A highly sensitive near-infrared fluorescent probe for cysteine and homocysteine in living cells

Article abstract of DOI:10.1039/c3cc45519j

A near-infrared fluorescent probe (Cy-O-CHO) for the detection of endogenous Cys/Hcy in living cells was designed and synthesized. Cy-O-CHO exhibited high sensitivity and good selectivity to Cys/Hcy under physiological conditions with a detection limit of 7.9 nM for Cys.

Full text of DOI:10.1039/c3cc45519j

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
A highly sensitive near-infrared fluorescent probe for
cysteine and homocysteine in living cells†
Cite this: Chem. Commun., 2013,
49, 9176
Fanpeng Kong, Renpu Liu, Ranran Chu, Xu Wang, Kehua Xu* and Bo Tang*
Received 21st July 2013,
Accepted 7th August 2013
DOI: 10.1039/c3cc45519j
www.rsc.org/chemcomm
A near-infrared fluorescent probe (Cy–O–CHO) for the detection of alkaline conditions (pH 9.5). Subsequently, based on the same
endogenous Cys/Hcy in living cells was designed and synthesized. principle, some probes for Cys/Hcy under physiological pH
Cy–O–CHO exhibited high sensitivity and good selectivity to conditions16 were developed. Unfortunately, most of them
Cys/Hcy under physiological conditions with a detection limit of operated in pure organic solvents or solutions with a high level
7.9 nM for Cys.
of organic solvents, such as DMF,17 DMSO,18 acetonitrile,19 and
ethanol.20 Although a few probes have been reported and
Cysteine (Cys) and homocysteine (Hcy) are considered to be successfully applied to intracellular Cys/Hcy imaging,21–23 the
related to a wide range of physiological processes.1–3 The detection wavelengths of these probes are located in the visible
deficiency of Cys can cause slowed growth, hair depigmentation, range, in which the detection would be interfered with cell
edema, lethargy, liver damage, muscle and fat loss, skin lesions, and autofluorescence. In a word, all of the abovementioned fluorescent
weakness.4 Elevated Hcy in the blood is a well-known risk factor for recognitions of Cys/Hcy were realized under non-physiological
a number of diseases, such as Alzheimer’s,5 cardiovascular diseases6 conditions or performed in the visible wavelength range, which
and osteoporosis.7 However, the mechanisms underlying their made them not ideal candidates for cell-imaging applications.
deleterious influences have not yet been elucidated8 due to lack of Besides, these probes are also not suitable for deep tissue imaging.
an available method to measure Cys and Hcy quantitatively in living
cells, especially in tissues.
As it is known, near-infrared (NIR) dyes, wavelengths of
which are located in the 650–900 nm range, have the unique
In recent years, a wide variety of sensing mechanisms have advantage of tracing molecular activity in vivo because their NIR
been used in the design of Cys/Hcy fluorescent probes, such as photons can penetrate relatively deeply into tissues with a low
Michael addition,9 cleavage of sulfonamide and sulfonate auto-fluorescence background. Moreover, the NIR radiation
ester,10 metal complexes-displace coordination,11 and cleavage shows less damage to biological samples.24 Therefore, it is vital
of disulfide.12 However, these methods were usually hampered to develop a highly sensitive NIR fluorescent probe for detecting
by interference from structurally related thiols, especially Cys/Hcy under physiological conditions. In 2012, Park and Yoon’s
glutathione (GSH), which is the most abundant intracellular group25 reported a ratiometric fluorescent probe CyAC for detecting
nonprotein thiol.13
Cys, and the probe has been successfully applied to image Cys in
In order to develop highly selective methods for Cys/Hcy, living cells. With the addition of Cys, the fluorescence intensity at
Strongin and coworkers14,15 first constructed two xanthene- 780 nm (lex = 720 nm) decreased significantly. In contrast, a new
based fluorescent probes for selective detection of Cys and fluorescence emission peak at 570 nm (lex = 520 nm) was observed,
Hcy based on the cyclization reaction of aldehydes with Cys/ which corresponds to the reaction product CyAK. And in their cell
Hcy. However, these two probes exhibited fluorescence quenching imaging experiments, the excitation and emission wavelengths of
towards Cys/Hcy, and all experiments were carried out under detection were 560 nm and 590 nm, respectively.
To date, there is no genuine NIR probe for direct detection
of endogenous Cys/Hcy in vivo. Herein, a highly sensitive
College of Chemistry, Chemical Engineering and Materials Science,
Cy7-based probe (Cy–O–CHO) for the detection of endogenous
Cys/Hcy was designed and synthesized based on the unique
cyclization between Cys (or Hcy) and aldehydes. Heptamethine
cyanine dye (Cy) with a high molar absorption coefficient and
NIR emission was selected as the fluorophore. Cy–O–CHO was
prepared by the reaction of Cy.7.Cl with 4-hydroxybenzaldehyde
in the presence of sodium hydride and purified using standard
Engineering Research Center of Pesticide and Medicine Intermediate Clean Production,
Ministry of Education, Key Laboratory of Molecular and Nano Probes,
Ministry of Education, Shandong Normal University, Jinan 250014, P.R. China.
E-mail: tangb@sdnu.edu.cn; Fax: +86-0531-86180017; Tel: +86-0531-86180010
† Electronic supplementary information (ESI) available: Experimental proce-
dures, synthesis of the probe, HR-MS, 1H NMR and 13C NMR data, optimum
experiments, photo-bleaching experiments and MTT assay. See DOI: 10.1039/
c3cc45519j
c
9176 Chem. Commun., 2013, 49, 9176--9178
This journal is The Royal Society of Chemistry 2013

View Article Online
Communication
ChemComm
Scheme 1 Synthesis of Cy–O–CHO.
column chromatography (Scheme 1). The structure of Cy–O–
CHO was fully characterized by HR-MS, 1H NMR, 13C NMR and
IR (ESI,† Fig. S7a–d).
Upon excitation at 730 nm, Cy–O–CHO displayed faint
fluorescence at 778 nm and a low fluorescence quantum yield
(FF) of 0.036 in PBS buffer (40 mM, pH 7.4). Fig. 1a showed the
fluorescence responses of Cy–O–CHO to different concentrations
of Cys. The emission intensities at 778 nm increased significantly
with the increasing concentration of Cys. A marked enhancement
in the fluorescence quantum yield (from 0.036 to 0.089) upon
addition of Cys to Cy–O–CHO was observed. These results should
be ascribed to the Cys-triggered conversion of the aldehyde group
to the thiazoline derivative, which resulted in the fluorescence
recovery of the cyanine dye.
When more than 2.0 mM (1 equiv.) of Cys was added, the
enhancement of the fluorescence intensity reached a maximum
without further variation (Fig. 1b), which indicated that Cy–O–
CHO could detect Cys quantitatively. The emission intensity of
778 nm showed a good linear relationship with Cys concentrations
(0.03 to 2.0 mM). The regression equation was F = 1043.59 + 101.83 Â
[Cys] (10À7 M) with a linear coefficient of 0.9941 and a detection
limit of 7.9 Â 10À9 M, respectively. To the best of our knowledge,
the detection limit is lower than those of the formerly reported
fluorescence probes (ESI,† Table S1).
To validate the recognition mechanism of Cy–O–CHO to Cys,
high resolution mass spectrometry experiments have been
carried out (Fig. 2). The probe showed a characteristic peak of
m/z at 597.3464. For the mixture of Cy–O–CHO and Cys, a new
peak of m/z at 750.3573 was observed, which corresponded to
the cyclization product (Cy–O–Th). The result confirmed the
sensing mechanism of the probe toward Cys, which resulted in
the formation of the thiazoline derivative. The recognition
mechanism toward Hcy was also confirmed by the HR-MS
method (ESI,† Fig. S3).
Fig. 2 Proposed recognition mechanism of Cy–O–CHO toward Cys.
The selectivity of Cy–O–CHO toward Cys/Hcy was testified.
As can be seen in Fig. 3, no visible changes in the spectra were
observed upon the addition of 20 equiv. of other amino acids.
Notably, 10 mM of GSH did not interfere with the fluorescence
(Fig. 3a). The results verified that Cy–O–CHO was highly
selective toward Cys/Hcy over GSH and other amino acids.
Considering the possible interactions of metal ions, the
responses of Cy–O–CHO to a series of metal ions were also
tested. Fig. 3b showed that no significant changes in the
fluorescence were observed in the presence of metal ions, such
as K+, Zn2+, and Cu2+ (400 mM, 200 equiv.); Cd2+, Mn2+, Ca2+, and
Al3+ (800 mM, 400 equiv.); Mg2+ (2 mM, 1000 equiv); Na+ (3.2 mM,
1600 equiv.). Therefore, Cy–O–CHO could be used as a highly
selective NIR fluorescent sensor for Cys/Hcy.
To access the feasibility of Cys/Hcy detection in living cells,
the confocal imaging of intracellular Cys/Hcy was performed. In
previous reports, additional stimulation25 or exogenous Cys26
was necessary for cell imaging because of the low sensitivity of
probes. In the present study, Cy–O–CHO with the detection limit
of 7.9 nM was successfully used for bioimaging of endogenous
Cys/Hcy in living cells. In order to prove that the probe was
specific to the intracellular Cys/Hcy, a control experiment was
Fig. 3 Fluorescence responses of Cy–O–CHO (2.0 mM) to various analytes in PBS
(pH 7.4, 40 mM). Light gray bars represent the addition of analytes, black bars
represent the subsequent addition of 2.0 mM Cys to the mixture. (a) Relative
fluorescence intensity (F À F0) of Cy–O–CHO upon addition of 20 equiv. of various
amino acids, 5000 equiv. of GSH or 1 equiv. of H2S and Na2SO3. (b) Relative
fluorescence intensity (F À F0) of Cy–O–CHO upon addition of K+ (400 mM), Na+
(3200 mM), Ca2+ (800 mM), Mg2+ (2000 mM), Cu2+ (400 mM), Zn2+ (400 mM), Al3+
(800 mM), Cd2+ (800 mM), and Mn2+ (800 mM). The data were acquired 2 h after
Fig. 1 (a) Fluorescence responses of 2 mM Cy–O–CHO to different concentra-
tions of Cys (0, 0.03, 0.06, 0.1, 0.2, 0.4, 0.8, 1.2, 1.5, 2.0 mM). The curves were
acquired 2 h after the addition of Cys in 40 mM PBS buffer (pH 7.4) at 37 1C.
(b) Response of the probe fluorescence intensity to Cys concentration. The curve
was plotted with the fluorescence intensity at 778 nm with Cys concentration.
Inset: a linear correlation between emission intensities at 778 nm and concen-
trations of Cys (lex = 730 nm).
the addition of the analytes in 40 mM PBS buffer (pH 7.4) at 37 1C (lex/lem
=
730/778 nm).
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 9176--9178 9177

View Article Online
ChemComm
Communication
3 R. O. Ball, G. Courtney-Martin and P. B. Pencharz, J. Nutr., 2006,
136, 1682S.
4 S. Shahrokhian, Anal. Chem., 2001, 73, 5972.
5 S. Seshadri, A. Beiser, J. Selhub, P. F. Jacques, I. H. Rosenberg,
R. B. D’Agostino, P. W. F. Wilson and P. A. Wolf, New Engl. J. Med.,
2002, 346, 476.
6 S. R. Lentz and W. G. Haynes, Cleveland Clin. J. Med., 2004, 71, 729.
7 J. B. J. van Meurs, R. A. M. Dhonukshe-Rutten, S. M. Pluijm, M. van
der Klift, R. de Jonge, J. Lindemans, L. C. de Groot, A. Hofman,
J. C. Witteman, J. P. van Leeuwen, M. M. Breteler, P. Lips, H. A. Pols
and A. G. Uitterlinden, New Engl. J. Med., 2004, 20, 2033.
8 (a) S. Sengupta, C. Wehbe, A. K. Majors, M. E. Ketterer, P. M. DiBello
and D. W. Jacobsen, J. Biol. Chem., 2001, 276, 46896; (b) A. K. Majors,
S. Sengupta, B. Willard, M. T. Kinter, R. E. Pyeritz and D. W.
Jacobsen, Arterioscler., Thromb., Vasc. Biol., 2002, 22, 1354;
(c) D. Ingrosso, A. Cimmino, A. F. Perna, L. Masella, N. G. De Santo,
M. L. De Bonis, M. Vacca, M. D’Esposito, M. D’Urso and P. Galletti,
Lancet, 2003, 361, 1693.
9 (a) H. Kwon, K. Lee and H.-J. Kim, Chem. Commun., 2011, 47, 1773;
(b) H. S. Jung, K. C. Ko, G.-H. Kim, A.-R. Lee, Y.-C. Na, C. Kang,
J. Y. Lee and J. S. Kim, Org. Lett., 2011, 13, 1498.
10 (a) J. Shao, H. Sun, H. Guo, S. Ji, J. Zhao, W. Wu, X. Yuan, C. Zhanga
and T. D. James, Chem. Sci., 2012, 3, 1049–1061; (b) J. Lu, C. Sun,
W. Chen, H. Ma, W. Shi and X. Li, Talanta, 2011, 83, 1050.
11 (a) K. H. Leung, H. Z. He, P. Y. Ma, D. S. H. Chan, C. H. Leung and
D. L. Ma, Chem. Commun., 2013, 49, 771–773; (b) C. Luo, Q. Zhou,
B. Zhang and X. Wang, New J. Chem., 2011, 35, 45–48.
12 (a) M. H. Lee, J. H. Han, P.-S. Kwon, S. Bhuniya, J. Y. Kim,
J. L. Sessler, C. Kang and J. S. Kim, J. Am. Chem. Soc., 2012,
134, 1316; (b) C. S. Lim, G. Masanta, H. J. Kim, J. H. Han,
H. M. Kim and B. R. Cho, J. Am. Chem. Soc., 2011, 133, 11132.
13 C. Hwang, A. J. Sinskey and H. F. Lodish, Science, 1992, 257, 1496.
14 (a) O. Rusin, N. N. S. Luce, R. A. Agbaria, J. O. Escobedo, S. Jiang,
I. M. Warner, F. B. Dawan, K. Lian and R. M. Strongin, J. Am. Chem.
Soc., 2004, 126, 438; (b) W. Wang, O. Rusin, X. Xu, K. K. Kim,
J. O. Escobedo, S. O. Fakayode, K. A. Fletcher, M. Lowry,
C. M. Schowalter, C. M. Lawrence, F. R. Fronczek, I. M. Warner
and R. M. Strongin, J. Am. Chem. Soc., 2005, 127, 15949.
15 (a) P. Schubert, J. Biol. Chem., 1936, 114, 341; (b) P. Schubert, J. Biol.
Chem., 1937, 121, 539.
Fig. 4 Bright-field and fluorescence images of living HepG2 cells. (a) Fluores-
cence and (b) bright-field image of cells pretreated with 1 mM NEM for 30 min,
and then incubated with 2.0 mM probe for 15 min. (c) Fluorescence and
(d) bright-field image of cells incubated with 2.0 mM probe for 15 min.
performed by removing intracellular thiols using 1 mM
N-methylmaleimide (NEM, a thiol-blocking reagent) before
incubation of the cells with Cy–O–CHO. A marked fluorescence
quenching was observed (Fig. 4a). Living HepG2 cells were
incubated with Cy–O–CHO (2 mM) in PBS buffer solution
(pH 7.4) for 15 min at 37 1C and then washed thrice with PBS
buffer solution. The bright-red fluorescence image corresponding
to the adduct of Cy–O–CHO and Cys/Hcy was obtained (Fig. 4c).
These results revealed that the probe could indeed react with
intracellular Cys/Hcy to produce fluorescence, which further
confirmed that Cy–O–CHO can detect and image intracellular
endogenous Cys/Hcy, which is promising.
In summary, we have developed a Cy7-based fluorescent
probe (Cy–O–CHO) for detecting Cys/Hcy in living cells. The
probe (lex/em = 730/780 nm) has good water solubility,
membrane permeability, and little cytotoxicity. Cy–O–CHO
featured excellent sensitivity and high selectivity to Cys/Hcy
over other amino acids and thiols, especially GSH, under
physiological conditions. Importantly, the detection limit of
the probe is in the nanomolar range (7.9 nM), which is lower
than those of the reported probes. Furthermore, Cy–O–CHO
has been successfully used to image endogenous Cys/Hcy in
living HepG2 cells. We anticipate that this probe will be of great
benefit to biomedical researchers for studying the physiological
and pathological functions of Cys/Hcy in biological systems.
This work was supported by the 973 Program (2013CB933800),
National Natural Science Foundation of China (21227005, 21035003,
21275092), Key Natural Science Foundation of Shandong Province
of China (No. ZR2011BZ006), Specialized Research Fund for the
Doctoral Program of Higher Education of China (20113704130001,
20123704110004), Program for Changjiang Scholars and Innovative
Research Team in University.
16 D. Zhang, M. Zhang, Z. Liu, M. Yu, F. Li, T. Yi and C. Huang,
Tetrahedron Lett., 2006, 47, 7093.
17 (a) W. Lin, L. Long, L. Yuan, Z. Cao, B. Chen and W. Tan, Org. Lett.,
2008, 10, 5577–5580; (b) M. Zhang, M. Li, Q. Zhao, F. Li, D. Zhang,
J. Zhang, T. Yi and C. Huang, Tetrahedron Lett., 2007, 48, 2329.
18 (a) H. Chen, Q. Zhao, Y. Wu, F. Li, H. Yang, T. Yi and C. Huang,
Inorg. Chem., 2007, 46, 11075; (b) P. Das, A. K. Mandal,
N. B. Chandar, M. Baidya, H. B. Bhatt, B. Ganguly, S. K. Ghosh
and A. Das, Chem.–Eur. J., 2012, 18, 15382.
19 (a) K. Huang, H. Yang, Z. Zhou, H. Chen, F. Li, T. Yi and C. Huang,
Inorg. Chim. Acta, 2009, 362, 2577; (b) Y. Yue, Y. Guo, J. Xua and
S. Shao, New J. Chem., 2011, 35, 61.
20 H. Li, J. Fan, J. Wang, M. Tian, J. Du, S. Sun, P. Sun and X. Peng,
Chem. Commun., 2009, 5904–5906.
21 M. Zhang, M. Yu, F. Li, M. Zhu, M. Li, Y. Gao, L. Li, Z. Liu, J. Zhang,
D. Zhang, T. Yi and C. Huang, J. Am. Chem. Soc., 2007, 129, 10322.
22 L. Yuan, W. Lin and Y. Yang, Chem. Commun., 2011, 47, 6275.
23 P. Wang, J. Liu, X. Lv, Y. Liu, Y. Zhao and W. Guo, Org. Lett., 2012,
14, 520.
Notes and references
24 (a) R. Weissleder and V. Ntziachristos, Nat. Med., 2003, 9, 123;
(b) L. Yuan, W. Lin, K. Zheng, L. He and W. Huang, Chem. Soc. Rev.,
2013, 42, 622.
1 (a) Z. A. Wood, E. Schroder, H. J. Robin and L. B. Poole, Trends
Biochem. Sci., 2003, 28, 32; (b) H. Refsum, A. D. Smtth, P. M. Ueland,
E. Nexo, R. Clarke, J. Mcpartlin, C. Johnston, F. Engbaek,
J. Schneede, C. McPartlin and J. M. Scott, Clin. Chem., 2004, 50, 3.
25 Z. Guo, S. W. Nam, S. Park and J. Yoon, Chem. Sci., 2012, 3, 2760.
2 H. Refsum, P. M. Ueland, O. Nygrd and S. E. Vollset, Annu. Rev. Med., 26 L. Y. Niu, Y. S. Guan, Y. Z. Chen, L. Z. Wu, C. H. Tung and
1998, 49, 31.
Q. Z. Yang, Chem. Commun., 2013, 49, 1294.
c
9178 Chem. Commun., 2013, 49, 9176--9178
This journal is The Royal Society of Chemistry 2013