A ratiometric fluorescent probe for rapid and sensitive visualization of hypochlorite in living cells

Article abstract of DOI:10.1039/c4ra07009g

A ratiometric fluorescent probe for ClO^- has been developed based on coumarin-hemicyanine, which displayed a colorimetric and fluorescence response to ClO^- with high selectivity, fast response (within 2 min) and an extremely low dete

Full text of DOI:10.1039/c4ra07009g

RSC Advances
View Article Online
View Journal | View Issue
COMMUNICATION
A ratiometric fluorescent probe for rapid and
sensitive visualization of hypochlorite in living
Cite this: RSC Adv., 2014, 4, 43110
cells†
a
b
b
ab
b
Received 12th July 2014
Accepted 26th August 2014
*
*
and Xichao Hu
Jiayu Zha, Boqiao Fu, Caiqin Qin, Lintao Zeng
DOI: 10.1039/c4ra07009g
www.rsc.org/advances
A ratiometric fluorescent probe for ClOꢀ has been developed based thioether,11 oxime,12 and hydroxamic acid.13 However, most of
on coumarin–hemicyanine, which displayed a colorimetric and fluo- these probes just show changes in emission intensity, which are
rescence response to ClOꢀ with high selectivity, fast response (within usually inuenced by environmental conditions, probe distri-
2 min) and an extremely low detection limit (0.08 mM). This probe was bution, and instrumental efficiency.14 By contrast, a ratiometric
successfully used to visualize hypochlorite in living cells.
probe which utilizes the ratio of two emissions at different
wavelengths as the detecting signal,15 can provide a built-in
correction for the above mentioned factors and thus allow more
accurate analysis. The uorescence resonance energy transfer
(FRET) mechanism provides an effective approach for
construction of a ratiometric uorescent probe. This strategy
has been successfully employed to design a few ratiometric
uorescent probes for ClOꢀ and has achieved good perfor-
mance.16,17 Nevertheless, it is difficult to construct such a system
because of the complicated synthetic routes as well as the
requirement for strong spectral overlap between emission of
donor and absorption of acceptor. Thus, it is highly desirable to
develop a ratiometric probe for ClOꢀ based on a simple and
efficient approach.
Hypochlorite anion (ClOꢀ)/hypochlorous acid (HOCl), a bio-
logically important reactive oxygen species (ROS), is generally
produced in living organisms from the myeloperoxidase (MPO)-
mediated peroxidation of chloride ions and hydrogen peroxide.1
The hypochlorite anion (ClOꢀ) plays a key role in the human
immune defense system and inammation by destruction of
the invading bacteria and pathogens.2 However, excessive or
misplaced production of ClOꢀ can lead to tissue damage and
diseases, such as atherosclerosis,3 arthritis,4 cancer,1b and
neurodegeneration.5 Therefore, it is of great importance to
investigate the complex contributions of HOCl/ClOꢀ to our
health and study the mechanism of action and specic func-
tions of HOCl in living organisms. Scientists have conducted
extensive research to elucidate the mechanism by which HOCl
kills bacteria and destroys human tissue.6 However, the detailed
understanding of HOCl formation during pathogenic biological
events still remains a challenge due to the lack of methods for
monitoring HOCl in living organisms.7
Diethylamino-coumarin dye possesses several favourable
uorescence properties, such as excitation and emission wave-
lengths in the visible region as well as a high uorescence
quantum yield, which are desirable for the intended biological
applications of the probe. In this work, we chose diethylamino-
coumarin aldehyde as one building block, and then modied it
by 1,2,3,3-tetramethyl-3H-indolium iodide to produce a water
soluble CMCY with long emission wavelength (shown in
Fluorescence-based assays are useful tools for real-time
sensing and for visualizing some biologically important species
in living organisms because of their high sensitivity, and high
temporal and spatial resolution.8 Several uorescent probes for
the detection and visualization of HOCl in living cells have been
developed on the basis of the HOCl-mediated oxidation reaction
of various functional groups such as p-methoxyphenol,9 ether,10
1
Scheme 1). The structure of CMCY was conrmed by H NMR,
13C NMR and HR-MS. It is well known that hypochlorite anion
(ClOꢀ)/hypochlorous acid (HOCl) have strong reactivity to double
bonds. We expect that ClOꢀ will react with the double bonds and
destroy the large p-conjugation of the probe, giving rise to a
colorimetric and ratiometric uorescence response to ClOꢀ. This
hybrid coumarin–hemicyanine probe exhibited an emission
maximum at 631 nm. Upon addition of ClOꢀ, the colour of the
resultant solution changed from purple to colourless and the
uorescence of the solution changed from red to blue.
aSchool of Chemistry & Chemical Engineering, Tianjin University of Technology,
Tianjin 300384, PR China. E-mail: zlt1981@126.com; Fax: +86 22 60214252
bDepartment of Chemistry and Material Sciences, Hubei Engineering University,
Hubei, Xiaogan 432000, PR China. E-mail: hxc30@163.com; Fax: +86 712 2345265
† Electronic supplementary information (ESI) available: Synthetic details and
characterization data. See DOI: 10.1039/c4ra07009g
To examine the response of CMCY to ClOꢀ, UV-vis absorp-
tion spectra titrations were performed in PBS/MeOH (pH 7.4,
43110 | RSC Adv., 2014, 4, 43110–43113
This journal is © The Royal Society of Chemistry 2014

View Article Online
Communication
RSC Advances
Scheme 1 Synthesis of probe.
Fig. 3 (a) Time-dependent fluorescence responses of CMCY (10 mM)
to ClOꢀ (200 mM) in PBS solution (50 mM, pH 7.4); (b) The ratiometric
fluorescence responses of probe (10 mM) to ClOꢀ (200 mM) at different
pH values.
As shown in Fig. 1b, CMCY displayed a characteristic emis-
sion band centred at 631 nm. Upon addition of increasing
amounts of ClOꢀ, the uorescence intensity at 631 nm gradu-
ally decreased and this was accompanied by the appearance of a
new emission band centred at 480 nm. Consequently, the
uorescence of CMCY solution changed from red to blue. In the
presence of 20 equiv. ClOꢀ, the ratio of emission intensity
Fig. 1 (a) UV-vis absorption responses of CMCY (10 mM) to ClOꢀ (0 to
200 mM) in 50 mM PBS solution (pH 7.4). Inset: The colour of CMCY
solution (10 mM) changed from purple to colourless; (b) Fluorescence
responses of CMCY (10 mM) to ClOꢀ (0 to 200 mM) in 50 mM PBS
solution (pH 7.4), lex ¼ 460 nm. Inset: The fluorescence of CMCY
(10 mM) changed from red to blue.
(I480/I631
) showed a drastic enhancement (ca. 400-fold
enhancement), establishing that CMCY could serve as a ratio-
metric uorescent probe for ClOꢀ. It is worth noting that such a
huge change of signal ratio at two wavelengths is highly desir-
able for ratiometric uorescent probes, as the sensitivity and
the dynamic range of ratiometric probes are controlled by the
ratios. By plotting the ratio of emission intensity (I480/I631) versus
v/v ¼ 9 : 1) at ambient temperature. As shown in Fig. 1, free
probe displayed a strong absorption band centred at 570 nm.
Upon addition of ClOꢀ, the absorption band centred at 570 nm
gradually decreased and a new absorption band at 392 nm
emerged with a well-dened isosbestic point at 455 nm. As a
result, an obvious colour change from purple to colourless was
observed, allowing colorimetric detection of ClOꢀ by the naked
eye (Fig. 1a inset). When the amount of ClOꢀ reached
20 equivalents with respect to the probe, these changes were
found to reach a plateau. A linear calibration of the UV-vis
absorption response to ClOꢀ concentrations from 0 to 73 mM
was obtained (Fig. 2a), indicating that CMCY could be poten-
tially used to quantitatively detect ClOꢀ concentrations. We
further examined the time course of the ratiometric response of
CMCY to ClOꢀ (Fig. 3a). CMCY was highly stable under the
assay conditions. However, upon addition of 200 mM ClOꢀ, a
dramatic colour change from purple to colourless and a large
increase in the ratio were observed within 2 min. This nding
suggested that CMCY was a fast-response probe for ClOꢀ and
might be suitable for real-time sensing of ClOꢀ in living cells.
the concentration of ClOꢀ, a good linear relationship (R2
¼
0.9932) was obtained with ClOꢀ concentrations ranging from
0 to 73 mM (shown in Fig. 2b). The detection limit of probe
CMCY for ClOꢀ was determined to be 0.08 mM according to the
signal-to-noise ratio (S/N ¼ 3), which is comparable to the
previously reported ratiometric uorescent probes for ClOꢀ.16,18
The ratiometric uorescence responses of CMCY to ClOꢀ at
different pH values were also investigated, as shown in Fig. 3b.
In the absence of ClOꢀ, CMCY did not display any uorescence
changes over a wide range of pH values from 4.0 to 7.8.
However, in the presence of 200 mM ClOꢀ, a very large ratio-
metric uorescence enhancement was observed from pH 6.5 to
7.8, which indicated that CMCY would be suitable for bio-
applications at physiological conditions.
Good selectivity is a crucial requirement for all kinds of
detection methods. To test the selectivity, CMCY (10 mM) was
incubated with 10 equiv. of representative species including ROS,
anions and ions. As shown in Fig. 4, a large ratiometric signal
with I480/I631 ¼ 15.7 was observed in the presence of 10 equiv. of
ClOꢀ. By contrast, other ROS (hydrogen peroxide, hydroxyl
radical, superoxide, t-BuOOH and ONOOꢀ) induced a very weak
ratiometric response with I480/I631 < 0.5. Some common aꢀnions
(CH3COOꢀ, Fꢀ, ClO4ꢀ, PO43ꢀ, HCO3ꢀ, NO3ꢀ, SO32ꢀ, NO2 and
SO42ꢀ) and heavy metal ions (Hg2+, Cu2+) caused only negligible
changes in the emission ratio (I480/I631 < 0.2). These results
suggest that the probe has excellent selectivity for ClOꢀ over other
anions and ROS. Although ozone is a very strong oxidation
reagent and can break alkene to give aldehyde and carboxylic
acid, it also easily oxidizes many biological species including
Fig. 2 (a) Absorbance changes and (b) fluorescence intensity changes
of CMCY (10 mM) versus concentrations of ClOꢀ in PBS solution (pH
7.4). The data were collected 3 minutes after ClOꢀ was added into the
CMCY (10 mM) solution.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 43110–43113 | 43111

View Article Online
RSC Advances
Communication
Fig. 4 Fluorescence intensity ratio (I480/I631) of CMCY (10 mM in PBS
solution, pH 7.4) in the presence of various species (100 mM). 1, blank;
2, ClOꢀ; 3, HCO3ꢀ; 4, NO3ꢀ; 5, CH3COOꢀ; 6, SO32ꢀ; 7, Fꢀ; 8, ClO4ꢀ; 9,
PO43ꢀ; 10, NO2ꢀ; 11, Cu2+; 12, Hg2+; 13, GSH; 14, Cys; 15, t-BuOOH;
16, hydroxyl radicals; 17, ONOOꢀ; 18, H2O2; 19, O2ꢀ; lex ¼ 460 nm.
Fig. 5 Confocal fluorescence images of HeLa cells. Cells were incu-
bated with CMCY (5 mM) for 30 min (a)–(c). Images of cells after
treatment with CMCY (5 mM) for 30 min and subsequent treatment of
the cells with 100 mM ClOꢀ for 30 min (d)–(f). Scale bar: 10 mm.
GSH, protein and antibody. As we known, the content of GSH,
protein and antibody are very rich in living cells. As a result,
ozone has been largely consumed by these biological species, and
thus will cause little interference to our probe. Therefore, the
In summary, we have designed a water-soluble red-emitting
probe CMCY has potential applications for ClOꢀ detection in probe based on coumarin–hemicyanine, which displayed a
complex biological environments.
noticeable colorimetric and uorometric dual response to ClOꢀ
To get an insight into the sensing mechanism, CMCY was on the basis of a simple alkene cleavage reaction. CMCY
treated with 20 equiv. ClOꢀ in PBS for 30 min, and then the exhibited a fast response to ClOꢀ in physiological conditions
major uorescent product was separated by silica gel column with high sensitivity and selectivity, and an extremely low
for MS analysis. The ESI-MS (+mode) showed the presence of a detection limit (0.08 mM). By plotting the ratio of emission
dominant peak at m/z ¼ 358.0976 and a noticeable isotopic peak intensity (I480/I631) versus the concentration of ClOꢀ, a good
at 360.0947, which corresponded to a coumarin derivative linear relationship (R2 ¼ 0.9932) was obtained with ClOꢀ
shown in Scheme 2 and Fig. S2.† Hence, spectral changes in the concentrations ranging from 0 to 73 mM. Cell staining results
presence of ClOꢀ originated from the oxidative cleavage of indicated CMCY was cell membrane permeable and could be
CMCY by ClOꢀ.
used for visualizing hypochlorite in living cells.
Furthermore, we explored the potential application of CMCY
for sensing ClOꢀ in living cells. HeLa cells incubated with
CMCY (5 mM) for 30 min exhibited a clear cell prole with red
Acknowledgements
colour (Fig. 5b), which indicated that CMCY has penetrated into This work was nancially supported by NSFC (no. 21203138,
the cells and been retained. When the HeLa cells were further 31371750), the Natural Science Foundation of Tianjin
treated with 100 mM ClOꢀ for 30 minutes, blue uorescence (13JCQNJC05300), Hubei Provincial Educational Department
from the HeLa cells was observed (shown in Fig. 5e). Mean- (D20132702), and Hubei Co-Innovation Center for Utilization of
while, the original red uorescence from CMCY was not Biomass Waste.
detected, implying that CMCY had been consumed by ClOꢀ and
produced some products with blue uorescence. Cell staining
Notes and references
results indicated that probe CMCY was cell membrane perme-
able and capable of responding to ClOꢀ in the living cells.
1 (a) Y. W. Yap, M. Whiteman and N. S. Cheung, Cell.
Signalling, 2007, 19, 219; (b) D. I. Pattison and M. J. Davies,
Biochemistry, 2006, 45, 8152.
2 (a) D. Roos and C. C. Winterbourn, Science, 2002, 296, 669;
(b) F. C. Fang, Nat. Rev. Microbiol., 2004, 2, 820.
3 S. Sugiyama, K. Kugiyama, M. Aikawa, S. Nakamura,
H. Ogawa and P. Libby, Arterioscler., Thromb., Vasc. Biol.,
2004, 24, 1309.
4 M. J. Steinbeck, L. J. Nesti, P. F. Sharkey and J. J. Parvizi,
J. Orthop. Res., 2007, 25, 1128.
5 D. I. Pattison and M. J. Davies, Chem. Res. Toxicol., 2001, 14,
1453.
6 (a) M. D. Rees, C. L. Hawkins and M. J. Davies, J. Am. Chem.
Soc., 2003, 125, 13719; (b) L. J. Marnett, J. Org. Chem., 2012,
77, 5224; (c) J. Park, H. Kim, Y. Choi and Y. Kim, Analyst,
2013, 138, 3368.
Scheme 2 The proposed mechanism for sensing of ClOꢀ.
43112 | RSC Adv., 2014, 4, 43110–43113
This journal is © The Royal Society of Chemistry 2014

View Article Online
Communication
RSC Advances
7 K. Setsukinai, Y. Urano, K. Kakinuma, H. J. Majima and 13 Y.-K. Yang, H. J. Cho, J. Lee, I. Shin and J. Tae, Org. Lett.,
T. Nagano, J. Biol. Chem., 2003, 278, 3170. 2009, 11, 859.
8 (a) J. Chan, S. C. Dodani and C. J. Chang, Nat. Chem., 2012, 4, 14 (a) K. Komatsu, Y. Urano, H. Kojima and T. Nagano, J. Am.
973; (b) Y. Yang, Q. Zhao, W. Feng and F. Li, Chem. Rev., 2013,
113, 192; (c) L. Zeng, N. Fan, J. Zha, X. Hu, B. Fu, C. Qin and
L. Wang, Analyst, 2013, 138, 7083.
9 Z.-N. Sun, F.-Q. Liu, Y. Chen, P. K. H. Tam and D. Yang, Org.
Lett., 2008, 10, 2171.
10 J. Shepherd, S. A. Hilderbrand, P. Waternan, J. W. Heinecke,
R. Weissleder and P. Libby, Chem. Biol., 2007, 14, 1221.
11 (a) S. Kenmoku, Y. Urano, H. Kojima and T. Nagano, J. Am.
Chem. Soc., 2007, 129, 13447; (b) D. Srikun, E. W. Miller,
D. W. Domaille and C. J. Chang, J. Am. Chem. Soc., 2008,
130, 4596; (c) L. Long, D. Zhang, X. Li, J. Zhang, C. Zhang
and L. Zhou, Anal. Chim. Acta, 2013, 775, 100; (d) Y. Zhou,
J. Y. Li, K. H. Chu, K. Liu and C. Yao, Chem. Commun.,
2012, 48, 4677.
15 J. V. Mello and N. S. Finney, Angew. Chem., Int. Ed., 2001, 40,
1536.
Chem. Soc., 2007, 129, 7313; (b) Y. Koide, Y. Urano, 16 (a) L. Yuan, W. Lin, J. Song and Y. Yang, Chem. Commun.,
K. Hanaoka, T. Terai and T. Nagano, J. Am. Chem. Soc.,
2011, 133, 5680.
2011, 47, 12691; (b) L. Yuan, W. Lin, Y. Xie, B. Chen and
J. Song, Chem.–Eur. J., 2012, 18, 2700.
12 (a) W. Lin, L. Long, B. Chen and W. Tan, Chem.–Eur. J., 2009, 17 (a) X. Wu, Z. Li, L. Yang, J. Han and S. Han, Chem. Sci., 2013,
15, 2305; (b) X. Cheng, H. Jia, T. Long, J. Feng, J. Qin and
Z. Li, Chem. Commun., 2011, 47, 11978; (c) G. Cheng,
J. Fan, W. Sun, K. Sui, X. Jin, J. Wang and X. Peng, Analyst,
2013, 138, 6091.
4, 460; (b) G. Chen, F. Song, J. Wang, Z. Yang, S. Sun, J. Fan,
X. Qiang, X. Wang, B. Dou and X. Peng, Chem. Commun.,
2012, 48, 2949.
18 S. Chen, J. Lu, C. Sun and H. Ma, Analyst, 2010, 135, 577.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 43110–43113 | 43113