A simple and sensitive fluorescent probe for specific detection of cysteine

Article abstract of DOI:10.1007/s12039-017-1326-y

Abstract: A fluorescent probe, with simplicity of structure and convenience of synthesis, is capable of detecting cysteine over other biothiols, such as homocysteine and glutathione. The detection limit of 60?nM and 190-fold increase in fluorescence intensity offer advantage for detection of Cys in biological systems. Furthermore, the probe 1 could be applied in bioimaging. Graphical abstract: We designed and synthesized the fluorescent probe 1, which displayed a highly selective and sensitive response to Cys over Hcy/GSH. The detection limit of 60?nM and 190-fold increase in fluorescence intensity offer advantage for detection of Cys in biological systems. Furthermore, the probe 1 could be applied in bioimaging. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s12039-017-1326-y

J. Chem. Sci.
© Indian Academy of Sciences
DOI 10.1007/s12039-017-1326-y
RAPID COMMUNICATION
A simple and sensitive fluorescent probe for specific detection
of cysteine
YAN-FEI KANG∗, HAI-XIA QIAO, YA-LI MENG, ZHEN-HUI XIN, LI-PING GE,
JIN-NAN ZHENG and YI-NA WANG
College of Laboratory Medicine, Hebei North University, 11 Diamond Street South, Zhangjiakou 075000,
Hebei Province, People’s Republic of China
E-mail: kangyanfei172@163.com
MS received 7 May 2017; revised 1 June 2017; accepted 13 June 2017
Abstract. A fluorescent probe, with simplicity of structure and convenience of synthesis, is capable of detecting
cysteine over other biothiols, such as homocysteine and glutathione. The detection limit of 60nM and 190-fold
increase in fluorescence intensity offer advantage for detection of Cys in biological systems. Furthermore, the
probe 1 could be applied in bioimaging.
Keywords. Fluorescence probe; cysteine; selectivity; bioimaging.
1. Introduction
addition-cyclization, cleavage reaction by thiols, thiol-
halogen nucleophilic substitution, disulfide exchange,
and others.4,11–16 However, selective detection of Cys
is difficult in view of structural similarity of Cys, Hcy
and GSH. Thus, development of probes to selectively
detect Cys also remains challenging for the scientific
community.
Among these approaches, the selective detection of
Cys based on the conjugate addition-cyclization of Cys
withacrylateshasproventobespecificallyeffective, this
strategy has attracted broad attention.17–24 Moreover,
coumarin, a well-known fluorophore, exhibits low cyto-
toxicity and possesses favorable photophysical proper-
ties such as a large Stokes shift, visible excitation and
emission wavelengths, good photostability, reasonable
fluorescent quantum yield and easy synthesis.25–27 Thus,
in this work, we designed and synthesized fluorescent
probe 1 (ethyl 7-(acryloyloxy)-2-oxo-2H-chromene-3-
carboxylate) with a latent coumarin fluorophore and a
receptorofacrylate. Weenvisionthattheprobe 1iscapa-
ble of detecting selectively Cys over Hcy and GSH.
Bio-thiols such as cysteine (Cys), homocysteine (Hcy),
and glutathione (GSH) play pivotal roles in various
biological processes including protein synthesis, detox-
ification, metabolism, signal transduction and gene
regulation.1,2 In addition, these bio-thiols are essen-
tial in regulating of the redox state of proteins and in
protein structure. More importantly, these species are
involved in many crucial functions, for example, cys-
teine (Cys) and homocysteine (Hcy) are related to vari-
ety of human diseases like neurotoxicity, skin lesions,
edema, muscle weakness, lethargy, slow growth, hair
depigmentation,3 cardiovascular4 and Alzheimer’s dis-
eases.5 Similarly, glutathione (GSH) is the most abun-
dant intracellular non-protein thiol and is considered
to be an indicator of oxidative stress.6,7 As a conse-
quence, detection of these low molecular weight thiols
is important and timely. Therefore, a great many of
detection methods were developed, but the fluores-
cence probe detection obtained considerable attention
on the basis of its simplicity, efficiency, low detection
limits and ease of handling.8–10 Up to now, the fluo-
rescence probes were designed and synthesized mainly
according to the different chemical reaction mecha-
nisms, such as Michael addition, cyclization, conjugate
2. Experimental
Unless otherwise noted, materials were purchased from
commercial suppliers and used without further purifica-
tion. All the solvents were purified and dried according to
*
For correspondence
Electronic supplementary material: The online version of this article (doi:10.1007/s12039-017-1326-y) contains supplementary material,
which is available to authorized users.

Yan-Fei Kang et al.
Scheme 1. Synthesis of probe 1.
Figure 1. (a) With addition of Cys (100 µM), UV-Vis absorption spectra of probe 1
(10 µM) with increase of incubation time from 0 to 15 min. (b) Fluorescence spectra of
probe 1 (10µM) in the presence of various analytes (100 µM) in an aqueous PBS buffer (pH
7.4, containing 20% acetonitrile); λex = 406 nm. Inset shows a picture of probe 1 without
and with Cys under ultraviolet radiation.
general methods. Synthesis and Characterization of probe an excitation at 406 nm. However, other amino acids did
1, and other experimental details are given in Supporting
Information.
not cause distinct change of fluorescence intensity; Hcy
and GSH showed slight interference, however. There-
fore, the probe 1 could selectively detect Cys level.
In order to explore the effects of other analytes for
detection of Cys, we carried out the competiton experi-
ment (Figure S1 in Supplementary Information). The
3. Results and Discussion
Keeping the following considerations in mind, namely, results indicated that fluorescence intensity of probe
acrylates can recognize Cys based on conjugate 1 for Cys did not change clearly in the presence of
addition-cyclization and coumarin is a well-known flu- other amino acids or metal ions, even Hcy and GSH.
orophore, probe 1 was synthesized (Scheme 1, for Moreover, there is only 20% decrease in fluorescence
details and characterization see the Supporting Infor- intensity of probe 1 toward Cys in the presence of GSH
mation) and structure confirmed by NMR and HRMS /Hcy (200 µM). Thus, these experimental results fur-
(ESI). Initially, weexploredtheUV-Visabsorptionspec- ther showed that the probe 1 could distinguish Cys
trum of probe 1 (10 µM) in phosphate buffer (PBS pH from other amino acids. In addition, the fluorescence
7.4, containing 20% acetonitrile). Probe 1 had obvi- intensity was enhanced gradually with the increase of
ous absorption peaks at 300 and 335 nm. However, Cys content from 1 equiv. to 10 equiv. (Figure 2a).
with addition of Cys (100 µM), the absorption bands Subsequently, we further investigated the properties of
at 300 and 335 gradually decreased with concomitant the probe 1. To investigate the fluorescence spectral
growth of a new band at 406 nm (Figure 1a). Thus, 406 response of probe 1 for Cys, we extended the action
nm was identified as excitation spectrum wavelength. time of Cys to 20 min. The result displayed that the
Subsequently, the fluorescence intensity of probe1 was increase of fluorescence intensity reached the plateau
screened with the different amino acids (L-Cys, Hcy, stage after 10 min (Figure 2b). Thus, this response is
GSH, Arg, Asp, Tyr, Pro, Lys, Gly, Ala, Val, Phe, Thr, faster than those reported previously.24 Furthermore,
DL-Met, His, Ser, Leu, Trp, Ile, L-Met, Glu, at100 µM)in the fluorescence intensity of probe1 toward Hcy/GSH
phosphate buffer (PBS pH 7.4, containing 20% acetoni- increased with extension of response time. However,
trile) (Figure 1b). Interestingly, a remarkable 190-fold this enhancement is slower and weaker than of probe1
enhancement in fluorescence intensity was observed toward Cys. Meanwhile, the probe 1 did not induce any
with the addition of Cys and the solution, after 5 min obvious change by itself, which meant the probe 1 was
incubation time, showed strong blue fluorescence with stable under this reaction condition. On the same basis,

Fluorescent probe for detection of cysteine
Figure 2. (a) Emission spectra of probe 1 (10 µM) with increase of Cys concentration
(from 0 to 10 equiv.) after incubation of 5 min at room temperature. (b) Time-dependent
fluorescence intensity of probe 1 (10 µM) in the presence and absence of Cys (10 equiv.),
λex = 406 nm.
Scheme 2. Proposed reaction mechanism of probe 1 with Cys.
the detection limit (3 s/m, n = 20) of Cys was evaluated first-order rate constant kꢀ at room temperature. The
to be 60 nM (Figure S2 in Supplementary Informa- pseudo-first-order rate constants for Cys, Hcy, and GSH
tion).28,29 More importantly, the fluorescence intensity were measured as kꢀ = 1.2x10−2 s−1, 1.0x10−3 s−1,
of probe 1 was weak in pH 3.0–9.0; however, with addi- 1.5x10−3 s−1, respectively (Figure S5 in Supplemen-
tion of Cys, the fluorescence intensity was enhanced tary Information). Thus, Cys induced a rapid cyclization
obviously in pH 6.0-9.0. Thus, probe 1 is capable of reaction of a stable seven-membered ring, which is
detecting selectively Cys level under the conditions of kinetically favorable. This difference of reaction rate
physiological pH (Figure S3 in Supplementary Infor- made that the probe 1 could selectively and quickly
mation).
detect Cys over Hcy/GSH.
We further investigated the reaction mechanism
Lastly, we studied the application of probe 1 in live-
between probe 1 and Cys. Cys (100 µM) was added cell imaging. Before the cell imaging was investigated,
to the solution of probe 1 (10 µM), and we completed we first measured the cytotoxic activity of probe 1 by
high resolution mass spectrometry analysis of the mix- MTT method.30 Interestingly, the results displayed that
ture. The peak at m/z = 257.0421, corresponding to the probe 1 (20 µM) did not induce apparent cell death
product 2 (M+Na) was clearly discovered (Figure S4 within 24 h of incubation time (Figure 6 in Supple-
in Supplementary Information). Therefore, we propose mentary Information). Subsequently, we recorded the
a probable reaction mechanism (Scheme 2). The initial bioimaging of probe 1 (Figure 3). When the HeLa
conjugate addition of Cys to acrylate moiety and subse- cells were incubated with probe 1 (10 µM) for 1 h,
quent intramolecular cyclization afforded the final flu- they became blue fluorescent. More importantly, when
orescent compound, which caused increase of fluores- the cells were pretreated with thiol scavenger (N-ethyl
cence intensity. Meanwhile, we measured the pseudo- maleimide, NEM, 1 mM) for 30 min, the blue fluores-

Yan-Fei Kang et al.
Figure 3. Bioimaging of HeLa cells. (A) Blank; (B) Cells after incubation with DMSO for 1 h; (C, D)
Cells after incubation with probe 1 (10 µM) for 1h; (E, F) cells which were pre-incubated with NEM (1mM)
for 30 min. λex = 406 nm.
cence did not appear with cells incubated by probe 1. 1621122H), and Guide Projects of Department of Education
of Hebei Province (No. Z2017026).
Therefore, probe 1 can be applied for sensing Cys in
living cells.
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Supplementary Information (SI)
All additional information pertaining to the characteri-
zation of compounds using 1H NMR, 13C MNR spectra,
and HRMS, and experimental methods are given in the
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