A novel rhodamine-based fluorescent probe for selective detection of ClO- and its application in living cell imaging

Article abstract of DOI:10.1139/cjc-2018-0123

A novel fluorescent rhodamine-based probe L for selective responding to ClO^- has been synthesized and characterized. The spectroscopy showed that probe L can detect ClO^- in aqueous solution without interaction with other interfering ions, and the detection is also evident by the colour change from colourless to reddish purple under white light. The remarkable fluorescence enhancement showed the high selectivity and sensitivity of probe L for the detection of ClO^-. Furthermore, probe L was applied to intracellular fluorescent imaging of HeLa cells treated with ClO^- and MTT assay showed nontoxicity in living cells.

Full text of DOI:10.1139/cjc-2018-0123

Page 1 of 22
A novel rhodamine-based fluorescent probe for selective detection of ClO- and
its application in living cells imaging
Changping Yang,#,a Mingliang Zuo,#,b Xiaoli Hu,a Xuelin Chen,a Duoduo Zhang,a Zhenping Qi,a
Xiaoyan Zhao,a Hua Zuo*,a
aCollege of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
bDepartment of Cardiovascular Ultrasound and Non-invasive Cardiology, Sichuan Academy of Medical
Sciences & Sichuan Provincial People’s Hospital, Chengdu 610072, China
Authors’ e-mail addresses:
Changping Yang: 1042792801@qq.com
Mingliang Zuo: zuoml@hotmail.com
Xiaoli Hu: 2461094457@qq.com
Xuelin Chen: 1591428605@qq.com
Duoduo Zhang: 1852435126@qq.com
Zhenping Qi: 1114068980@qq.com
Xiaoyan Zhao: zhaoxiaoyan67@163.com
Corresponding author: H. Zuo, Email: zuohua@swu.edu.cn
# These authors made equal contribution to this work.
1

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Abstract: A novel fluorescent rhodamine-based probe L for selective responding to ClO- has
been synthesized and characterized. The spectroscopy showed that probe L can detect ClO- in
aqueous solution without interaction with other interfering ions, and the detection is also evident
by the color change from colorless to red purple under white light. The remarkable fluorescence
enhancement showed the high selectivity and sensitivity of probe L for the detection of ClO-.
Furthermore, probe L was applied to intracellular fluorescent imaging of Hela cells treated with
ClO-, and MTT assay showed nontoxicity in living cells.
Keywords: Rhodamine B; fluorescent probe; ClO-; Hela cells; bioimaging
2

Page 3 of 22
Introduction
Hypochlorite/hypochlorous acid (ClO-/HClO), one of the reactive oxygen species (ROS),1
plays an important role in human immune defense system, such as killing a wide range of
bacteria and pathogens in cells.2, 3 Due to the good antibacterial effects and cheap price, HClO
has been widely used as disinfectant for tap water.4 However, high concentrations of
hypochlorous acid solution may damage the respiratory system,5 and many studies suggest that
the oxidative stress mediated by HClO participates in kinds of pathophysiological processes such
as atherosclerosis,6, 7 apoptosis and cellular senescence, which may be potential harm to human
health.8–11
Compared with the traditional sensing techniques such as atomic absorption spectroscopy,
inductively coupled plasma-mass spectroscopy and inductively coupled plasma-atomic emission
spectroscopy,12, 13 and the recent development for the novel sensing techniques,14–16 fluorescence
chemosensors17–20 turn out to be quite cost-efficient.
A hypochlorite fluorescent probe rhodamine 6G hydrazide has been synthesized for the
detection of HClO in tap water and fluorescence imaging in Hela cells.21 For the detection of
hypochlorous acid produced by human neutrophil cells, a highly selective fluorescent probe was
synthesized.22, 23 In the pH detection of physiological environment, this probe showed higher
selectivity on the HClO than other reactive oxygen species.23, 24 These probes showed the
"off-on" mechanism for the fluorescence and luminous reactions of HClO through spirolactam
3

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ring opening.25–27
Because of the great importance of hypochlorous acid/hypochlorite and the fluorescent method
used in cell imaging superior to many classical cell sensing methods28–30 in view of the easy use
and low cost, we herein developed a new rhodamine B hydrazide derivative as a fluorescent
probe for ClO- detection. Rhodamine fluorophores are known for their high fluorescence
quantum yield, low sensitivity to pH, excellent optical properties and cell membrane penetrating
ability, low toxic effects of organism,31–33 we therefore speculate the probe is highly selective and
sensitive, and thus could be applied in bioimaging in living cells.
Experiment sections
Materials and methods
All used reagents from commercial suppliers were analytical grade, untreated and used directly.
-
-
-
2-
Deionized water was used in the whole study. Cl-, SiO32-, S2O32-, H2PO4 , NO3 , MnO4 , SO4
were respectively prepared from NaCl, Na2SiO3, Na2S2O3, NaH2PO4, NaNO3, KMnO4, Na2SO4
dissolved in deionized water. Ac- and ClO- were respectively prepared from acetic acid and
·-
1
sodium hypochlorite. O2 was prepared by KO2 dissolved in DMSO34 and O2 was prepared by
the reaction of NaClO and 2 equiv. of H2O2.35 ·OH was prepared by the reaction of ammonium
ferrous sulfate and hydrogen peroxide.36, 37 Phosphate buffer saline (PBS buffer) contained 2 mM
potassium dihydrogenphosphate, 10 mM disodium hydrogen phosphate, 137 mM sodium
chloride and 2.7 mM potassium chloride in deionized water at pH 7.4, adjusted by hydrochloric
acid. The probe was dissolved in N, N-dimethylformamide as stock solution with a final
4

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concentration at 100 μmol/L. The test solution was mixed by 2.0 mL PBS buffer (0.01 M, pH
7.4), 1.6 mL DMF and 0.4 mL stock solution.
13
1H NMR (400 MHz) and C NMR (100 MHz) spectra were recorded on a Bruker Advance
400 FT spectrometer, using CDCl3 as solvent with tetramethylsilane (TMS) as internal standard.
Chemical shifts were reported in parts per million. Mass spectra (MS) were measured by the ESI
method on an Agilent 6510 Q-TOF mass spectrometer. UV-Vis spectra were measured by a
UV-2550 spectrometry (Shimadzu, Japan). Fluorescent measurements were recorded on an
F-4500 FL spectrophotometer (Hitachi, Japan). All reactions were monitored by TLC (thin-layer
chromatography) using 0.25 mm silica gel plates with UV indicator (Shanghai Jiapeng
Technology Co. Ltd., China). The pH was determined with PHS-3B pH meter (Shanghai Ruixi
equipment Co. Ltd., China). Images of Hela cells were captured by a laser confocal microscope
(Nikon A1/A1R+, Japan). Melting point (uncorrected) was determined on a micro melting point
apparatus (Shanghai Shenguang Instrument Co. Ltd., China).
Synthesis of probe L
The synthetic route for probe L is shown in Scheme 1. The reaction mixture of rhodamine
hydrazide 2 (1.17 mmol, 1.0 equiv.),38 chloroacetyl chloride (2.32 mmol, 2.0 equiv.) and
triethylamine in 17.5 mL dichloromethane was stirred at room temperature for 4 h and quenched
by excess triethylamine (11.67 mmol, 10.0 equiv.). The solvent was evaporated and to the residue
30 mL deionized water was added. The mixture was filtered and the filter cake was dried to give
the compound 3 without further purification.39
5

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To obtain probe L, the mixture of intermediate 3 (0.91 mmol, 1.0 equiv.), potassium carbonate
(2.09 mmol, 2.3 equiv.), and N-methyl piperazine (0.90 mmol, 1.0 equiv.) in acetonitrile (12 mL)
was heated to reflux for 18 h. The reaction was monitored by TLC. When the reaction was
completed, the solvent was removed under vacuum and water was added to the residue. The
crude product was extracted by ethyl acetate for three times. The combined organic layer was
dried and filtered, and the filtrate was evaporated to give the crude product. The crude product
was further recrystallized from acetonitrile to afford probe L as a white solid in 60% yield. m.p.
1
176.6–178.0 °C. H NMR (CDCl3, 400 MHz) δ: 8.33 (s, 1H), 7.98–7.95 (m, 1H), 7.52–7.47 (m,
2H), 7.14 (s, 1H), 6.66 (s, 1H), 6.64 (s, 1H), 6.32 (dd, J = 4.7, 2.3 Hz, 4H), 3.37–3.30 (m, 8H),
13
2.97 (s, 2H), 2.35 (s, 4H), 2.18 (s, 3H), 1.89 (s, 4H), 1.16 (t, J = 7.0 Hz, 12H) ppm; C NMR
(CDCl3, 100 MHz) δ: 12.66, 44.35, 45.89, 52.98, 55.20, 60.31, 66.05, 97.38, 104.10, 108.14,
123.45, 124.07, 128.34, 129.43, 133.09, 149.00, 151.42, 153.60, 165.17, 167.61 ppm. HRMS
(ESI): Calcd. for [M + H]+ (C35H45N6O3) m/z 597.3548, found m/z 597.3562.
Cell culture and imaging
Hela cells were cultured in dulbecco's modified eagle medium (DMEM, Gibco) containing
10% fetal bovine serum. All cells were cultured at 37 °C in an ESCO cell incubator (Esco Micro
Pte.
Ltd.,
Singapore).
Cells
were
digested
with
0.25%
trypsin
(w/v)
and passaged at approximately 90% confluence every 2–3 days, and then cultured for 12 h in
glass bottom culture dishes (NEST Biotech Co. Ltd., Hong Kong). The cells were washed three
times by PBS buffer solution (pH 7.2). The ClO- dissolved in DMEM was then added into the
6

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dishes, at the final concentration at 200, 400, and 800 μM, respectively. The cells with ClO- were
incubated for 1.5 h, and washed three times by PBS buffer. And then the Hela cells were
incubated for 1.5 h with probe L in PBS/DMF (v/v = 5: 5, pH 7.2), which reached to a final
concentration at 10 μM. The Hela cells were washed by PBS buffer for three times, dissolved in 1
mL PBS and then monitored by a laser confocal microscopy (Nikon A1/A1R+, Japan). In the
experiments, the control group only contained the Hela cells and DMEM. The probe group gave
red fluorescence emission under confocal microscopy with the excitation wavelength at 565 nm.
Results and discussion
Synthesis of probe L
As depicted in Scheme 1, the reaction of rhodamine B and hydrazine hydrate obtained
rhodamine hydrazide 2, which then reacted with chloroacetyl chloride and triethylamine to afford
the intermediate 3. The probe L was obtained from N-alkylation of N-methyl piperazine with
intermediate 3. Probe L was characterized by 1H NMR, 13C NMR and HRMS. The chemical shift
for the protons of CH2CH2 and CH3 from N-methyl piperazine appeared in the 1H NMR spectrum
(Fig. S3.), and the new peaks at 165.17, 167.61 from the two carbonyl groups in 13C NMR
spectrum (Fig. S4.) confirmed the formation of probe L.
Spectroscopic properties of probe L and the detection of ClO-
The solution of the probe L (10 μM) in PBS/DMF (5: 5, v/v, pH 7.4) was colorless and had
no significant UV absorption at 400–700 nm and fluorescence at 450–700 nm. However, the
color of the solution changed from colorless to purple under visible light with the addition of
different concentrations of hypochlorite, and a significant absorption peak appeared at 565 nm in
7

Page 8 of 22
UV spectrum and a strong emission at 576 nm in fluorescence spectrum were observed. We
further found that the UV absorbance and fluorescence intensity of probe L increased
significantly when ClO- varied from 0–30 equiv., as shown in Figure 1. The linear correlations of
probe L were shown in Fig. S1 and Fig. S2.
In order to explore probe L as an efficient and selective probe, the fluorescence spectrum
2-
of probe L was examined with different anions and reactive oxygen species (Ac-, Cl-, SiO3 ,
SO4 , S2O3 , H2PO4 , NO3 , H2O2, MnO4 , ·OH, O2 , 1O2) in PBS/DMF solution (5:5, v/v, pH 7.4)
(Figure 2). All species except hypochlorite ion only induced weak emissive peaks for probe L
while probe L showed a very strong emissive peak in the presence of ClO-. Although these
interfering anions at high concentration above 14 equiv. of probe L did not affect the probe for
sensing hypochlorite, they had an impact at low concentrations less than 14 equiv. For this reason,
increasing the concentration of the probe can be helpful for the detection. And under visible light,
the color change of the solution of probe L with these interfering anions was not observed. These
fluorescence spectra results suggested that this probe might be a desirable probe for selectively
detecting hypochlorite ions.
2-
2-
-
-
-
·-
We next investigated the effect of the interfering anions on probe L for selective detection of
-
-
-
ClO-. After the addition of Ac- (1 mM), Cl- (1 mM), ClO3 (1 mM), H2PO4 (1 mM), MnO4 (20
-
μM), NO3 (1 mM), SO42- (1 mM), S2O32- (1 mM), SiO32- (1 mM), O2·- (1 mM KO2), 1O2 (1 mM
8

Page 9 of 22
Na2MoO4 + 2 mM H2O2) respectively to the solution of probe L containing 20 equiv.
hypochlorite, the fluorescence intensity did not significantly change, which indicated that these
anions had no effect on the selectivity of probe L, as illustrated in Figure 3. Interestingly, the
·
fluorescence intensity was slightly increased in the presence of H2O2 (1 mM) or OH (100 mM
Fe2+ + 1 mM H2O2). These two are active oxygen species and hypochlorite ion is also a reactive
oxygen species, and we thus speculate that H2O2 or ·OH results in a synergistic effect on ClO- for
the fluorescence intensity of this probe.
We next explored the effect of pH value on the probe L for detecting ClO-. The fluorescence
intensity of the probe with 20 equiv. hypochlorite under different pH value was increased
significantly. The intensity did not change markedly as pH converted from 6.76 to 7.68, whereas
a variation appeared when the pH was over 7.68 (Figure 4).
Fluorescent imaging of intracellular ClO-
Hela cells were incubated with probe L and hypochlorite at the concentration of 0.2, 0.4 and
0.8 mM, respectively, for intercellular ClO- imaging of Hela cells by a laser confocal microscope.
The cells only with the probe (final concentration at 10 μM) incubated at 37 °C for 1.5 h did not
show fluorescence, in agreement with our previous study, while the cells and probe L in the
presence of hypochlorite manifested strong red fluorescence. The intracellular fluorescence
9

Page 10 of 22
images were shown in Figure 5, which indicated that the probe could penetrate into Hela cells
and can detect hypochlorite ions in viable cells. In addition, Hela cells can be pierced by this
probe within 1.5 h, which is more practical than other low-permeability probes. We also found
the fluorescence intensity in cells increased when the concentration of hypochlorite increased.
MTT assay for toxicity of probe L
Then MTT assay has been carried out to assay the toxicity of probe L to cells. After incubation
of Hela cells with probe L at 5, 10, 15, 20, 25 μM, respectively for 24 h, the cell viability
remained similar to the control group, as shown in Figure 6. Higher concentration at 25 μM did
not decrease the viability, however, the survival rate of Hela cells was slightly higher than that of
the untreated group. Therefore, probe L was non-toxic to Hela cells and thus expected to be
safely applied in cell marking and clinical use in physiological conditions.
Conclusions
In summary, the synthesized rhodamine-based probe L for fluorescent recognition of ClO- in
living cells has been developed in the present study. The probe is highly selective and sensitive to
ClO- without the interference from other anions, and also has the property of fast color change,
easy detection, and direct observation through naked eyes. Probe L is nontoxic and has good
penetrability to Hela cells, and has been successfully applied to fluorescent imaging of living
10

Page 11 of 22
cells by sensing hypochlorite.
Acknowledgements
We would like to thank the Southwest University Undergraduate Training Programs for
Innovation and Entrepreneurship, China (20152902005), the Fundamental Research Funds for the
Central Universities, P. R. China (XDJK2016A015), Technology and Program Project of the
Science and Technology Department of Sichuan Province, China (2015FZ0070) for financial
support.
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Schemes and figures captions:
Scheme 1. The synthetic route of probe L. Reagents and conditions: (a) ethanol, reflux, 24 h; (b)
Et3N, CH2Cl2, r.t., 4 h; (c) K2CO3, CH3CN, reflux, 18 h.
Figure 1. The UV spectrum (a) and fluorescence spectrum (b) of L (10 μM) in PBS/DMF
solution (v/v = 5: 5, pH 7.4) in the presence of ClO- (0–30 equiv.). λex = 565 nm, λem = 576 nm.
Figure 2. The fluorescence spectra of L (10 μM) in PBS/DMF solution (v/v = 5: 5, pH 7.4) with
-
20 equiv. ClO- or other anions, λex = 565 nm. The anions were Ac- (1 mM), Cl- (1 mM), ClO3 (1
-
-
-
2-
2-
2-
mM), H2PO4 (1 mM), MnO4 (20 μM), NO3 (1 mM), SO4 (1 mM), S2O3 (1 mM), SiO3 (1
·-
·
mM), O2 (1 mM KO2), 1O2 (1 mM Na2MoO4 + 2 mM H2O2), H2O2 (1 mM) or OH (100 mM
Fe2+ + 1 mM H2O2).
Figure 3. Fluorescence intensity of probe L (10 μM) in PBS/DMF solution (v/v = 5: 5, pH 7.4)
with 20 equiv. ClO- ion with or without the interfering ions, λex = 565 nm, λem = 576 nm.
Figure 4. Fluorescence intensity at 576 nm of probe L in different pH PBS/DMF solution (v/v =
5: 5) before and after the addition of 20 equiv. ClO-. λex = 565 nm, λem = 576 nm.
Figure 5. Fluorescent imaging of ClO- in Hela cells incubated with probe L. (a–c) Hela cells only
incubated with 10 μM probe for 1.5 h at 37 °C. (d–f) Hela cells incubated with 0.2 mM ClO- in
14

Page 15 of 22
the growth medium for 1.5 h and then incubated with 10 μM probe for 1.5 h at 37 °C. (g–i) Hela
cells incubated with 0.4 mM ClO- in the growth medium for 1.5 h and then incubated with 10 μM
probe for 1.5 h at 37 °C. (j–l) Hela cells incubated with 0.8 mM ClO- in the growth medium for
1.5 h and then incubated with 10 μM probe for 1.5 h at 37 °C; (a), (d), (g) and (j) are the
fluorescent images. (b), (e), (h) and (k) are the images of the bright field. (c), (f), (i) and (l) are
merged images for (a) and (b), (d) and (e), (g) and (h), (j) and (k), respectively. Scale bar: 100
μm.
Figure 6. The viability of Hela cells after cultured with different concentrations of probe L (5, 10,
15, 20, 25 μM) for 24 h.
15

Page 16 of 22
O
COOH
NH2NH2.H2O
a
N
NH2
Cl
N
O
N
N
O
N
2
1
CH3
N
O
O
Cl
O
N
O
N
O
HN
N
CH3
Cl
N
NH
NH
Cl
c
b
N
O
N
N
O
N
3
L

Page 17 of 22
0.6
3000
2500
2000
1500
1000
500
a)
b)
0.5
0.4
0.3
0.2
0.1
0.0
30 equiv.
0 equiv.
30 equiv.
0 equiv.
0
550
575
600
625
650
450
500
550
600
650
Wavelength (nm)
Wavelength (nm)

Page 18 of 22
1800
1600
1400
1200
1000
800
600
400
200
0
-
ClO
-
MnO4
only L,
other anions
550
575
600
625
650
Wavelength (nm)

Page 19 of 22
3000
2500
2000
1500
1000
500
L + ions
-
L + ClO + other ions
0
-
-
-
-
2
-
-
-
2
-
-
-
-
2

4
3
2
l
3
4
2
3
4
2
3
c

O
O
O
2
O
O
O
P
2
O
1
C
O
i
O
l

l
O
O

A
n
N
2
S
C
H
C
S
S
M
H

Page 20 of 22
2000
1800
1600
1400
1200
1000
800
only L
L+20 equiv. ClO
-
600
400
200
0
6.0
6.5
7.0
7.5
8.0
pH

Page 21 of 22

Page 22 of 22
120
80
40
0
Ctr
5
10
15
20
25
Concentration of probe L ()