A sensitive BODIPY-based fluorescent probe suitable for hypochlorite detection in living cells

Article abstract of DOI:10.1016/j.jphotochem.2017.10.037

A fluorescent probe composed of BODIPY and 2,4-dinitrophenylhydrazine was designed for the detection of ClO^?. The oxidizing reaction between probe and ClO^? resulted in a distinct fluorescence enhancement together with a color variation from pink to orange. This probe showed excellent selectivity to ClO^? among various ions including common reactive oxygen species and high sensitivity with a detection limit of 228 nM. A fast response (7 min) was observed, which made the probe a promising method in real-time detection. Furthermore, this probe was successfully applied to monitor ClO^? in real-life water and living cells.

Full text of DOI:10.1016/j.jphotochem.2017.10.037

Journal of Photochemistry and Photobiology A: Chemistry 352 (2018) 65–72
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Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Full length article
A sensitive BODIPY-based fluorescent probe suitable for hypochlorite
detection in living cells
c
b
Yuanyuan Lia, Yong Tangb, Mengmeng Gaob, Yun Wangb, , Juan Han , Jinchen Xia ,
*
Lei Wangb, Xu Tangb, Liang Nib
a
Jingjiang College, Jiangsu University, Zhenjiang 212013, PR China
School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, PR China
b
c
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, PR China
A R T I C L E I N F O
A B S T R A C T
Article history:
A fluorescent probe composed of BODIPYand 2,4-dinitrophenylhydrazine was designed for the detection
of ClOꢀ. The oxidizing reaction between probe and ClOꢀ resulted in a distinct fluorescence enhancement
together with a color variation from pink to orange. This probe showed excellent selectivity to ClOꢀ
among various ions including common reactive oxygen species and high sensitivity with a detection limit
of 228 nM. A fast response (7 min) was observed, which made the probe a promising method in real-time
detection. Furthermore, this probe was successfully applied to monitor ClOꢀ in real-life water and living
cells.
Received 6 July 2017
Received in revised form 19 October 2017
Accepted 21 October 2017
Available online 28 October 2017
Keywords:
Fluorescent probe
Hypochlorite
BODIPY
© 2017 Elsevier B.V. All rights reserved.
Bioimaging
1. Introduction
like amide, thiosemicarbazone, p-methoxyphenol, oxime have
been developed to detect ClOꢀ [12–16]. Nonetheless, some
Hypochlorite (ClOꢀ), as a biologically significant part of the
reactive oxygen species (ROS), plays a vital role in various
physiological processes [1]. Endogenous hypochlorite, which is
produced mainly from the reactivity of H2O2 and Clꢀ by
myeloperoxidase(MPO) in leukocytes, has antibacterial effects
on the biological immune-defense system [2]. A low concentration
of hypochlorite can devastate invasive bacteria and pathogens,
which is particularly crucial to physiological balances of proteins,
DNA and RNA [3,4]. Unfortunately, the excessive hypochlorite may
cause tissue damage and a variety of human diseases, including
atherosclerosis, cardiovascular diseases, osteoarthritis, cystic
fibrosis cancers and pulmonary lesions [5–9]. Hypochlorite is also
widely used as disinfector in our daily life, for example, in
swimming pool water and drinking water [10]. Owing to the
biological importance of ClOꢀ, the development of detection
methods for ClOꢀ in both real-life water and living cells has
become an important issue.
limitations of these probes appeared during the detection process,
such as delayed response time, low quantum yield, poor
biocompatibility.
In this work, a new fluorescent probe (Probe 1) based on C N
¼
bond transformation was designed and synthesized to detect ClOꢀ.
BODIPY was selected for the basic fluorophore in consideration of
excellent light stability, low molecular weight, high fluorescence
quantum yield, admirable cells’ penetration [17]. Probe 1 was non-
fluorescent because C N isomerization [18,19] quenched the
¼
fluorescence of fluorophore, but exhibited significant fluorescence
enhancement in response to ClOꢀ. Excellent properties like high
sensitivity and selectivity, rapid response, make Probe
1 a
promising method for ClOꢀ detection in neutral water and
biological cells.
2. Experimental
Fluorescent probe is a kind of powerful tool for ion detection,
due to the advantages of nontoxicity, spatial resolution capability,
sensitivity, simple operation, potential biological value [11].
Recently, some fluorescent probes containing functional groups
2.1. Chemicals and instruments
All chemicals used were of analytical grade, purchased form
Aldrich and Sinopharm Chemical Reagent Co., Ltd. (Shanghai,
China). Unless otherwise stated, these reagents were used without
further purification and redistilled water were the main solvent.
Mass spectrometry data were obtained with a Bruker ESQUIRE
HPLC–MS AB 4000Q mass spectrometer. Ultraviolet-visible
* Corresponding author: Tel: +86 158 9638 5156; Fax: +86-0511-88791800.
E-mail address: yunwang@ujs.edu.cn (Y. Wang).
https://doi.org/10.1016/j.jphotochem.2017.10.037
1010-6030/© 2017 Elsevier B.V. All rights reserved.

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Y. Li et al. / Journal of Photochemistry and Photobiology A: Chemistry 352 (2018) 65–72
Scheme 1. Synthesis of Probe 1.
spectrum data were obtained with UV-2550 spectrophotometer
and 1H NMR spectra were recorded on an AVANCE II 400 MHz
spectrometer (Bruker Bio Spin). Varian Cary Eclipse Fluorescence
spectrophotometer was used to measure fluorescence spectra. An
Olympus Zeiss 710 laser scanning confocal microscopy was used
for Fluorescence image of cells. All the samples were tested in
DMF-PBS solution (1:1 v/v, 0.01 M PBS, pH 7.4) at room tempera-
ture.
buffered saline, pH 7.4) (1:1, v/v) for the spectral analysis. The
stock solutions of some ions (10 mM for Fꢀ, Clꢀ, NO2ꢀ, ClO4
,
ꢀ
HCO3ꢀ, H2PO4ꢀ, SO42ꢀ, S2O32ꢀ, CO32ꢀ, Fe3+, Cu2+, H2O2, ONOOꢀ,
ROOꢁ, ꢁOH, NOꢁ and ClOꢀ) and the blank solution were prepared in
deionized water. NaOCl and H2O2 stock solutions were respec-
tively obtained by diluting the commercialization solutions
(14.5% and 30%). Peroxynitrite (ONOOꢀ) was provided by nitro-
zation of NaNO2 and H2O2. Peroxyl radicals (ROOꢁ) was prepared
from 30 min stirring of 2,20-Azobis (2-amidinopropane) dihydro-
chloride(AAPH) solution at 25 ꢂC. Hydroxyl radical (ꢁOH) was
derived from Fenton reaction with ferrous chloride and 10 equiv.
H2O2 [20]. Nitric oxide (NOꢁ) was obtained from sodium
nitroferricyanide (III) dehydrate (SNP).
2.2. Synthesize of Probe 1
The intermediate products, Compound 1 and 2 were prepared
according to our previous work [17]. As illustrated in Scheme 1,
69.3 mg(0.35 mmol)of 2,4-dinitrophenylhydrazine and 105.5 mg
(0.3 mmol)of Compound 2 were added in the mixed solution (ethyl
alcohol-ethyl acetate, v:v, 1:1). Then 5 drips of acetic acid were
added and the mixture was stirred for 2 h at room temperature. The
reaction progress was monitored by TLC (petroleum ether- ethyl
acetate, v:v, 1:2) until Compound 2 was consumed. The crude
product was recrystallized from ethyl alcohol and further purified
by column chromatography (petroleum ether-dichloromethane,
v: v, 1:1) to give Probe 1 (139.6 mg, 87.3%). 1H NMR (400 MHz,
2.4. Calculation of fluorescent quantum yield [21]
Rhodamine B dissolved in EtOH (quantum yield
Ks = 0.97) was
used as the standard. The quantum yields of probe in the absence
and presence of NaClO were estimated according to the following
equations:
ꢀ
ꢁꢀ ꢁꢀ
ꢁ
2
AS
AX
FX
FS
nX
nS
FX
¼
Fs
CDCl3, ESI)
d 11.21 (s, 1H), 9.12 (dd, J = 12.7, 2.5 Hz, 1H), 8.30
(dd, J = 9.6, 2.4 Hz, 1H), 8.09 (s, 1H), 7.80 (d, J = 9.6 Hz, 1H), 7.63-7.49
(m, 3H), 7.31 (dd, J = 6.5, 2.9 Hz, 2H), 6.11 (s, 1H), 2.84 (s, 3H), 2.62
(s, 3H), 1.60 (s, 3H), 1.41 (s, 3H). MS (ESI): 533.70 [M + H]+.
Where
K is the quantum yield; A is absorbance of excitation
wavelength; F is integrated area under the corrected emission
spectra; the subscript S and X stand for the standard and for the
unknown, respectively.
2.3. Preparation of test solutions
2.5. Measurement of the detection limit [22]
The corresponding amount of Probe 1 solid was dissolved in
DMF to prepare a stock solution (1 mM). The test solutions were
diluted with the mixture of DMF and 0.01 M PBS (phosphate-
The detection limit (D) based on the fluorescence titrations was
estimated according to the following equations: Detection limit =
Fig. 1. (a) Fluorescence response of Probe 1 (10
the absence and presence of 400 ex = 470 nm.
M ClOꢀ
m mM) in
M) in presence of different concentrations of ClOꢀ; (b) Effect of pH value on the fluorescence intensity of Probe 1 (10
m
. l

Y. Li et al. / Journal of Photochemistry and Photobiology A: Chemistry 352 (2018) 65–72
67
3
s
/k, where
s
is the standard deviation of the blank measure-
another 10 min incubation. Finally, HepG2 cells were rinsed by the
ment, and k is the slope for the fluorescence intensity versus the
concentration of target ion. To gain the standard deviation of the
blank measurements, the S/N ratio of Probe 1 was not obtained
until the emission intensity of Probe 1 were measured in the
absence of target ions by twenty times.
same way for fluorescence imaging.
3. Results and discussion
3.1. Effects of pH and time
We sought optimal [H+] concentration while monitoring
fluorescence intensity. Fig. 1b exhibited the maximum fluores-
cence intensity of Probe 1 solution in the absence and presence of
ClOꢀ. Probe 1 solution was found almost non-fluorescent at pH
4–12 while the mixed solution of Probe 1 and ClOꢀ showed the
maximum fluorescence at pH range from 6 to 8. This phenomenon
revealed that the decreasing reactivity of ClOꢀ under acidic or
alkaline conditions could lead to the decline of fluorescence
intensity. Hence, the pH 7.4 was chosen to be the test condition
throughout the following experiment, which was appropriate for
ClOꢀ detection in bioimaging. Time dependence is another key
factor and five types of solutions were tested. As shown in Fig. 1a,
Probe 1 had no fluorescence even though the time was extended to
900 s. After the addition of ClOꢀ, the intensity was dramatically
increased and then held wave nearby a constant value within
7 min, which illustrated that Probe 1 could respond to ClOꢀ in real
time and certified the stability of Probe 1 under the same
condition.
2.6. Practical application
To explore the applicability of Probe 1 for the detection of ClOꢀ,
filter papers (1 ꢃ1.5 cm2) were immersed in the solution of Probe 1
(10 mM) for 20 min and then desiccated in air to prepare test strips.
Finally, various concentrations of ClOꢀ were added dropwise to
these strips and dried up for observation.
2.7. Cell incubation and fluorescence imaging
HepG2 cells seeded into 24-well plates were incubated in
Dulbecco's modified Eagle's medium (DMEM) with 10% FBS (fetal
bovine serum), and maintained in a humidified atmosphere
containing 5% CO2 at 37 ꢂC for 24 h. Subsequently, Probe 1
(2
the residue probe was washed by PBS buffer for 3 times. Then
sodium hypochlorite (80 M) was added into some plates for
mM) was cultured with the well-grown cells for 30 min and
m
Fig. 2. (a) Absorbance spectra of Probe 1 (10
1 (10 M) with 400 M of other ions, the illustration is the photo of these solutions under UV light (365 nm) illumination; (c) Fluorescence spectra response
M of ClOꢀor400
of Probe 1 (10 M) in the presence of other anions (400
M) to ClOꢀ (400 M). The order of the ions is: 1 blank, 2 Fꢀ, 3 Clꢀ, 4 ClO4ꢀ, 5 HCO3ꢀ, 6 S2O32ꢀ, 7 Fe3+, 8 CO32ꢀ, 9H2O2,
10 ONOOꢀ, 11 ROOꢁ, 12 ꢁOH, 13 NOꢁ, 14 ClOꢀ
ex = 470 nm.
mM) with 400 m
M of ClOꢀ or other ions, the illustration is the photo of these solutions; (b) Fluorescence emission spectra of Probe
m
m
m
m
m
m
.
l

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Y. Li et al. / Journal of Photochemistry and Photobiology A: Chemistry 352 (2018) 65–72
Fig. 3. (a) Fluorescence emission spectra of Probe 1 (10
m
M) with increasing amount of ClOꢀ (0ꢀ380
mM). The illustration is the photo of the solutions under UV light
(365 nm) illumination. (b) The fluorescence intensity of Probe 1 (10
m
M) at 515 nm versus the ClOꢀ concentration (0ꢀ380
mM). The illustration shows the linearity relative to
the ClOꢀ concentration: intensity = 2.70 [ClOꢀ]ꢀ6.23, R2 = 0.9980.
lex = 470 nm.
Table 1
A comparison of related probes (structures and statistics) reported for ClOꢀ fluorescence detection.
Probe
Naked-eye
None
Response time
None
Bioimaging
None
Detection limit
6.4 nM
Reference
Analyst, 2017 [23]
None
None
7 min
3 min
HepG2
L929
356 nM
Chem.Commun., 2015 [24]
3800 nM
Sen. Actuators B: Chem.,2016 [25]
Colourless to yellow
None
None
2000 nM
Tetrahedron, 2016 [26]
None
None
10 min
90 min
None
Hela
1400 nM
200 nM
J. Fluoresc., 2017 [27]
Talanta, 2017 [28]
None
5 min
HepG2
A549
600 nM
Sen. Actuators B: Chem.,2017 [29]
Sen. Actuators B: Chem.,2017 [30]
Yellow to none
10 min
2660 nM
Pink to
orange
7 min
HepG2
228 nM
This work

Y. Li et al. / Journal of Photochemistry and Photobiology A: Chemistry 352 (2018) 65–72
69
Scheme 2. Specific reaction process of Probe 1 with ClOꢀ
.
3.2. Selectivity studies
the colorimetric and fluorescent sensing analysis of ClOꢀ and been
applied in the multitudinous actual systems successfully.
The absorption and fluorescence spectra of Probe 1 were
measured with addition of 12 kinds of ions (the first one is the
blank) to Probe 1 solution, in order to evaluate the selectivity of
Probe 1. In the UV–vis absorption spectrum (Fig. 2a), the maximum
absorption of the blank and other competing ions emerged at
543 nm. Upon addition of ClOꢀ, the absorption band at 543 nm for
3.3. Sensitivity studies
The titration experiments with addition of ClOꢀ revealed the
relationship between the emission intensity and the concentration
of ClOꢀ. Probe 1 solution was non-fluorescent (
K1 = 0.059). With
Probe
1 decreased and became blue-shifted (505 nm). The
ClOꢀ added, the emission intensity of Probe 1 solution dramatically
illustration showed that Probe 1 solutions in the presence of
competing ions were pink while the solution with an addition of
ClOꢀ was orange. This phenomenon illustrated that Probe 1 could
be utilized for visual detection of ClOꢀ. Fig. 2b showed the emission
of these solutions. The addition of competing ions (40 equiv.) did
not cause obvious emission changes of the fluorescence. In
contrast, the fluorescence intensity of the samples with 40 equiv.
of ClOꢀ dramatically increased and the solution emitted green
fluorescence (illuminated by UV light at 365 nm in the illustration).
Further studies was carried out by adding 40 equiv. of ClOꢀ to other
13 kinds of solutions and measured the maximum fluorescence
emission intensity. It could be found in Fig. 2c that the intensity
was almost the same with the blank. That is to say, the fluorescence
response to ClOꢀ was impervious to other ions. Both the UV
absorption spectrum and the fluorescence emission spectrum
demonstrated that Probe 1 was able to selectively detect ClOꢀ
among many common ions and ROS. All in all, Probe 1 has realized
went up (K2 = 0.56). Moreover, the fluorescence was gradually
enhanced with the increasing concentration of ClOꢀ (Fig. 3a).
When the concentration of ClOꢀ becomes 36 equiv. compared to
Probe 1, the fluorescence intensity attains saturation. This
fluorescent variation is visible under the UV light at 365 nm in
the illustration of Fig. 3b. Besides, in the range of NaOCl 0–340 mM,
the fluorescent intensity showed a pretty good linear relationship
with the ClOꢀ concentration and the linear coefficient was 0.998
(Fig. 3b). Detection limit based on this linearity was calculated to
be 228 nM, which was more sensitive than most reported probes.
These results demonstrated that the probe could be used for the
accurate quantitative analysis.
Comparing to the chemosensors listed in Table 1 and reported
in some other studies [14,15], Probe 1 exhibited tremendous
superiorities in those aspects of visual inspection, response time,
detection limit and bioimaging. The probe also realized the
recognition of hypochlorite by dual methods. Accompanied by the
Fig. 4. Mass spectrum for the product from the reaction of Probe 1 and ClOꢀ
.

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Y. Li et al. / Journal of Photochemistry and Photobiology A: Chemistry 352 (2018) 65–72
Fig. 5. Photos of test strips for detecting ClOꢀ in aqueous solution, the samples are:
1 blank, 2 tap water + 30
m
M ClOꢀ, 3 tap water + 100
M ClOꢀ
m mM
M ClOꢀ, 4 tap water + 300
ClOꢀ, 5 tap water + 400
m
.
Fig. 6. Cell viability of Probe 1 by CKK-8 assay.
research of C N bond transformation, the choices of recognition
¼
site and fluorophore were the dominant key to synthesize the
better chemosensor. Besides, the probe was designed for ClOꢀ
detection with the colorimetric and fluorescent sensing methods,
rapid response, good selectivity, excellent sensitivity and great
biocompatibility.
353 [M + H]+ was clearly observed, which was in accordance with
the proposed product.
3.5. Application test in test strips
For visual recognition of ClOꢀ, test strips based on Probe 1 were
3.4. Proposed reaction mechanism
applied to tap water. Distilled deionized water/tap water, 30
ClOꢀ/tap water, 100 M ClOꢀ/tap water, 300 M ClOꢀ/tap water
and 400
M ClOꢀ/tap water were added into the test paper and
mM
m
m
The proposed reaction mechanism is illustrated in Scheme 2.
m
The C
fluorophore [13,14]. So Probe 1 exhibited no fluorescent on account
of the existence of C N
N bond. Upon ClOꢀ added, the C
¼
N isomerization is thought to quench fluorescence of many
dried at room temperature. The color of paper in Fig. 5 changed
from pink to orange and green fluorescence appeared under UV
light (365 nm) illumination. After adding a higher concentration of
ClOꢀ, the paper revealed stronger fluorescence. Through visual
observation, the ClOꢀ concentration could be estimated roughly
without fluorescent instruments. This obvious phenomenon
¼
¼
isomerization was removed by oxidation reaction between Probe 1
and ClOꢀ. As a result, the product recovered the strong fluores-
cence of BODIPY group. To confirm the mechanism, the reaction
product was proved by mass spectrometry (Fig. 4). The peak at m/z
Fig. 7. Confocal fluorescence images of HepG2 cells. (a) and (b) are the bright-field images of HepG2 cells loaded with 2
ClOꢀ (80
M) for another 10 min. (c) and (d) represent the fluorescence images of (a) and (b), respectively.
mM Probe 1 for 30 min in the absence and presence of
m

Y. Li et al. / Journal of Photochemistry and Photobiology A: Chemistry 352 (2018) 65–72
71
demonstrated that Probe 1 is applicable for visual detection of ClOꢀ
in real water samples.
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3.7. Cellular imaging
To further investigate the practical applicability of Probe 1 in
biological researches, the fluorescence imaging was carried out in
HepG2 living cells. As expected, the HepG2 cells (Fig. 7c) loaded
only with Probe 1 (2
kept consistent with the fluorescence selectivity studies. Then the
HepG2 cells were incubated with sodium hypochlorite (80 M) for
another 10 min under the same condition. These cells exhibited
obvious green fluorescence (Fig. 7d), verifying excellent cell
permeability and stable fluorescence behavior of Probe 1 in
biosystem. Hence, these results indicated evidently that Probe 1
was cell-permeable and capable of imaging exogenous ClOꢀ in
living HepG2 cells.
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m
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In summary, a novel fluorescent probe based on the integration
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synthesized to monitor ClOꢀ. Upon adding ClOꢀ to Probe 1
solution, a naked-eye visible variation from pink to orange with a
remarkable fluorescence increase were observed. Moreover, Probe
1 was suitable for the quantitation in complex ionic environment
with high sensitivity and rapid response. With those attractive
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Nos. 31470434, 21576124, 21507047 and
21676124), China Postdoctoral Science Foundation funded project
(No. 2017M610308), Zhenjiang Social development project (No.
SH2016019), and the Project supported by the Science Foundation
of Jiangsu Entry-exit Inspection Quarantine Bureau(Nos. 2016KJ51
and 2017KJ47).
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