A simple probe with visible color change for selective detection of cysteine

Article abstract of DOI:10.1080/00387010.2020.1821063

In this work, we synthesized a simple fluorescent probe, which can selectively detect cysteine. With addition of cysteine, the probe solution showed marked color change from pale yellow to orange color by naked eye. There was an excellent linear relations

Full text of DOI:10.1080/00387010.2020.1821063

Spectroscopy Letters
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A simple probe with visible color change for
selective detection of cysteine
Hai-Feng Yin , Meng-Jiao Gao , Wen-Jing Jiang , Yu-Han Gan , Chen Li , Yan-Fei
Kang , Ya-Li Meng & Zhen-Hui Xin
To cite this article: Hai-Feng Yin , Meng-Jiao Gao , Wen-Jing Jiang , Yu-Han Gan , Chen Li ,
Yan-Fei Kang , Ya-Li Meng & Zhen-Hui Xin (2020): A simple probe with visible color change for
selective detection of cysteine, Spectroscopy Letters, DOI: 10.1080/00387010.2020.1821063
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SPECTROSCOPY LETTERS
https://doi.org/10.1080/00387010.2020.1821063
A simple probe with visible color change for selective detection of cysteine
Hai-Feng Yina, Meng-Jiao Gaob, Wen-Jing Jiangb, Yu-Han Ganb, Chen Lib, Yan-Fei Kangb, Ya-Li Mengb and
Zhen-Hui Xinb
b
aCollege of Basic Medicine, Hebei North University, Zhangjiakou, People’s Republic of China; Hebei Key Laboratory of Quality &
Safety Analysis-Testing for Agro-Products and Food, Zhang Jiakou Key Laboratory of Organic Light Functional Materialsnd College of
Laboratory Medicine, Hebei North University, Zhangjiakou, People’s Republic of China
ABSTRACT
ARTICLE HISTORY
Received 30 July 2020
Accepted 4 September 2020
In this work, we synthesized a simple fluorescent probe, which can selectively detect cyst-
eine. With addition of cysteine, the probe solution showed marked color change from pale
yellow to orange color by naked eye. There was an excellent linear relationship between
fluorescence intensity of probe and cysteine concentration. Moreover, the probe displayed
satisfactory detection limit. Low toxicity and cellular imaging offered the important condi-
tions for detecting cysteine in biological system.
KEYWORDS
Bioimaging; cysteine;
fluorescence; probe;
selectivity
Introduction
detection of Cys in biological samples by con-
venient method is of great significance.
Biothiols, such as glutathione (GSH) and homo-
cysteine (Hcy), as well as cysteine (Cys) play crit-
ical role in maintaining redox homeostasis and
the regulation of important cellular processes.[1,2]
GSH is the most abundant reductant in the bio-
logical systems. GSH insufficiency can result in
oxidative stress in cells, which is linked to neuro
degenerative disorders. In addition, abnormal
level of GSH and Hcy are also related to cardio-
vascular disease, cancers and AIDS.[3,4] Among
them, Cys, as an important amino acid, partici-
pates in many enzymatic reactions, regulates a lot
of important cellular processes, and plays a crit-
ical role in the cellular antioxidant system.[5,6]
Furthermore, the abnormal level of Cys may
cause many human diseases. For example, ele-
vated levels of Cys are closely related to cardio-
vascular complications, Parkinson’s disease, and
Alzheimer’s disease, while low levels of Cys are
associated with slow growth rate, hair depigmen-
tation, muscle and fat loss, liver damage, skin
lesions, weakness.[7–11] Therefore, selective
Among the various detection methods, the
fluorescent probe detection has attracted much
attention due to its high sensitivity and selectiv-
ity, and ease of handling.[12–14] With the efforts
of researchers, many fluorescence probes have
been developed for detection of biothiols. The
reaction mechanisms included Michael addition,
nucleophilic substitution, cyclization reaction,
conjugate addition, disulfide exchange, and
others.[15–25] However, it is difficult to distinguish
Cys from Hcy and GSH because they have the
same recognition site and similar chemical reac-
tion activity. Although many fluorescence probes
toward Cys were reported,[26–32] they more or
less had some limitations in the practical applica-
tion including low selectivity, poor sensitivity, use
of surfactant, undesired spectra overlap.
Moreover, shorter excitation and emission spectra
all resulted in interferences for detection of Cys
in biological systems. Therefore, development of
fluorescence probes for efficient detection of Cys
is urgently needed.
CONTACT Ya-Li Meng
limengyajiajia@163.com; Zhen-Hui Xin 331747894@qq.com
Hebei Key Laboratory of Quality & Safety Analysis-Testing for
Agro-Products and Food, Zhang Jiakou Key Laboratory of Organic Light Functional Materialsnd College of Laboratory Medicine, Hebei North University,
Zhangjiakou, People’s Republic of China.
Supplemental data for this article can be accessed here.
ß 2020 Taylor & Francis Group, LLC

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H.-F. YIN ET AL.
Scheme 1. Synthesis of probe 1.
Acrylate, as the recognition site, can selectively
recognize Cys over Hcy and GSH by addition-
cyclization mechanism. Therefore, in this work,
we designed and synthesized the fluorescent
Suppementary Material) and confirmed by NMR
and HRMS (ESI). Keeping the probe 1 in hand,
next we explored detailedly the optical properties
of probe 1. Firstly, we measured the absorption
spectra of the probe 1 in presence or not of Cys
to determine the excited wavelength (Fig. S1).
The result showed that the probe 1 had strong
absorption at 420 nm with the addition of Cys.
Subsequently, we investigated the influence of
solvent type (Fig. S2) and content (Fig. S3) on
the fluorescence intensity of free probe 1 and
probe 1 toward Cys. On the basis of fluorescence
intensity (I) and relative value (I/I0), we selected
the aqueous solution (pH 7.4 PBS, containing
20% acetonitrile) as the spectral response.
Subsequently, we surveyed the fluorescence
spectrum response of probe 1 (20 mM) in the
presence of different amino acids (L-Cys, alanine
(Ala), arginine (Arg), Aspartate (Asp), methio-
nine (DL-Met), Glutamate (Glu), glycine (Gly),
histidine (His), isoleucine (Ile), methionine (L-
Met), leucine (Leu), lysine (Lys), N-acetylcysteine
(NAC), phenylalanine (Phe), proline (Pro), serine
(Ser), threonine (Thr), tryptophan (Trp), valine
(Val), Homocysteine (Hcy), 200 mM; glutathione
(GSH), 1 mM) with incubation for 10 min at
37 ꢀC (Fig. 1a). The results displayed the fluores-
cence intensity of probe solution was clearly
strengthened at 580 nm with the addition of Cys.
However, other analytes didn’t cause obvious
change of fluorescence intensity. Although Hcy
induced the slight enhancement of fluorescence
intensity, which was negligible compared with
the fluorescence intensity increasement caused by
Cys. More importantly, 1 mM GSH also didn’t
change distinctly the fluorescence intensity. The
probe showed a large Stokes shift (160 nm),
which greatly reduces the interference of absorp-
tion spectrum to fluorescence intensity. Thus,
probe
1
((E)-4-(2-(3-(dicyanomethylene)-5,5-
dimethylcyclohex-1-en-1-yl)vinyl)phenyl acrylate)
for selective detection of Cys based on acrylate as
the recognition site. Firstly, the probe showed a
large Stokes shift (160 nm), which greatly reduces
the interference of absorption spectrum to fluor-
escence intensity. In addition, larger emission
wavelength (580 nm) reduces background inter-
ference of biological system. Meanwhile, the
results showed that the probe 1 was able to deter-
mine selectively Cys over Hcy and GSH, and
accompanied by color change by the naked eye,
which facilitated the Cys identification. Low tox-
icity and cellular imaging offered the potential
conditions for detecting cysteine in bio-
logical system.
Experimental
1H/13C NMR spectra were recorded on a Bruker
AVIII-400/600 MHz spectrometer. High reso-
lution mass spectra (HRMS) were measured with
Thermo (orbitrap Elite). Absorption spectra were
measured using a Thermo (BioMate 3S) UV/Vis
spectrophotometer. Fluorescence measurements
were carried out with a F97pro fluorospectropho-
tometer. Unless otherwise noted, materials were
purchased from commercial suppliers and used
without further purification. All the solvents were
purified and dried according to general methods.
Results and discussion
The probe 1 was synthesized (Scheme 1, for
details and characterization see the the

SPECTROSCOPY LETTERS
3
Figure 1. (a) Fluorescence spectra of probe 1 (20 mM) in the presence of various analytes in an aqueous PBS buffer (pH 7.4, con-
taining 20% acetonitrile), kex ¼ 420 nm; inset: color change of probe solution and probe toward Cys by naked eye. (b)
Fluorescence intensity of probe 1 (20mM) with various analytes (200 mM) in an aqueous PBS buffer (pH 7.4, containing 20% aceto-
nitrile), kex ¼ 420 nm. Black bar: probe 1 þ analytes; Gray bar: probe 1 þ Cys þ analytes.
Figure 2. (a) Time-dependent fluorescence spectra of probe 1 (20 mM) in the presence of Cys/Hcy/GSH. (b) Fluorescence spectra
response with increase of Cys concentration (from 0 to 10 equiv.) over probe 1 (20 mM) within incubation of 10 min at 37 ꢀC; inset:
Cys concentration-dependent change of fluorescence intensity.
probe 1 can detect selectively Cys. In addition,
with the addition of Cys, the solution of probe 1
changed from pale yellow to orange color by
naked eye, which provided the advantageous and
convenient condition for practical application.
Encouraged by the above results, we carried out
the competition experiment to investigate the
interferences of other concomitant analytes for
probe 1 toward Cys (Fig. 1b). The results showed
the existence of other analytes didn’t change obvi-
ously fluorescence intensity of probe 1 toward Cys,
even including GSH and Hcy. In addition, we fur-
ther lengthened the reaction time of probe 1 for
Cys/GSH/Hcy (Fig. 2a). The fluorescence intensity
increased gradually in presence of Cys and reached
the plateau within 15 min, which is faster than that
of previous reports.[33–35] Moreover, the fluores-
cence intensity enhanced slowly with increasement
of GSH/Hcy incubation time, but the fluorescence
intensity is far less than that of Cys. Therefore, the
probe 1 is able to detect selectively Cys in view of
above results.
We further explored the relationship between
fluorescence intensity and Cys concentration by
titration experiment (Fig. 2b). Results displayed
the fluorescence intensity firstly heightened and
arrived at saturation state with increase of Cys
concentration from 0 to 200 mM. Meanwhile,
there was excellent linear relation (R2 ¼ 0.9963)
between fluorescence intensity and Cys

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H.-F. YIN ET AL.
Figure 3. (a) pH-Dependent fluorescent intensity changes of probe 1 and probe 1 toward Cys at room temperature, kex ¼
420 nm. (b) Concentration-dependent fluorescence intensity response of probe 1 for Cys in an aqueous PBS buffer (pH 7.4, contain-
ing 20% acetonitrile) kex ¼ 420 nm.
Figure 4. Cell imaging of probe 1. (A) Hela cells; (B) Hela cells were incubated with probe 1 (20mM) for 30 min; (C) Hela cells
were pre-incubated with Cys (100 mM) for 30 min and then incubated with probe 1 (20 mM) for another 30 min; (D) Hela cells were
pre-incubated with NEM (1 mM) for 30 min and then incubated with probe 1 (20mM) for another 30 min. (kex ¼ 400–460 nm,
kem ¼ 590 nm).
concentration from 0 to 60 mM, which offered
possibility to analyze quantitatively the Cys con-
tent. On this same basis, the detection limit (Fig.
3b, 3s/m, n ¼ 20) of Cys was evaluated to be
0.17 mM, which further guaranteed the practical
application of the probe 1 in the biological sys-
tem. Therefore, it is possible that probe 1 can
detect Cys in biological systems.
Subsequently, we discussed the effect of pH
value for fluorescence intensity (Fig. 3a). The
result indicated the fluorescence intensity change
of probe 1 toward Cys is obvious in pH 6.0–9.0,
which is critical to the application of probe under
the condition of physiological pH value. More
interestingly, the fluorescence intensity of probe
didn’t present significant changes in pH 3.0–10.0,

SPECTROSCOPY LETTERS
5
Funding
which meant the probe 1 was stable adequately
for application in biological systems.
This work was supported by the Key Projects of Hebei
Educational Committee [No. ZD2020148], Hebei North
University Fund [No. YB2018002; No. QN2018008; No.
QN2018012; JYT2019011], Hebei Province Three Three
Talent Project [No. A201901062], Youth Foundation of
Hebei Educational Committee [QN2020402].
We also investigated the reaction mechanism
of probe 1 and Cys (Scheme S1). The reaction
mechanism between probe 1 and Cys undergo
conjugate addition and subsequent intramolecular
cyclization
two-step reaction.[36–39]
which
afforded the compound 2, corresponding mass
spectrum was observed (Fig. S4). Reaction of
probe 1 and Cys formed seven numbered rings,
which is more preponderant than that of Hcy
and GSH in thermodynamics. Therefore, advan-
tage of reaction rate played important role for
selective detection of Cys.
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Disclosure statement
The authors have declared no conflicting interests.

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