatives of biologically significant thiols, ho-
mocysteine (Hcy), cysteine (Cys) and γ-Lglutamyl-L-cysteinylglycine
(glutathione, GSH) exert various important effects on human health [1,
2]. Cys plays a vital role in regulation of protein function [3]. On the
other hand, Cys exhibits neurotoxicity [4] and may induce senescence
and decelerate cell growth in melanoma [5]. Thus efficient methods for
detection of biothiols with both high sensitivity and selectivity and for
specific sensing of Cys over other biothiols are in urgent demand. As one
of the best choices for biothiols detection, fluorescent sensors derived
from different fluorophore platforms receive extensive attention and
application in view of their short response time, low detection limit, and
high sensitivity and selectivity [6–16]. Considering that the sulfhydryl
group in biothiol molecules is strongly nucleophilic and shows strong
binding affinity to mercury and copper ions, several strategies for
reasonable design of biothiol sensors have been developed including
Michael addition [17–21], cyclization reaction with cyano group [22] or
aldehyde [23,24], conjugate addition to an acrylate followed by
[41,42],
nucleophilic
substitution
[43‒45],
metal
complex-displacement coordination [46‒48], and indicator displace-
ment [49,50]. As a typical and excellent Michael acceptor, maleimide
group can be attached to various fluorophores to construct fluorescent
sensors for specific sensing of thiols. Following the pioneering work
reported by Sippel [51,52], fluorogenic chemosensors based on different
fluorophores functionalized with maleimide were designed and syn-
thesized on the purpose of biothiol sensing [53‒60]. It is found that most
of these sensors are efficient for detecting Cys, Hcy and GSH simulta-
neously, only a small part of them can be utilized for discriminating Cys
from Hcy and GSH. Specific detection of Cys by using the fluorescent
chemosensors is limited due to the drawbacks including the introduction
of heavy metal ions, complicated synthetic procedures and a relatively
long response time from 10 min to 60 min. Development of ultrasensi-
tive fluorogenic chemosensors to distinguish Cys from other biothiols is
in high demand and remains to be a huge challenge as a result of the
similar structures and comparable reactivities between Cys and other
* Corresponding author.
** Corresponding author.
E-mail address: hongqili@dhu.edu.cn (H. Li).
https://doi.org/10.1016/j.ab.2021.114138
Received 24 November 2020; Received in revised form 29 January 2021; Accepted 12 February 2021
Available online 24 February 2021
0003-2697/© 2021 Elsevier Inc. All rights reserved.

L. Hu et al.
Analytical Biochemistry 620 (2021) 114138
biothiols. With it in mind that maleimide-based fluorogenic sensors
respond to biothiols very rapidly [54,56] and following the efforts in
construction of highly selective fluorescent biothiol chemosensors in our
group [46,61], we developed a new structurally simple yet ultrasensitive
and selective fluorometric chemosensor for efficiently discriminating
Cys from Hcy and GSH by taking advantage of the easy response be-
tween thiols and maleimide which is incorporated into coumarin, the
excellent fluorophore for construction of fluorescent sensors [62,63]. In
this paper we report the design, synthesis, structural characterization,
responsive performance, and application in drug analysis and bio-
imaging of the coumarin-maleimide dyad-based specific chemosensor
for Cys.
the resulted yellow powder was filtered out. Recrystallization of the
crude product with ethanol as solvent gave compound 1 (6.0 g, 90%) as
yellowish solid with m.p. 202–204 ◦C (literature value: 203–204 ◦C
[64]).
2.3. Synthesis of ethyl 6-amino-2-oxo-2H-chromene-3-carboxylate
(compound 2)
Compound 1 (2.63 g, 10 mmol) and stannous chloride (9.5 g, 50
mmol) were added to a flask. Dry ethanol (30 mL) was used to dissolve
the mixture. The resulted solution was refluxed and stirred for 1 h. After
the reaction had finished, the solution was evaporated under reduced
pressure to remove the solvent. Ethyl acetate (300 mL) was used to
dissolve the residue. NaHCO3 solution (400 mL) was added for washing.
Then the solution was dried over anhydrous MgSO4. After filtration the
solution was evaporated for crystallization. The solid was collected and
was recrystallized from EtOH to give compound 2 (1.5 g, 64%) as red-
dish brown powder with m.p. 179–181 ◦C (literature value: 180–182 ◦C
[64]).
2. Experimental part
2.1. Chemicals and measurements
The chemicals and solvents for synthesis were analytically pure and
were used as received unless otherwise stated. The drug Super-Bio L-
CYSTEINE was produced in Hubei Shubang Pharmaceutical Co., Ltd. An
WRS-2A melting apparatus was utilized to determine melting points. An
2.4. Synthesis of N-(3-ethoxycarbonyl-2-oxo-2H-chromen-6-yl)
Bruker ACF-500 spectrometer was used for recording 1H NMR and 13
C
maleimide (A1)
NMR spectra with.CD3Cl and Me4Si as solvent and the internal refer-
ence, respectively. FT-IR spectra were recorded on an Bruker Tensor 27
spectrophotometer. An Agilent 1100 Series LC/MSD Trap mass spec-
trometer was used for mass spectra recording by using electronspray
ionization (ESI). An 7600CRT spectrophotometer and an FS5 spectro-
fluorometer were utilized for measuring UV–vis absorption spectra and
fluorescence spectra, respectively.
Compound 2 (223 mg, 1 mmol), glacial acetic acid (10 mL) and
maleic anhydride (108 mg, 1.1 mmol) were added to a flask. The
mixture was stirred then the solution was refluxed for 6 h. After reaction
the mixture was cooled to ambient temperature. Evaporation under
reduced pressure to remove solvent yielded light yellow solid, which
was submitted to column chromatography for purification. Petroleum
ether/ethyl acetate (1:1, v/v) was used as eluent to produce sensor A1
◦
2.2. Synthesis of ethyl 6-nitro-2-oxo-2H-chromene-3-carboxylate
(0.17 g, 53%) as light yellow powder with m.p. 178–180 C. IR (KBr
(compound 1)
pellet): ν = 3085, 3045, 2966, 1747, 1708, 1569, 1489, 1430, 1371,
1222, 1133, 1024, 865, 816 cmꢀ 1. 1H NMR (400 MHz, CDCl3) δ 8.45 (s,
1H), 7.65–7.61 (m, 2H), 7.38 (d, J = 9.6 Hz, 1H), 6.85 (s, 2H), 4.35 (q, J
= 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ
Diethyl malonate (4.40 g, 27.5 mmol) and 5-nitrosalicylaldehyde
(4.19 g, 25 mmol) were added to a flask and then dissolved in anhy-
drous ethanol (50 mL), followed by addition of catalytic amount of
piperidine. The solution was stirred and refluxed for 2 h. After the re-
action had finished the solution was cooled to ambient temperature and
–
–
–
–
167.91 (C O), 161.64 (C O), 155.14 (C O), 152.77 (Ph‒C), 146.74
–
–
(=C), 133.42 (=C), 130.27 (=C), 127.01 (Ph‒C), 125.01 (Ph‒C), 118.16
(Ph‒C), 117.11 (Ph‒C), 116.62 (Ph‒C), 61.12 (CH2), 13.19 (CH3). ESI-
Scheme 1. Synthetic route to sensor A1.
Fig. 1. Color of sensor A1 solution (20 μM in DMF) before and after addition of 1.0 equivalent of Cys, Hcy, GSH, Gly, Glu, Val, Tyr, Arg, Trp, Lys, His, Leu, Phe, Asp
or Met under UV irradiation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
2

L. Hu et al.
Analytical Biochemistry 620 (2021) 114138
MS: m/z 313 (M+).
2.5. Measurements of the absorption and fluorescence emission spectra
UV–vis and fluorescence spectra were determined in N,N-dime-
thylformamide (DMF) or DMF/H2O (9:1, v/v) at ambient temperature
by using sensor A1 solution of 20 μM concentration. The excitation
wavelength was 369 nm with both the excitation and emission slit width
being 10 nm in all the fluorescence titration experiments. The analytes
were added to a cuvette of 2 mL volume by a microliter syringe.
2.6. Preparation of drug samples for determination
One pill of the drug Super-Bio L-CYSTEINE (18%) containing 0.400 g
drug powder and 72 mg (0.595 mmol) of the effective constituent Cys
was dissolved in ultrapure water (297.5 mL) to obtain the drug sample
solution (Cys concentration 2 mM). Then 25 μL, 50 μL or 75 μL of the
above solution was measured with a pipette and was diluted 10 times to
obtain the drug samples with a concentration of 5
respectively.
μM, 10 μM or 15 μM,
Fig. 2. Fluorescence emission spectra of sensor A1 (20 μM) before and after
addition of 20 μM of Cys, Hcy, GSH, Gly, Glu, Val, Tyr, Arg, Trp, Lys, His, Leu,
Phe, Asp or Met in DMF with λex of 369 nm.
2.7. Cell imaging
Hela cells were supplied by the American Type Culture Collection.
Storage of all the cells was achieved in a 100% humidity atmosphere of
37 ◦C and 5% CO2. Firstly, Hela cells were cultured in the flasks, sup-
plemented with DMEM, 10% fetal bovine serum and 1% antibiotics in a
100% humidity atmosphere of 37 ◦C and 5% CO2. Then sensor A1 (20
μ
M in DMSO) was incubated with Hela cells for 30 min at 37 ◦C as well
as washed with phosphate-buffered saline three times after incubation.
In control group, Hela cells were incubated with N-ethylmaleimide (0.1
mM) for 30 min at 37 ◦C. Next, the incubation solution was washed with
phosphate-buffered saline (2 mL × 3) to remove the excess N-ethyl-
maleimide. Sensor A1 (20 μM in DMSO) was incubated with above N-
ethylmaleimide pre-treated Hela cells for 30 min at 37 ◦C and washed
with phosphate-buffered saline three times soon after before the cell
imaging.
3. Results and discussion
Fig. 3. Fluorescence intensity at 502 nm of A1 (20 μM) upon addition of 20 μM
3.1. Preparation of sensor A1
of Hcy, GSH, Gly, Glu, Val, Tyr, Arg, Trp, Lys, His, Leu, Phe, Asp or Met without
(black bars) or with Cys (red bars) in DMF. (For interpretation of the references
to colour in this figure legend, the reader is referred to the Web version of
this article.)
The designed sensor A1 was synthesized by utilizing a three-step
protocol and the synthetic route was illustrated in Scheme 1. Com-
pound 1 was prepared from the raw materials diethyl malonate and 5-
nitrosalicylaldehyde in 90% yield by Knoevenagel condensation
Fig. 4. Fluorescence intensity at 502 nm of A1 (20
μ
M) upon addition of 100
μ
M of Hcy, GSH, Gly, Glu, Val, Tyr, Arg, Trp, Lys, His, Leu, Phe, Asp or Met without
(black bars) or with Cys (red bars) in DMF. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3

L. Hu et al.
Analytical Biochemistry 620 (2021) 114138
Fig. 5. Fluorescence emission spectra of sensor A1 (20
Phe, Asp or Met in DMF with λex being 369 nm.
μ
M) obtained immediately after addition of 20
μM of Cys, Hcy, GSH, Gly, Glu, Val, Tyr, Arg, Trp, Lys, His, Leu,
Fig. 6. Changes in the fluorescence intensity at 502 nm of A1 (20 μM) upon addition of 1 equivalent of Cys in DMF with time (λex = 369 nm).
Fig. 7. (a) Fluorescence spectra of A1 (20
μ
M) upon addition of different concentration of Cys (2–20
M) in DMF.
μM) in DMF. (b) Linear relationship between the fluorescence
intensity at 502 nm of A1 (20 M) and Cys concentration (0–20
μ
μ
4

L. Hu et al.
Analytical Biochemistry 620 (2021) 114138
reaction. Piperidine and ethanol were used as the catalyst and solvent,
respectively. Reduction of compound 1 with stannous chloride in
ethanol gave compound 2 in 64% yield. Imidation reaction between
compound 2 and maleic anhydride afforded sensor A1 in 53% yield. This
condensation-reduction-imidation protocol is efficient for convenient
and straightforward synthesis of divergent coumarin-based chemo-
sensors substituted with maleimide at different position since various
nitrosalicylaldehydes are available. The chemical structure of the sensor
was characterized by IR spectrum (Fig. S1), 1H NMR (Fig. S2), 13C NMR
(Fig. S3) and MS (Fig. S4).
in Fig. 5. It is apparent that sensor A1 responds to Cys very rapidly and
adding Cys to sensor A1 solution induces the fluorescence intensity
enhancement by 170-fold instantly. Then the time-dependency of the
fluorescence emission of sensor A1 (20 μM in DMF) at 502 nm after
adding Cys (1 equivalent) excited at 369 nm was investigated. Changes
in the fluorescence emission intensity at 502 nm of sensor A1 upon
addition of 1 equivalent of Cys with time were recorded and showed in
Fig. 6. The results revealed clearly that after Cys had just been added, the
fluorescence intensity increased immediately to a high point. When the
3.2. Sensing behavior of A1 in DMF
Sensor A1 shows bad solubility in water but can be dissolved readily
by organic solvent DMF and acetonitrile. Considering that sensor A1
exhibits the strongest absorption and fluorescence emission in DMF,
sensing performance of A1 was investigated in two media: DMF and
DMF/H2O (9:1, v/v).
The absorption spectra of sensor A1 (20 μM solution in DMF) were
measured before and after addition of different amino acids including
Arg, Asp, Cys, Glu, Gly, GSH, Hcy, His, Leu, Lys, Met, Phe, Trp, Tyr, and
Val (20 μM) (Fig. S5). No obvious spectral change was observed after
addition of the amino acids. If the solution of sensor A1 or A1/amino
acid (20 M in DMF) was irradiated with a UV lamp (λ = 365 nm), it
μ
could be observed that sensor A1 displayed extremely weak fluorescence
and the solution containing sensor A1 and Cys presented strong bright
blue fluorescence. The fluorescence did not change markedly upon
addition of other amino acids as showed in Fig. 1. Thus A1 can be uti-
lized for visual distinguishing of Cys from Hcy, GSH and other amino
acids under UV irradiation.
Fluorescence spectra of sensor A1 (20
μM solution in DMF) were
measured before and after addition of 20 M of Arg, Asp, Cys, Glu, Gly,
μ
GSH, Hcy, His, Leu, Lys, Met, Phe, Trp, Tyr, and Val as showed in Fig. 2.
It was visible that a broad emission band appeared at around 502 nm
after addition of Cys. This means that a red shift of 87 nm compared to
A1 has been induced by addition of Cys with 320-fold enhancement of
the fluorescence intensity. Addition of other amino acids including Gly
and biothiols Hcy and GSH also caused enhancement of the fluorescence
but the intensities were quite small compared with that of Cys. It can be
considered that the addition of amino acids other than Cys induce
negligible changes in the fluorescence emission spectra of A1. The re-
sults demonstrate the high selectivity of sensor A1 toward Cys over other
common competitive amino acids and biothiols including Hcy, GSH,
Gly, Glu, Val, Tyr, Arg, Trp, Lys, His, Leu, Phe, Asp and Met.
The competitive experiments were conducted in the presence of Cys
over other amino acids. Fluorescence intensity at 502 nm of A1 (20 μM)
upon addition of Arg, Asp, Hcy, His, Glu, Gly, GSH, Leu, Lys, Met, Phe,
Trp, Tyr or Val without or with Cys (20 μM in DMF) were recorded in
Fig. 3. The fluorescence intensity of A1 with Cys was not influenced
substantially by the addition of competitive amino acids including Hcy,
GSH and Gly. To further demonstrate the excellent anti-interference
feature of sensor A1, fluorescence intensity at 502 nm of A1 (20
μM)
upon addition of 5 equivalent (100
μM) of Hcy, GSH, Gly, Glu, Val, Tyr,
Arg, Trp, Lys, His, Leu, Phe, Asp or Met without or with 1 equivalent of
Cys in DMF were measured and showed in Fig. 4. Though the addition of
a large amount of competitive amino acids especially GSH brought
about the fluorescence intensity decrease of A1 with Cys, the changes
were small and could not influence substantially the fluorescence turn-
on detection of Cys by A1. This means the fluorescent sensor A1 exhibits
excellent selectivity and anti-interference ability. Therefore sensor A1 is
applicable for highly selective fluorogenic discrimination of Cys from
other common amino acids and biothiols.
Response time of sensor A1 toward Cys was investigated. Fluores-
cence of sensor A1 solution (20 μM in DMF) measured immediately after
addition of Arg, Asp, Cys, Glu, Gly, GSH, Hcy, His, Leu, Lys, Met, Phe,
Trp, Tyr or Val (20 M) excited at a wavelength of 369 nm were showed
Scheme 2. Structures of reported fluorescent sensors for specific detection
μ
of Cys.
5

L. Hu et al.
Analytical Biochemistry 620 (2021) 114138
Table 1
Performance comparison of typical fluorescent sensors for specific detection of
Cys.
Sensor
Limit of detection
Response time
Reference
3
0.657
μ
M
40 min
10 min
10 min
30 min
20 min
12 min
40 min
4 min
[25]
[66]
[29]
[30]
[67]
[33]
[32]
[28]
[26]
[27]
[18]
[68]
[69]
[70]
[17]
[71]
[72]
[21]
[39]
[53]
[57]
[73]
[54]
[56]
This work
4
60 nM
5
0.2
μ
M
6
47.7 nM
70 nM
40 nM
7
8
9
0.307
μ
M
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
25
A1
50 nM
5.08
84 nM
μ
M
40 min
5 min
14
μM
10 min
30 min
30 min
3 min
22 nM
60 nM
0.15 μM
11.1 nM
25 nM
3 min
1 min
46.3
0.48
μ
M
M
30 min
15 min
60 min
4 s
μ
Fig. 9. Fluorescence emission spectra of sensor A1 (20 μM) before and after
1.4
μM
addition of 20 μM of Cys, Hcy, GSH, Gly, Glu, Val, Tyr, Arg, Trp, Lys, His, Leu,
38 nM
14 nM
13.7 nM
4.8 nM
2.0 nM
4.7 nM
Phe, Asp or Met in DMF/H2O (9:1, v/v) with λex of 369 nm.
20 min
35 min
10 min
10 min
5 min
Cys. Performance comparison of typical fluorescent sensors for specific
detection of Cys was summarized in Table 1. Several points are note-
worthy from the data in the table. LOD values of the sensors range from
2.0 nM to 0.657 μM while the response time is in the range of 4 sec–60
time of the reaction between Cys and sensor A1 was prolonged, the
fluorescence intensity increased gradually and much slowly and leveled
off to a saturation value in 5 min. It is concluded that sensor A1 exhibits
extremely rapid response to Cys.
min. Coumarin serves a favorable fluorophore for construction of ul-
trasensitive fluorescent Cys sensors with short response time (e.g. sensor
12, 16–18, 22 and A1). The acrylate group is the most widely employed
reaction functionality for Cys due to its high reactivity and specificity.
Sensors based on the acrylate recognition site (sensor 3–12) show LOD
Titration experiments of Cys were performed to investigate the
sensitivity of sensor A1. The fluorescence emission of sensor A1 solution
from 40 nM to 0.657 μM, inferior to the maleimide-based counterparts
(20 μM in DMF) after adding different concentration (2–20 μM) of Cys
(sensor 22–25 and A1) which exhibit low LOD values of 2.0–38 nM.
Sensor A1 is undoubtedly among the best in respect of its low LOD (4.7
nM), rapid response (several sec‒5 min), extraordinary fluorescence
enhancement (320-fold), and facile synthesis.
was showed in Fig. 7a. And the concentration-dependent responses of
sensor A1 to Cys in DMF were showed in Fig. 7b. With the increment of
Cys concentration, the fluorescence intensity at 502 nm increased
gradually. The fluorescence intensity is linearly proportional to the
concentration of added Cys and the linear fitting equation can be
depicted as Y = 919911 + 194309X with R2 = 0.9921. The limit of
detection (LOD) of A1 for Cys was found to be 4.7 nM, based on the
definition L = 3S/K of IUPAC, where L is the detection limit, S denotes
the standard deviation of fluorescence intensity of blank, and K is slope
of the calibration curve [65].
3.3. Sensing behavior of A1 in DMF/H2O (9:1, v/v)
No substantial UV–vis spectral change was observed after addition of
amino acids including Arg, Asp, Cys, Glu, Gly, GSH, Hcy, His, Leu, Lys,
Met, Phe, Trp, Tyr and Val (20
μ
M) to sensor A1 solution (20
μ
M) in
M) in DMF/
H2O (9:1, v/v) is non-fluorescent and upon addition of Cys (20 M)
To compare the sensing performance of sensor A1 with other re-
ported examples, chemical structures of typical fluorescent sensors for
specific detection of Cys have been outlined in Scheme 2.
DMF/H2O (9:1, v/v) (Fig. S6). Solution of sensor A1 (20 μ
μ
strong bright blue fluorescence was visible under UV irradiation (λ =
365 nm). Adding other amino acids or biothiols (Arg, Asp, Glu, Gly,
GSH, Hcy, His, Leu, Lys, Met, Phe, Trp, Tyr and Val) failed to cause
distinct change in the fluorescence, as showed in Fig. 8. Thus A1 can be
utilized for visual discriminating Cys from Hcy, GSH and other amino
acids under UV irradiation in a partially aqueous solution.
Among the various fluorogenic Cys sensors, the fluorophore mainly
concentrates on coumarin, naphthalimide, rhodamine, BODIPY, and
naphthalene. The sensing mechanisms include Michael addition, con-
jugate addition–cyclization reaction with an acrylate, and cleavage of
sulfonamide. Acrylate and maleimide groups, as typical Michael ac-
ceptors, exhibit superior features and contribute heavily to construction
of highly sensitive fluorescent chemosensors for specific detection of
Changes in the fluorescence (excited at 369 nm) of sensor A1 solu-
tion (20 μM) caused by addition of amino acids Arg, Asp, Cys, Glu, Gly,
Fig. 8. Color of sensor A1 solution (20 μM) in DMF/H2O (9:1, v/v) before and after addition of 1 equivalent of Cys, Hcy, GSH, Gly, Glu, Val, Tyr, Arg, Trp, Lys, His,
Leu, Phe, Asp or Met under UV irradiation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
6

L. Hu et al.
Analytical Biochemistry 620 (2021) 114138
Fig. 10. Fluorescence intensity at 502 nm of A1 (20 μM) upon addition of 20
μ
M of Hcy, GSH, Gly, Glu, Val, Tyr, Arg, Trp, Lys, His, Leu, Phe, Asp or Met
Fig. 13. Linear relationship between the fluorescence intensity at 502 nm and
without (black bars) or with Cys (red bars) in DMF/H2O (9:1, v/v). (For
interpretation of the references to colour in this figure legend, the reader is
referred to the Web version of this article.)
Cys concentration (2–20 μM) in DMF/H2O (9:1, v/v).
Fig. 11. Fluorescence intensity at 502 nm of A1 (20 μM) upon addition of 100
μ
M of Hcy, GSH, Gly, Glu, Val, Tyr, Arg, Trp, Lys, His, Leu, Phe, Asp or Met
Fig. 14. Changes in the fluorescence intensity at 502 nm of A1 (20 μM) upon
without (black bars) or with Cys (red bars) in DMF/H2O (9:1, v/v). (For
interpretation of the references to colour in this figure legend, the reader is
referred to the Web version of this article.)
addition of 1 equivalent of Cys in DMF/H2O (9:1, v/v) with time (λex
=
369 nm).
GSH, Hcy, His, Leu, Lys, Met, Phe, Trp, Tyr or Val (20 μM) in DMF/H2O
(9:1, v/v) were determined as showed in Fig. 9. It was visible that a
broad emission band appeared at around 502 nm upon adding Cys and
the emission intensity was hugely enhanced. Negligible change in
fluorescence emission at 502 nm was observed after addition of other
amino acids including Hcy and GSH. The results demonstrate the high
selectivity of sensor A1 toward Cys over other common competitive
amino acids and biothiols including Hcy, GSH, Gly, Glu, Val, Tyr, Arg,
Trp, Lys, His, Leu, Phe, Asp and Met in a partially aqueous solution
DMF/H2O (9:1, v/v).
To investigate the anti-interference property of sensor A1 for selec-
tive sensing Cys in an aqueous medium, competitive experiments were
conducted in the presence of Cys over other amino acids. Fluorescence
intensity at 502 nm of A1 (20 μM) upon addition of 1 equivalent (20 μM)
of Arg, Asp, Glu, Gly, GSH, Hcy, His, Leu, Lys, Met, Phe, Trp, Tyr or Val
without or with Cys in DMF/H2O (9:1, v/v) were measured as showed in
Fig. 10. The fluorescence intensity of A1 did not change substantially
upon adding competitive analytes including Hcy, GSH and Gly.
Furthermore, fluorescence intensity at 502 nm of A1 (20
addition of 5 equivalent (100
Trp, Lys, His, Leu, Phe, Asp or Met without or with 1 equivalent of Cys in
μ
M) upon
μ
M) of Hcy, GSH, Gly, Glu, Val, Tyr, Arg,
Fig. 12. Fluorescence response of A1 (20
(2–20 M) in DMF/H2O (9:1, v/v).
μM) with increasing amount of Cys
μ
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L. Hu et al.
Analytical Biochemistry 620 (2021) 114138
Fig. 15. (a) Schematic illustration of application of probe A1 in drug analysis. Changes in fluorescence emission intensity at 502 nm (λex = 369 nm) of probe A1 (20
μ
M) upon addition of different concentrations of drug L-CYSTEINE. (b) 5 μM. (c) 10 μM. (d) 15 μM and (e) cell bioimaging with A1 probe, fluorescence image at the
blue channel. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
8

L. Hu et al.
Analytical Biochemistry 620 (2021) 114138
observed in the cells through the blue channel after 30 min incubation.
However, in the control group, we selected a traditional thiol-blocking
reagent called N-ethylmaleimide to pre-treat the cells. The probe A1
Table 2
Quantitative analytical results of Cys concentration in drug Super-Bio L-
CYSTEINE by using sensor A1.
(20 μM in DMSO) was incubated with N-ethylmaleimide (0.1 mM) pre-
Added/
Determined/
μM
Average/
RSD/
%
μM
μM
treated cells and no fluorescence signal could be observed after the
treatment with A1 probe (20 μM in DMSO) for 30 min, validating that
5
5.25
5.12
9.97
14.25
5.37
9.95
14.24
4.87
5.49
5.22
4.4
4.1
5.9
10
15
10.79
14.18
10.32
14.13
11.04
13.82
10.41
14.12
A1 probe is more sensitive to the endogenous Cys in living cells with
satisfactory cell membrane penetration ability.
3.5. Sensing mechanism of A1
DMF/H2O (9:1, v/v) were determined as showed in Fig. 11. Though the
addition of a large amount of competitive amino acids brought about
decrease in the fluorescence intensity, enhancement in the fluorescence
emission of sensor A1 by addition of Cys was still obvious and the
fluorescence turn-on detection of Cys by A1 was not influenced sub-
stantially. It can be concluded that sensor A1 exhibits superior selec-
tivity and anti-interference ability. Therefore A1 may be applicable for
highly selective fluorescence turn-on discriminating Cys from Hcy, GSH
and other common amino acids in a partially aqueous solution.
A reasonable sensing mechanism of sensor A1 for fluorescence
detection of Cys was proposed, as showed in Scheme 3. Sensor A1 has a
3-ethoxycarbonyl-coumarin- 6-maleimide structure with the maleimide
group as the specific reaction site for recognizing Cys. This maleimide
group was linked directly to the coumarin skeleton and triggered an
intramolecular charge transfer (ICT) effect. Sensor A1 showed non-
fluorescence due to a dual-quenching mechanism—ICT and an effi-
cient photoinduced electron transfer (PET) quenching effect involving
the Lowest Unoccupied Molecular Orbital (LUMO) of the maleimide
moiety [56]. Upon Michael addition between Cys and the maleimide
group, this orbital disappeared and the ICT process in the system was
also switched off. Thus fluorescence emission of the system was recov-
ered to generate a fluorescence turn-on response for sensor A1 toward
Cys. This will be a sufficient way for design and development of Cys
sensors applicable in environmental science and life science with high
sensitivity and selectivity.
For investigating the sensitivity and the detection limit of sensor A1
for Cys in an aqueous medium, titration experiments of Cys were per-
formed and the fluorescence spectra of A1 (20
μ
M) in DMF/H2O (9:1, v/
v) upon addition of Cys with different concentration (2–20
μ
M) were
determined (Fig. 12). The corresponding concentration-dependent re-
sponses of sensor A1 to Cys in DMF/H2O (9:1, v/v) were showed in
Fig. 13. It is visible that the fluorescence intensity at 502 nm increased
gradually with the increment of Cys concentration from 2 μM to 20 μM.
The fluorescence intensity is linearly proportional to the concentration
of added Cys and the linear fitting equation can be obtained as Y =
841398 + 179471X with R2 = 0.9946. The LOD of A1 for Cys was
calculated to be 14 nM.
4. Conclusion
In conclusion, a novel fluorescent sensor A1 containing a maleimide
group as the recognition site was developed. It is applicable for fluo-
rescence turn-on detection of Cys in both DMF and DMF/H2O (9:1, v/v)
media. Addition of Cys to A1 solution in DMF induced bright blue
fluorescence and emission intensity enhancement by 320-fold while
other amino acids including Hcy, GSH, Gly, Glu, Val, Tyr, Arg, Trp, Lys,
His, Leu, Phe, Asp and Met did not bring about remarked change. Sensor
A1 responds to Cys in seconds by sharp enhancement in the fluorescence
emission. Immediately after addition of Cys to sensor A1 solution in
DMF the fluorescence intensity increased by 170-fold and attained the
maximum value in 5 min. The detection limit of sensor A1 toward Cys is
4.7 nM in DMF. Quantitative determination of Cys concentration is
available by fluorescence titration experiments on sensor A1 in both
DMF and partially aqueous (DMF/H2O = 9:1, v/v) solution. Sensor A1
responds to Cys in DMF/H2O (9:1, v/v) solution with a response time of
Response time of sensor A1 toward Cys in DMF/H2O (9:1, v/v) was
investigated. The time-dependency of the fluorescence intensity at 502
nm of A1 (20 μM) upon addition of Cys (1 equivalent) in DMF/H2O (9:1,
v/v) was investigated. Changes in the fluorescence intensity at 502 nm
of sensor A1 (20 M) in DMF/H2O (9:1, v/v) upon addition of 1
μ
equivalent of Cys with time were recorded and showed in Fig. 14. From
the results it is found that upon addition of Cys the fluorescence intensity
was enhanced sharply by ca. 180-fold in 1 min then the increase in the
fluorescence intensity became slow. The fluorescence intensity
approached to a saturation value in 7 min. The results reveal the rapid
response of sensor A1 to Cys in a partially aqueous medium.
3.4. Application of A1 in drug analysis and bioimaging
It has been ascertained that the emission of sensor A1 at 502 nm
increased gradually with the increment of Cys concentration and the
fluorescence intensity is linearly proportional to the concentration of
added Cys within a range of 2–20 μM. Therefore the potential of probe
A1 for quantitative analysis of Cys content in drugs containing Cys was
explored. Changes in fluorescence emission intensity at 502 nm (λex
369 nm) of probe A1 (20 M) upon addition of the drug Super-Bio L-
CYSTEINE containing different concentrations of Cys (5 M, 10 M or
15 M) were measured and showed in Fig. 15a,b,c,d. The analytical
=
μ
μ
μ
μ
results are listed in Table 2. It can be seen that reliable data have been
obtained and the relative standard deviation (RSD) of this fluorescence
method is less than 6%. These test results demonstrate that the designed
sensor A1 is applicable for the quantitative analysis of Cys in real sam-
ples like drugs.
As mentioned above, the as-synthesized probe A1 shows good
applicability for quantitative analysis of Cys in drugs, which drives us to
explore further potential utilization of A1 probe in detecting Cys in
living cells. The bioimaging experiments were conducted in living Hela
cells under confocal fluorescence microscope. Firstly, Hela cells were
cultured with A1 probe at a concentration of 20
μM in DMSO. As showed
Scheme 3. Plausible sensing mechanism of sensor A1 towards Cys.
in Fig. 15e, there is a stronger fluorescence signal which could be
9

L. Hu et al.
Analytical Biochemistry 620 (2021) 114138
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Authors’ contributions
Conceiving and designing of the experiments: Y.Song, H.Li and Y.
Sun; Performing research and analyzing the data: L.Hu, T.Zheng, J.Fan
and R.Zhang; Writing of the paper: L.Hu, T.Zheng and H.Li.
All authors read and approved the final manuscript.
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Appendix A. Supplementary data
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