A near-infrared fluorescent probe for selective detection of HClO based on Se-sensitized aggregation of heptamethine cyanine dye

Article abstract of DOI:10.1039/c3cc47864e

A Se-containing heptamethine cyanine dye based fluorescent probe was successfully developed and used for HClO detection with rapid response and high selectivity based on aggregation behavior. The probe could react with HClO with significant change in its fluorescence profile, which makes it practical for detecting HClO in fetal bovine serum and in living mice. This journal is

Full text of DOI:10.1039/c3cc47864e

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A near-infrared fluorescent probe for selective
detection of HClO based on Se-sensitized
aggregation of heptamethine cyanine dye†
Cite this: Chem. Commun., 2014,
50, 1018
Received 13th October 2013,
Accepted 11th November 2013
Guanghui Cheng, Jiangli Fan,* Wen Sun, Jianfang Cao, Chong Hu and
Xiaojun Peng*
DOI: 10.1039/c3cc47864e
www.rsc.org/chemcomm
A Se-containing heptamethine cyanine dye based fluorescent probe was cyanine dye, has been widely utilized to explore chemosensors for
successfully developed and used for HClO detection with rapid response various inorganic and biologically related species.5 The self-aggregation
and high selectivity based on aggregation behavior. The probe could react of dyes is a frequently encountered phenomenon, especially in terms of
with HClO with significant change in its fluorescence profile, which makes dyes used in aqueous solutions.6 Though cyanine dyes belong to the
it practical for detecting HClO in fetal bovine serum and in living mice.
best known self-aggregating dyes, this behavior, to the best of
our knowledge, has seldom been applied in the design of HClO
As a kind of reactive oxygen species (ROS), hypochlorite is widely fluorescent probes. In addition, the selenium-containing structure
employed in our daily life, such as in disinfection of drinking water, which is reported to be selective to ROS is a good choice for developing
cooling-water treatment, household bleach, and as an antimicrobial.1 a HClO probe.7 It is noteworthy that selenium was introduced into the
However, once the used concentration of HClO cannot maintain the conjugated system of all such probes, which may contribute to
level within the range of 10À5–10À2 M, high concentration of the the relatively long response time to some extent. Therefore, we
hypochlorite solutions are a potential health hazard to plants, animals hypothesized that a non-conjugated-selenium structure may be more
and even to human beings. On the other hand, endogenous hypo- sensitive to HClO, and will speed up the response time.8
chlorite is essential to life and has important antibacterial properties.
Keeping all these ideas in mind, by introducing thiamorpholine
Maintenance of appropriate concentrations of HClO is of great and selenomorpholine into the structure of the heptamethine
significance to the biosystems because excessive or misplaced gen- cyanine dye, we present two novel HClO probes SCy7 and SeCy7
eration of HClO can lead to severe tissue damage and several kinds of based on the aggregation behavior of the heptamethine cyanine dye,
diseases (e.g. arthritis, lung injury, neuron degeneration and cancer2). using divalent sulfur and selenium as the redox-sensitive groups.
These findings highlight the importance of HClO homeostasis. To After examining the properties of the two compounds, we found that
determine the accurate concentration of HClO in environmental SeCy7 showed a much greater degree of aggregation, and was more
systems and monitor its in vivo site of action, fluorescent probes with sensitive to HClO. In this work, we report the synthesis of these two
high sensitivity, excellent selectivity and real-time capability to mea- probes and the application of SeCy7 in the measurement of HClO
sure HClO are quite desirable. To develop satisfactory HClO probes, concentrations in vitro and its utility in HClO imaging in living mice.
many research groups have come up with different frameworks to
The synthesis procedures of SCy7 and SeCy7 are shown in
design selective probes for HClO determination.3 Although these Scheme 1. SCy7 and SeCy7 were synthesized from the reaction of
fluorescent molecular probes are able to detect HClO specifically,
making HClO visible in living animals still arouses much attention.
Compared to light in the ultraviolet/visible (UV/vis) region, near-
infrared (NIR) light is much more favorable for in vivo imaging due to
its minimum photodamage to biological samples, good tissue pene-
tration, and weak autofluorescence interference from the complicated
living systems.4 As an important kind of NIR dye, heptamethine
State Key Laboratory of Fine Chemicals, Dalian University of Technology,
2 Linggong Road, Dalian 116024, P.R. China. E-mail: fanjl@dlut.edu.cn,
pengxj@dlut.edu.cn
† Electronic supplementary information (ESI) available: Synthesis; experimental
Scheme 1 Synthesis of SCy7, SeCy7 and the proposed recognition
mechanism of SeCy7 towards sodium hypochlorite.
details; characterization of new compounds. CCDC 957815. For crystallographic
data in CIF or other electronic format see DOI: 10.1039/c3cc47864e
1018 | Chem. Commun., 2014, 50, 1018--1020
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with the detection limit of 0.31 mM for HClO (3s/k), indicating that
SeCy7 is suitable for quantitative detection of HClO (Fig. S6, ESI†).
The unique sensing performance of SeCy7 to HClO drives us to
clarify the detection mechanism. We deduced that SeCy7 encountered
serious aggregation in aqueous solution, thus showing very weak
fluorescence. However, after the reaction with HClO, SeCy7 was
converted into SeOCy7. Meanwhile, the aggregated probe was gradu-
ally released and the fluorescence intensity boosted. To confirm the
Fig. 1 Crystal structure of compound SeCy7.
compound Cy7Cl with thiamorpholine and selenomorpholine respec- formation of quadrivalent selenium after addition of HClO to SeCy7
tively in anhydrous DMF. The final compounds together with all the solution, the 77Se NMR spectra of SeCy7 and the product after being
other intermediates (Fig. S13–S20, ESI†) were well characterized using treated with HClO are shown in Fig. S7 and S8 (ESI†). The resonance
1H NMR, 13C NMR, 77Se NMR (for SeCy7) and TOF-MS. And signal at 149 ppm was assigned to the bivalent selenium of SeCy7
fortunately, we also obtained single crystals of SeCy7 (CCDC number: (Fig. S7, ESI†). After reaction with HClO, the signal corresponding to
957815) by slowly evaporating its methanol solution at room tempera- the quadrivalent selenium of SeOCy7 at 783 ppm apparently emerged
ture (Fig. 1). We then tested the aggregation behaviour of the two new (Fig. S8, ESI†). TOF-MS has also been used to prove our hypothesis.
compounds as well as the intermediate Cy7Cl in aqueous solution. Fig. S9 (ESI†) indicated that the formula of the main product was
As expected, upon increase of the concentration (5–30 mM), all the C38H43N3OSe (m/z calc. 642.2963, found 642.2991), which could be
three dyes aggregated in PBS buffer (pH = 7.4, 20 mM), which could be corresponded to SeOCy7.
easily illustrated by the changes of their absorption and emission
Next, we examined the selectivity of SeCy7 towards HClO and
spectra (Fig. S1–S3, ESI†). Surprisingly, SeCy7 showed much weaker other reactive oxygen species (ROS) including hydrogen peroxide
fluorescence intensity than that of SCy7. Such a low background (H2O2), hydroxyl radicals (ꢀOH), nitric oxide (NOꢀ), tert-butyl hydro-
enables SeCy7 to be a better candidate for HClO detection.
peroxide (TBHP), tert-butoxy radical (TBOꢀ), superoxide (O2À), and
Considering the positive effects of aggregation, we tested the singlet oxygen (1O2). In 20 mM PBS buffer, 2.0 equiv. NaClO as well
optical properties of SeCy7 in PBS buffer (pH = 7.4, 20 mM). UV/vis as 20 equiv. other ROS were added respectively to a 30 mM solution
spectra of SeCy7 (30 mM) exhibited two absorption peaks at around of SeCy7. As shown in Fig. 3, SeCy7 features a robust fluorescence
695 nm and 752 nm, respectively. After being treated with NaClO turn-on response that is selective for HClO over other ROS. Other
(a commonly used HClO source, 0–2.0 equiv.), both of the two ROS including hydrogen peroxide, nitric oxide, tert-butyl hydroper-
absorption peaks decreased drastically (Fig. S4, ESI†), showing that oxide, tert-butoxy radical and superoxide did not lead to fluorescence
SeCy7 can react with HClO efficiently. The changes of the emission turn-on responses. The excellent selectivity for HClO over other ROS
spectra of SeCy7 in the presence of HClO were also studied. The shows that SeCy7 has the potential to detect HClO in a complex
probe exhibited very weak fluorescence in aqueous solution. Upon biological environment. The pH titration curves (Fig. S10, ESI†)
addition of NaClO, the emission spectra at about 786 nm enhanced also reveal that the fluorescence intensity of SeCy7 and the corre-
gradually and reached a plateau when 2.0 equiv. of NaClO was sponding product after the treatment of NaClO remain almost stable
added (Fig. 2). At this point, a 19.4-fold fluorescence enhancement at pH 4–10, demonstrating that SeCy7 can work in the biological pH
was observed. The whole recognition process finished within just range without being influenced.
dozens of seconds (Fig. S5, ESI†). Such a short response time will
The favourable fluorescence properties of SeCy7 for HClO
enable the real-time detection of HClO. Moreover, a good linear prompted us to further establish its utility in the determination
relationship (FL at 786 nm vs. HClO concentration) was obtained of HClO in biological samples. SeCy7 was first evaluated to
detect HClO in commercially available fetal bovine serum.
Bovine serum containing NaClO with different concentrations
Fig. 2 Fluorescence emission spectra of SeCy7 (30 mM) upon the addition
of increasing concentrations of sodium hypochlorite (0–2.0 equiv.) in
PBS buffer (pH 7.4, 20 mM). The arrows indicate the changes in the emis-
sion intensities with the increased sodium hypochlorite concentrations;
lex = 690 nm.
Fig. 3 Fluorescence responses of SeCy7 (30 mM) to NaClO (2.0 equiv.)
and other ROS (20.0 equiv. for H2O2, ꢀOH, NOꢀ, TBHP, TBOꢀ, O2À, 1O2) at
786 nm. Conditions: PBS buffer (pH 7.4, 20 mM). lex = 690 nm (three times
for each experiment).
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SeCy7 showed a rapid fluorescence response that was selective for
HClO over other related species. We have also compared the
aggregation behavior of SCy7 and SeCy7, and found that a
Se-containing compound (SeCy7) showed a much greater aggrega-
tion degree and was more sensitive to HClO. Moreover, SeCy7
could be used to detect HClO in commercial fetal bovine serum
and make HClO visible in living mice.
This work was financially supported by NSF of China
(21136002, 20923006 and 21376039), the National Basic
Research Program of China (2013CB733702), and the Ministry
of Education (NCET-12-0080).
Fig. 4 Representative fluorescence images (pseudocolor) of
a nude
mouse given a skin-pop injection of SeCy7 (25 mL, 60 mM, region A).
Representative fluorescence images (pseudocolor) of a nude mouse given
a skin-pop injection of LPS and PMA and a subsequent skin-pop injection
of SeCy7 (25 mL, 60 mM, region B). Images were taken after incubation for
0, 15, 30, 45 and 60 min, respectively. Images were taken using an
excitation laser of 690 nm and an emission filter of 820 Æ 20 nm.
Notes and references
1 (a) T. Aokl and M. Munemorl, Anal. Chem., 1983, 55, 209; (b) E. Hidalgo,
R. Bartolome and C. Dominguez, Chem.-Biol. Interact., 2002, 139, 265;
(c) D. I. Pattison and M. J. Davies, Biochemistry, 2005, 44, 7378;
(d) W. A. Rutala and D. J. Weber, Clin. Microbiol. Rev., 1997, 10, 597.
2 (a) M. J. Steinbeck, L. J. Nesti, P. F. Sharkey and J. Parvizi, J. Orthop.
Res., 2007, 25, 1128; (b) D. I. Pattison and M. J. Davies, Chem. Res.
Toxicol., 2001, 14, 1453; (c) S. A. Weitzman and L. I. Gordon, Blood,
1990, 76, 655.
(0, 5, 10, 15, 20, 25, 30, 35 and 40 equiv.) was prepared. These
serum solutions were then incubated with 60 mM SeCy7 at room
temperature. Fig. S11 (ESI†) shows that the fluorescence inten-
sity enhanced with the increased concentration of HClO, indi-
cating that SeCy7 is suitable for HClO detection in serum
without addition of any co-solvents or detergents.
3 (a) Y. Zhou, J.-Y. Li, K.-H. Chu, K. Liu, C. Yao and J.-Y. Li, Chem.
Commun., 2012, 48, 4677; (b) L. Yuan, W. Lin, Y. Yang and H. Chen,
J. Am. Chem. Soc., 2011, 134, 1200; (c) L. Yuan, W. Lin, J. Song and
Y. Yang, Chem. Commun., 2011, 47, 12691; (d) Y.-K. Yang, H. J. Cho,
J. Lee, I. Shin and J. Tae, Org. Lett., 2009, 11, 859; (e) Y. Yang, Q. Zhao,
W. Feng and F. Li, Chem. Rev., 2012, 113, 192; ( f ) Q. Xu, K.-A. Lee,
S. Lee, K. M. Lee, W.-J. Lee and J. Yoon, J. Am. Chem. Soc., 2013,
135, 9944; (g) X. Wu, Z. Li, L. Yang, J. Han and S. Han, Chem. Sci.,
2013, 4, 460; (h) D. Oushiki, H. Kojima, T. Terai, M. Arita,
K. Hanaoka, Y. Urano and T. Nagano, J. Am. Chem. Soc., 2010,
132, 2795; (i) Y. Koide, Y. Urano, S. Kenmoku, H. Kojima and
T. Nagano, J. Am. Chem. Soc., 2007, 129, 10324; ( j) Y. Koide,
Y. Urano, K. Hanaoka, T. Terai and T. Nagano, J. Am. Chem. Soc.,
2011, 133, 5680; (k) S. Kenmoku, Y. Urano, H. Kojima and T. Nagano,
J. Am. Chem. Soc., 2007, 129, 7313; (l) X. Cheng, H. Jia, T. Long,
J. Feng, J. Qin and Z. Li, Chem. Commun., 2011, 47, 11978;
(m) X. Chen, K.-A. Lee, E.-M. Ha, K. M. Lee, Y. Y. Seo, H. K. Choi,
H. N. Kim, M. J. Kim, C.-S. Cho, S. Y. Lee, W.-J. Lee and J. Yoon, Chem.
Commun., 2011, 47, 4373; (n) G. Cheng, J. Fan, W. Sun, K. Sui, X. Jin,
J. Wang and X. Peng, Analyst, 2013, 138, 6091.
We then evaluated the suitability of the probe for visualizing
HClO in living animals. Institute of cancer research (ICR) mice were
selected. The mouse was given an s.p. (skin-pop) injection of SeCy7
(60 mM, 25 mL DMSO) on the right leg, and 15 minutes later, the
mouse was given an s.p. injection of 2.0 equiv. of NaClO in the same
region. The mouse was imaged by using a NightOWL II LB983 small
animal in vivo imaging system with a 690 nm excitation laser and an
820 Æ 20 nm emission filter. Fig. S12 (ESI†) shows representative
fluorescence images of an ICR mouse at different times after the
injection of NaClO. It was demonstrated that the fluorescence
intensity enhanced gradually within 40 minutes, proving that SeCy7
can detect NaClO in vivo without the interference of background
signals. More importantly, all these images were obtained from
normal mice without unhairing because of the NIR absorption and
emission properties of the probe.
4 (a) X. Wu, S. Chang, X. Sun, Z. Guo, Y. Li, J. Tang, Y. Shen, J. Shi, H. Tian
and W. Zhu, Chem. Sci., 2013, 4, 1221; (b) S. A. Hilderbrand and
R. Weissleder, Curr. Opin. Chem. Biol., 2010, 14, 71; (c) J. M. Baumes,
J. J. Gassensmith, J. Giblin, J.-J. Lee, A. G. White, W. J. Culligan,
W. M. Leevy, M. Kuno and B. D. Smith, Nat. Chem., 2010, 2, 1025.
5 (a) Z. Guo, G.-H. Kim, I. Shin and J. Yoon, Biomaterials, 2012,
33, 7818; (b) T. Hirayama, G. C. Van de Bittner, L. W. Gray,
S. Lutsenko and C. J. Chang, Proc. Natl. Acad. Sci. U. S. A., 2012,
109, 2228; (c) K. Xu, L. Sun, S. P. Li, L. Li, M. Qiang and B. Tang,
Chem. Commun., 2012, 48, 684; (d) N. Karton-Lifshin, E. Segal,
L. Omer, M. Portnoy, R. Satchi-Fainaro and D. Shabat, J. Am. Chem.
Soc., 2011, 133, 10960; (e) F. Yu, P. Li, P. Song, B. Wang, J. Zhao and
K. Han, Chem. Commun., 2012, 48, 2852; ( f ) L. Yuan, W. Lin,
K. Zheng, L. He and W. Huang, Chem. Soc. Rev., 2013, 42, 622.
6 (a) A. Mishra, R. K. Behera, P. K. Behera, B. K. Mishra and
G. B. Behera, Chem. Rev., 2000, 100, 1973; (b) Y. Xu, Z. Li,
A. Malkovskiy, S. Sun and Y. Pang, J. Phys. Chem. B, 2010, 114, 8574.
7 (a) G. Li, D. Zhu, Q. Liu, L. Xue and H. Jiang, Org. Lett., 2013, 15, 2002;
(b) S.-R. Liu and S.-P. Wu, Org. Lett., 2013, 15, 878; (c) F. Yu, P. Li,
B. Wang and K. Han, J. Am. Chem. Soc., 2013, 135, 7674; (d) K. Xu,
M. Qiang, W. Gao, R. Su, N. Li, Y. Gao, Y. Xie, F. Kong and B. Tang,
Chem. Sci., 2013, 4, 1079; (e) B. Wang, P. Li, F. Yu, J. Chen, Z. Qu and
K. Han, Chem. Commun., 2013, 49, 5790.
Finally, we investigated the possibility of SeCy7 detecting the
endogenous HClO in living mice. In this case, nude mice were
selected as our model. According to the previous literature,
the synergistic effect of lipopolysaccharide (LPS) and phorbol
12-myristate 13-acetate (PMA) can stimulate cells to produce
HClO.9 A solution of LPS (1 mg mLÀ1) was injected into the back
(region B) of the mouse, and 12 hours later, PMA was then
injected into the same region. The probe (60 mM, 25 mL DMSO)
was injected into the back of the mouse (region A and B) after
30 min of the above disposal. The pictures were taken under the
imaging system after the mice were incubated for 0, 15, 30, 45
and 60 min, respectively. As shown in Fig. 4, the fluorescence
intensities of region A were stable, while the signals at region B
became stronger gradually within 60 min. The result established
that SeCy7 was a desired imaging agent for visualizing endogen-
ous HClO in vivo. Taking these results together, it is established
that SeCy7 is suitable for detecting HClO in living animals.
In summary, we have developed a selective NIR fluorescent 8 C. Storkey, D. I. Pattison, J. M. White, C. H. Schiesser and
M. J. Davies, Chem. Res. Toxicol., 2012, 25, 2589.
9 S. E. Gomez-Mejiba, Z. Zhai, M. S. Gimenez, M. T. Ashby,
J. Chilakapati, K. Kitchin, R. P. Mason and D. C. Ramirez, J. Biol.
probe SeCy7 for HClO based on the aggregation behavior of a
heptamethine cyanine dye derivative. Upon addition of HClO,
the fluorescence emission profile of SeCy7 changed significantly.
Chem., 2010, 285, 20062.
1020 | Chem. Commun., 2014, 50, 1018--1020
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