UV and fluorescent spectra study the reaction between 1, 8-Naphthalimide derivative and hypochlorite their applications

Article abstract of DOI:10.1016/j.saa.2016.11.019

Two simple, efficient turn-on fluorescent probes for hypochlorite have been rationally designed and developed by utilizing the oxidation of hypochlorite. Notably, probe 1 and 2 displayed rapid and remarkable turn-on responses to ClO^? in PBS buffer solution (pH 7.4). Further, the optical properties of two probes and their ClO^?-addition products were confirmed by density functional theory calculations. And detection limits of two probes for ClO^? based on the definition by IUPAC were calculated for 2.882 nM and 0.354 μM. More importantly, cell imaging experiments demonstrated that probe 1 was more suitable for detecting the ClO^? in living A549 cells. And both two probes had the possibility of potentially applied in practical applications such as detecting the hypochlorite concentration of tap water and river water.

Full text of DOI:10.1016/j.saa.2016.11.019

UV and fluorescent spectra study the reaction between 1, 8-Naphthalimide
derivative and hypochlorite their applications
Jiawei Li, Tao Liu, Fangjun Huo, Jianbin Chao, Yongbin Zhang, Caixia
Yin
PII:
DOI:
Reference:
S1386-1425(16)30673-4
doi:10.1016/j.saa.2016.11.019
SAA 14779
To appear in:
Received date:
Revised date:
Accepted date:
20 August 2016
2 November 2016
13 November 2016
Please cite this article as: Jiawei Li, Tao Liu, Fangjun Huo, Jianbin Chao,
Yongbin Zhang, Caixia Yin, UV and fluorescent spectra study the reaction be-
tween 1, 8-Naphthalimide derivative and hypochlorite their applications, (2016),
doi:10.1016/j.saa.2016.11.019
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1 -
UV and fluorescent spectra study the reaction between 1,
-Naphthalimide derivative and hypochlorite their applications
8
a
a
b,
b
b
§
Jiawei Li ,§Tao Liu, Fangjun Huo *, Jianbin Chao , Yongbin Zhang , Caixia Yin
a,
**
a
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key
Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Institute of
b
Molecular Science, Shanxi University, Taiyuan, 030006, China; Research Institute of Applied
Chemistry, Shanxi University, Taiyuan, 030006, China.
*Corresponding author: C.X. Yin, E-mail: yincx@sxu.edu.cn;
**Corresponding author: F.J. Huo, Email: huofj@sxu.edu.cn.
§Jiawei Li and Tao Liu contributed equally
Abstract: Two simple, efficient turn-on fluorescent probes for hypochlorite have
been rationally designed and developed by utilizing the oxidation of hypochlorite.
−
Notably, probe 1 and 2 displayed rapid and remarkable turn-on responses to ClO in
PBS buffer solution (pH 7.4). Further, the optical properties of two probes and their
−
ClO -addition products were confirmed by density functional theory calculations. And
−
detection limits of two probes for ClO based on the definition by IUPAC were
calculated for 2.882 nM and 0.354 μM. More importantly, cell imaging experiments
−
demonstrated that probe 1 was more suitable for detecting the ClO in living A549
cells. And both two probes had the possibility of potentially applied in practical
applications such as detecting the hypochlorite concentration of tap water and river
water.
Keywords: Turn-on; Fluorescent probes; Hypochlorite; Theory calculations; Cell
imaging

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1
. Introduction
A series of reactive oxygen species (ROS) can be produced in the normal
physiological metabolism of aerobic cells, which play an indispensable role in the
1
-3
metabolism of organisms and have a pivotal influence on the process of life. ROS at
a normal level are conducive to control and regulate a wide range of physiological
functions, but abnormal content of ROS can results in oxidative stress which are
4
,5
connected with the pathogenesis of many diseases. Hypochlorous acid (HOCl) and
−
its conjugate base hypochlorite (ClO ) are one type of important ROS. In living
organisms, the endogenous hypochlorite can be produced by peroxidation of chloride
6
,7
ions catalyzed the enzyme myeloperoxidase (MPO).
As important signaling
molecules, they can control and mediate a wide range of physiological and biological
process, such as control of cell life cycle, reparation of tissue as well as antimicrobial
activity in the defense system. Hypochlorite has an important antimicrobial effect in
living body, thus endogenous hypochlorite is very necessary for life. However, excess
concentration of hypochlorite can be implicated in many tissue damages and a variety
8
9
10
of human diseases, such as rheumatoid, cardiovascular diseases, lung injury,
1
1
12
13
hepatic ischemia-reperfusion injury, atherosclerosis, neuron degeneration and
1
4
even cancer. Additionally, hypochlorite is indispensable and essential for our daily
life, which can be often used as bleach, deodorant and disinfectant. Also, it is widely
used as a bleacher in paper pulp and textiles, a disinfectant in drinking water, milk
industries, a purifying treatment in swimming pool water, wastewater, cooling water
1
5,16
and a disinfectant agent in hospital and daily life and so on.
As a chlorinated

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disinfectant, its disinfection effect is done by the generation of hypochlorite. But
when hypochlorite as a chlorinated disinfectant is used, this process will produce
1
7
chlorate or chlorite, they have potential hazards to the human body. Thus all
countries in the world have already stipulated the use of the highest hypochlorite
concentration in drinking water. And our country has stipulated that hypochlorite is
handled as concentrated aqueous solutions, typically, in the concentration range of
−
5
−2
18
19
1
0 to 10 M, which is a potential health hazard to human and animals.
Therefore, to build and develop high efficient, selective and sensitive methods for
−
direct detection of ClO in environmental and other fields are of considerable
importance. Compared with traditional detection methods, fluorescent probes have
innate advantages including efficient selectivity, better sensitivity and quick response
time, some of which be allowed be used to monitor analyte at physiological levels in
20-23
vitro and in vivo.
Based on different fluorophores, fluorescent probes have been
developed based on coumarin, fluorescein, rhodamine, 1, 8-naphthalimide, BODIPY
2
4-28
and so on.
Generally speaking, 1, 8-naphthalimide type fluorescent materials are application
of a very wide range of important functional materials. From the molecular structure,
this kind of compound molecules have a rigid and good planarity naphthalene ring
structure unit, and it itself contains a strong electron acceptor and an electron donor
and exists an intramolecular charge transfer (ICT) process, so it has good
photoluminescence and electroluminescence properties. Because of these fascinating
and excellent properties such as high quantum yields, good photostability and large

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stokes shift, 1, 8-naphthalimide-based derivatives have been widely used in
fluorescence detection and imaging. By utilizing the virtue of its specific features, we
design and synthesized two simple, efficient 1, 8-naphthalimide-based fluorescent
−
probes for detecting ClO , which feature excellent selectivity and high sensitivity for
−
ClO over other analytes (Scheme 1).
Scheme 1 Synthesis of probe 1 and 2.
2
. Experimental
2
.1 Materials
Unless otherwise stated, all reagents and analytes were purchased from commercial
suppliers and were of analytical grade without further purification. H O was deionized
2

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and was used throughout all experiments. The solutions of anions were prepared from
their sodium salts, respectively. The spectroscopic measurements of probe 1 and 2
were performed in PBS buffer solution (pH 7.4) and PBS buffer solution (pH 7.4,
containing 20% DMSO), respectively.
2
.2. Instruments
A pH meter (Mettler Toledo, Switzerland) was used to determine the pH. Reactions
were carried out on the magnetic stirrers and their reaction processes were monitored
on thin layer chromatography (TLC). UV-Vis spectra and fluorescence spectra were
measured on a Cary 50 Bio UV-Visible spectrophotometer and a Hitachi F-7000
fluorescence spectrophotometer, respectively. A PO-120 quartz cuvette (10 mm) was
1
13
purchased from Shanhai Huamei Experiment Instrrument Plants, China. H NMR, C
NMR spectra were recorded on a Bruker AVANCE III-600 MHz and 150 MHz NMR
spectrometer, respectively (Bruker, Billerica, MA). MS determinations were carried
out on AB Triple TOF 5600plus System (AB SCIEX, Framingham, USA) and a
−
LTQ-MS (Thermo) instrument. The abilities of probes reacting to ClO in the living
cells were also evaluated by using a Leica DMi8 laser scanning microscope.
2
2
.3. Preparation and characterization of probe 1 and probe 2
.3.1. Preparation and characterization of A
2
9
The probes synthesis routes were using a modification of a literature method and
summarized in Scheme 1. A mixture of 4-bromo-1, 8-naphthalic anhydride (8.28 g, 30
mmol) and n-butylamine (2.19 g, 30 mmol) in 60 mL of ethanol was refluxed for 5 h.
After the reaction was completed, the reaction mixture was evaporated in vacuum and

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dried to obtain a solid product. Later on, the solid was purified by chromatography on
a silica gel column in dichloromethane resulted in a pale yellow solid product A (8.74
1
g) in 88% yield. H NMR (DMSO-d , 600 MHz): δ (ppm): 8.53 (dd, J = 7.9 Hz, 2H),
6
8
7
.30 (d, J = 7.6 Hz, 1H), 8.19 (d, J = 7.7 Hz, 1H), 7.98 (t, J = 7.8 Hz, 1H), 4.02 (t, J =
13
.3 Hz, 2H), 1.61 (m, 2H), 1.36 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H). C NMR
(
DMSO-d , 150 MHz): δ (ppm): 163.22, 163.17, 132.95, 131.94, 131.74, 131.32,
6
1
30.14, 129.51, 129.17, 128.61, 123.10, 122.32, 30.02, 20.27, 14.17. HR-MS of the
+
+
compound A: m/z: [compound A+ H] Calcd for C H BrNO 332.0281, Found
1
6
15
2
3
32.0286 (Fig. S1).
2
.3.2. Preparation and characterization of B
To a 100 mL radius flask, A (20 mmol, 6.62 g) and sodium methoxide (20 mmol,
1
.36 g) in methanol (40 mL) was refluxed for 8 h while CuSO ·5H O served as
4
2
catalyst. Then the crude product was evaporated in vacuum and the residue was added
HCl (1 mol/L, 30 mL) to crystallize out. Subsequently, a solid separated by filtration
was purified by chromatography on a silica gel column in dichloromethane to give a
1
white product B (4.93 g) in 87% yield. H NMR (DMSO-d , 600 MHz): δ (ppm): 8.49
6
(d, J = 8.3 Hz, 1H), 8.46 (d, J = 7.2 Hz, 1H), 8.42 (d, J = 8.2 Hz, 1H), 7.79 (t, J = 7.8
Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 4.12 (s, 3H), 4.01 (t, J = 7.4 Hz, 2H), 1.60 (m, 2H),
1
3
1
.35 (d, J = 14.8, 7.4 Hz, 2H), 0.93 (t, J = 7.4 Hz, 3H). C NMR (DMSO-d , 150
6
MHz): δ (ppm): 163.99, 163.36, 160.74, 133.70, 131.46, 128.97, 128.66, 126.83,
23.18, 122.34, 114.69, 106.71, 57.08, 30.18, 20.29, 14.20. HR-MS of the compound
1
+
+
B: m/z: [compound B+ H] Calcd for C H NO 284.1281, Found 284.1286 (Fig.
17
18
3

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7 -
S2).
2
.3.3. Preparation and characterization of C
Compound B (15 mmol, 4.25 g) was added to 55% HI solution with refluxing at
40°C for 7 h, and then the mixture solution was cooled to room temperature. After
1
that, the mixture was slowly poured into ice water, filtered and washed with distilled
water. The pale yellow solid thus obtained was dried under vacuum to give compound
1
C (3.75 g) in 93% yield. H NMR (DMSO-d , 600 MHz): δ (ppm): 11.88 (s, 1H), 8.54
6
(d, J = 8.3 Hz, 1H), 8.48 (d, J = 7.2 Hz, 1H), 8.36 (d, J = 8.1 Hz, 1H), 7.77 (t, J = 7.7
Hz, 1H), 7.16 (d, J = 8.1 Hz, 1H), 4.02 (t, J = 7.3 Hz, 2H), 1.60 (m, 2H), 1.34 (m, 2H),
1
3
0
1
3
.92 (t, J = 7.3 Hz, 3H). C NMR (DMSO-d , 150 MHz): δ (ppm): 164.13, 163.46,
6
60.70, 134.02, 131.59, 129.64, 129.34, 126.08, 122.84, 122.28, 113.08, 110.42,
+
0.22, 20.29, 14.21. HR-MS of the compound C: m/z: [compound C+ H] Calcd for
+
C H NO 270.1125, Found 270.1136 (Fig. S3).
16
16
3
2
.3.4. Preparation and characterization of D
To a stirred 20 mL trifluoroacetic acid, C (10 mmol, 2.69 g) and
hexamethylenetetramine (13 mmol, 1.82 g) was added, and then the mixture was
refluxed at 120 ℃ for 10 h. When the remained solution was cooled to room
temperature, it was slowly poured into the mixture solution of 50 mL CHCl and 50
3
mL HCl (1 mol/L) and then the mixture was warmed at room temperature whilst
stirring overnight. Followed that, separate the organic phase with a separatory funnel
and wash the aqueous phase three times with CHCl (20 mL×3). The merged organic
3
layer was dried with sodium sulfate and evaporated in vacuum to dry to a solid. Then

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the solid was purified by column chromatography on a silica gel (ethyl acetate:
1
petroleum ether = 1: 3) to get a pale yellow product D (1.66 g) in 56% yield. H NMR
(
DMSO-d , 600 MHz): δ (ppm): 10.37 (s, 1H), 8.70 (m, 2H), 8.57 (d, J = 7.3 Hz, 1H),
6
7
.87 (t, J = 7.8 Hz, 1H), 4.02 (t, J = 7.4 Hz, 2H), 1.60 (m, 2H), 1.35 (d, J = 14.8, 7.5
1
3
Hz, 2H), 0.93 (t, J = 7.4 Hz, 3H). C NMR (DMSO-d , 150 MHz): δ (ppm): 194.51,
6
1
65.10, 163.69, 163.02, 134.00, 133.21, 131.70, 130.50, 127.43, 124.03, 122.77,
117.59, 113.64, 30.13, 20.27, 14.20. HR-MS of the compound D: m/z: [compound D+
+
+
4
H] Calcd for C H NO 298.1074, Found 298.1080 (Fig. S4).
17
16
2
.3.5. Preparation and characterization of probe 1
A mixture of D (0.595 g, 2 mmol), hydroxylamine hydrochloride (0.555 g, 8 mmol)
and 30 μL piperidine in EtOH (25 mL) was stirred and refluxed at 80 ℃ over night.
After the reaction was complete, the mixture solution was then cooled to filtered and
washed with EtOH. The yellow solid thus obtained was dried under vacuum to give
1
probe 1 (0.562 g) in 90 % yield. H NMR (DMSO-d , 600 MHz): δ (ppm): 12.33 (s,
6
1
H), 11.89 (s, 1H), 8.75 (s, 1H), 8.63 (d, J = 8.3 Hz, 1H), 8.61 (s, 1H), 8.50 (d, J = 6.4
Hz, 1H), 7.84 (m, 1H), 4.05 (m, 2H), 1.61 (dt, J = 15.0, 7.5 Hz, 2H), 1.35 (m, 2H),
1
3
0
1
.93 (t, J = 7.4 Hz, 3H). C NMR (DMSO-d , 150 MHz) δ (ppm): 194.51, 165.10,
6
63.69, 163.02, 134.00, 133.21, 131.70, 130.50, 127.43, 124.03, 122.77, 117.59,
+
1
13.64, 30.13, 20.27, 14.20. ESI-MS of the probe 1: m/z: [Probe 1+ H] Calcd for
+
C H N O 313.12, Found 312.92 (Fig. S5).
17
17
2
4
2
.3.6. Preparation and characterization of probe 2
A mixture of D (0.595 g, 2 mmol), 1,4-dimethylpyridin-1-ium iodide (0.470 g, 2

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mmol) in EtOH (25 mL) was stirred and refluxed at 80 ℃ over night. After the
reaction was complete, the mixture solution was cooled to filtered and washed with
EtOH. The blackish green solid thus obtained was dried under vacuum to give probe 2
1
(
0.823 g) in 80 % yield. H NMR (DMSO-d , 600 MHz): δ (ppm): 8.53 (d, J = 6.2 Hz,
6
2
H), 8.48 (d, J = 7.2 Hz, 1H), 8.38 (s, 1H), 8.26 (d, J = 7.0 Hz, 1H), 8.12 (d, J = 15.4
Hz, 1H), 7.98 (d, J = 6.2 Hz, 2H), 7.90 (d, J = 15.3 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H),
1
3
4
.11 (s, 3H), 4.02 (m, 2H), 1.58 (m, 2H), 1.34 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H). C
NMR (DMSO-d , 150 MHz) δ (ppm): 164.57, 163.20, 155.64, 144.11, 142.81, 142.55,
6
1
2
3
2
32.43, 131.97, 130.88, 123.02, 121.70, 121.53, 118.83, 116.82, 101.45, 46.30, 30.48,
-
+
+
0.36, 14.30. HR-MS of the probe 2: m/z: [Probe 2-I ] Calcd for C H N O
3
24
23
2
87.1703, Found 387.1684 (Fig. S6).
.4. General UV-Vis and fluorescence spectra measurements
All UV-Vis spectra and Fluorescence spectra were recorded using a PO-120 quartz
cuvette (10 mm). Stock solutions (2 mM) of probe 1 and 2 were both prepared in 2
mL DMSO. The stock solutions of sodium hypochlorite (2 mM) were made by the
mixture of 5 mL H O and 1 mL sodium hypochlorite (13.4 mM). Other analytes
2
−
−
−
−
−
−
−
−
−
−
−
−
2
including ClO , ClO , ClO , F , Cl , Br , CN , SCN , MnO , N , NO , NO ,
2
3
4
4
3
3
2−
−
4−
3−
2−
−
CO , AcO , P O , PO , S and HSO were dissolved in deionized water for the
3
2
7
4
3
−
preparation of stock solutions (0.2 M). The stock solutions of ClO , H O and
2
2
2−
tBuOOH were using double distilled water to dilute and O was obtained by
dissolved KO in double distilled water. tBuO· was obtained by the reactions of H O
2
2
2
−
and tBuOOH with ferrous perchlorate, respectively. ONOO was obtained by the fast

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mixture of NaNO , H O and NaOH. In the investigated of the effect of pH on the
2
2
2
fluorescence spectra experiment, phosphoric acid (0.5 M) and sodium hydroxide
solution (0.5 M) were added sodium dihydrogen phosphate solutions (0.2 M) and
sodium hydrogen phosphate solutions (0.2 M) to adjust the different pH values (2-13).
The excitation wavelength of fluorescence spectra of probe 1 and 2 were 400 nm and
4
05 nm, respectively. And the excitation and emission slit widths of probe 1 and 2
were set to 2.5 nm and 5 nm, 5 nm and 5 nm, respectively.
2
.5. Imaging of A549 cells
The A549 cells were grown in Dulbecco’s Modified Eagle’s medium supplemented
with 12% Fetal Bovine Serum and 1% antibiotics at 37 ℃ in humidified
environment of 5% CO . Cells were plated on 6-well plate and allowed to adhere for
2
2
4 h. Before the experiments, cells were washed with PBS three times. The A549
were treated with 2 μM probe 1 (DMSO stock solution) in culture media for 30 min at
7 ℃ and then washed with PBS three times. For the control experiment, the cells
3
were treated with 5 mM NAC (N-acetyl-L-cysteine, it is a commonly used antioxidant)
in culture media for 30 min at 37 ℃. And then, the cells were washed with PBS three
times and incubated with 2 μM probe 1 in culture media for 30 min at 37 ℃. For
probe 2, the A549 were treated with 4 μM probe 2 in culture media for 30 min at
3
7 ℃ and then washed with PBS three times.
2
.6 Determination of quantum yield
Fluorescence quantum yield was defined as the ratio of the number of excited
molecules which were returned to the ground state by emitting fluorescence and the

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total number of excited state molecules. The quantum yields were determined using
3
0
quinoline sulfate (Φ = 0.58, 0.1 M H SO ) as a standard reference. The absorbance
r
2
4
values of probe 1 at 400 nm of samples were less than 0.02. Accordingly, the
absorbance values of probe 2 at 405 nm of samples were less than 0.02. The UV-Vis
absorption and accordingly emission spectra were recorded, respectively. And then,
the fluorescence quantum yield was calculated according to the formula as follow:
Among them, the subscripts of formula r and s represent standard reference and
sample, respectively.
3
. Results and Discussions
−
3
.1. UV-Vis and fluorescence spectra of detecting ClO
To investigate the sensitivity of two probes to hypochlorite, we first made UV-Vis
and fluorescence titration spectra experiments. As shown in Fig. 1a, the absorption
peaks of probe 1 were at 450 nm, 372 nm and 305nm. With increasing concentration
−
of ClO (0-147.4 μM) in PBS (pH 7.4) containing probe 1 (4 μM), the original
absorbance centered at 305 nm, 372 nm and 450 nm was gradually decreased with
obvious blue-shift from 450 nm to 421 nm. Accordingly, the visible color changed
from golden yellow to light green, which can be observed by the naked eye. For probe
−
2
, when addition of ClO (0-1.608 mM) in PBS buffer solution (pH 7.4, containing
2
0% DMSO) containing probe 2 (8 μM), the original absorbance centered at 349 nm
and 480 nm was gradually decreased with obvious blue-shift from 480 nm to 465 nm
Fig. 1b).
(

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−
Accordingly, upon addition of ClO (0-16 μM) in PBS (pH 7.4) containing probe 1
(2 μM), the fluorescence intensity at 515 nm (λex = 400 nm) increased with
remarkable fluorescence color change from colorless to green (Fig. 2a) and the
-
fluorescence quantum yield from Φf probe 1 = 0.004 to Φf probe 1-ClO = 0.423 with
quinoline sulfate as reference. As shown in Fig. 2b, the fluorescence intensity at 528
−
nm (λ = 405 nm) increased with increasing concentration of ClO (0-589.6 μM) in
ex
PBS buffer solution (pH 7.4, containing 20% DMSO) containing probe 2 (4 μM). And
-
fluorescence quantum yield from Φf probe 2 = 0.010 to Φf probe 2-ClO = 0.369 with
−
quinoline sulfate as reference. In addition, the probe 1, probe 1-ClO and probe 2,
−
probe 2-ClO had excellent photostability (Fig. S7).
(a)
(b)
Fig. 1. (a) The absorption spectra of the probe 1 (4 μM) in the presence of various concentrations
−
of ClO (0-147.4 μM) in PBS (pH 7.4); (b) The absorption spectra of the probe 2 (8 μM) in the
−
presence of various concentrations of ClO (0-1.608 mM) in PBS buffer solution (pH = 7.4,
containing 20% DMSO).

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13 -
(a)
(b)
Fig. 2. (a) Fluorescence spectra of probe 1 (2 μM) in the presence of various concentrations of
−
ClO (0−16 μM) in PBS (pH 7.4) (λ = 400 nm, λem = 515 nm, slit: 2.5 nm/5 nm). (b)
ex
−
Fluorescence spectra of probe 2 (4 μM) in the presence of various concentrations of ClO
(
0−589.6 μM) in PBS buffer solution (pH = 7.4, containing 20% DMSO) (λ = 405 nm, λ = 528
ex
em
−
nm, slit: 5 nm/5 nm). Inset: A visual fluorescence color change photographs for ClO under
illumination with a 365 nm UV lamp.
−
3
.2. The Selective response of probes to ClO
As everyone knows, good selectivity of probe is one of the most important criteria
for probe design. So, we validated the selectivity of probe 1 and 2 to hypochlorite by
using UV-Vis and fluorescence spectra. Fig. S8 showed the UV-Vis spectral changes
−
−
−
3
of probe 1 and 2 upon addition of various analytes including ClO , ClO , ClO ,
2
−
−
−
−
−
−
−
4
−
−
3
−
2−
−
ClO , H O , F , Cl , Br , CN , SCN , MnO , N , NO , NO , CO , AcO ,
4
2
2
3
2
3
4−
7
3−
2−
−
−
2−
P O , PO , S , HSO , tBuOOH, tBuO•, ONOO , O . As shown in Fig. S8a,
2
4
3
−
only ClO induced a decrease in the absorption peaks at 305 nm, 372 nm and 450 nm.
Similarly, for probe 2, the absorption peaks at 349 nm and 480 nm were decreased
−
when added ClO (Fig. S8b).
As shown in Fig. S9a, for probe 1, a significant increase in fluorescence intensity at
−
5
15 nm was observed only in the presence of ClO (16 μM). The other analytes (320
μM) exhibited little change on the fluorescence signal. The probe 2 (4 μM) also
displayed a fluorescence enhancement when added hypochlorite (589.6 μM), while

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the other analytes had no changes (Fig. S9b). In addition, selective and competitive
experiments indicated that analytes including various anions and other reactive
−
oxygen species do not disturb the determination of probe 1 and 2 for ClO by
fluorescence spectra (Fig. 3). These experiments contributed considerably to the high
−
selectivity of the two probes for ClO .
(a)
(b)
Fig. 3. (a) Competitive binding assay responses of probe 1 (2 μM) towards various analytes (16
−
μM for ClO and 320 μM for other analytes) in PBS (pH 7.4) (λ = 400 nm, λ = 515 nm, slit:
ex
em
2
.5 nm/5 nm); (b) Competitive binding assay responses of probe 2 (4 μM) towards various
−
analytes (589.6 μM for ClO and 11.79 mM for other analytes) in PBS (pH 7.4) (λ = 405 nm, λ
ex
em
=
528 nm, slit: 5 nm/5 nm).
−
3
.3. Time-dependence in the detection process of ClO
As seen from the above experiments, two probes have good sensitivity and high
−
selectivity for ClO . In order to determine the kinetics for the reaction of the probe 1
−
and 2 with ClO , time-dependent fluorescence measurements were carried out. We
examined the time courses of the fluorescence intensities of the probe 1 (2 μM) in the
−
presence of ClO (80 μM) in PBS (pH 7.4) and probe 2 (4 μM) in the presence of
−
ClO (589.6 μM) in PBS buffer solution (pH 7.4, containing 20% DMSO),
respectively. As shown in Fig. S10, the reactions of probe 1 and 2 were complete
−
−
within 50 s and 1s for ClO . It indicated that two probes reacted rapidly with ClO

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under the experimental conditions.
.4. pH dependence
Additionally, to evaluate the potential applications of probe in different biological
3
environments, we studied pH effects on fluorescence intensity of two probes. From
Fig. S11a, the fluorescence intensity of probe 1 appeared to be stable in the pH range
−
2
-13 was observed for the probe 1 itself. Upon addition of ClO , the system
containing probe 1 produced a distinct fluorescence increase in the pH range of
−
7
.0-12.0. And when the solution pH was between 2.0 and 6.0 or at 13.0, ClO induced
a small fluorescence increase for probe 1. Besides, for probe 2 (Fig. S11b), in the pH
−
range 7-10, the fluorescence intensity of probe 2-ClO had distinct enhancement. As
3
1
we know, HClO will decompose rapidly in strongly acidic solutions. In addition, the
phenolic proton of the product induced by hypochlorite is easily taken away by
hydroxyl ions in alkaline solution, so the phenolic group (OH) changes into oxygen
anion (O-) with rich electron cloud density and resulting in high fluorescence intensity.
Therefore, the pH ranges of 7.0-12.0 and 7-10 were effective for the probe 1 and 2,
respectively. And the neutral pH was used for further studies.
−
3
.5 The detection limit of probes for ClO
−
In addition, we investigated the detection limits of two probes for ClO . Probe 1 (2
−
μM) was treated with various concentrations of ClO (0-16 μM) and the fluorescence
−
intensity of probe at 515 nm was plotted as a function of the ClO concentration. Also,
−
probe 2 (4 μM) was treated with various concentrations of ClO (0-589.6 μM) and the
−
fluorescence intensity of probe at 528 nm was plotted as a function of the ClO

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concentration. We used the equation (CDL = 3 S /m) based on the definition by IUPAC
b
to calculate the detection limit, where S is the standard deviation of blank
b
measurements, m is the slope of the plot of the fluorescence intensity as a function of
−
the concentrations of ClO . Through calculation, the detection limits of probe 1 and 2
were found to be 2.882 nM and 0.354 μM (Fig. 4). The experiments showed that the
detection limit of probe 1 was much lower than the probe 2. Compared with other
−
−
reported ClO probes, two probes showed a high sensitivity towards ClO (Table S1).
3
2-36
(a)
(b)
Fig. 4. (a) Plot of the fluorescence intensity of probe 1 at 515 nm as a function of the
−
concentration of ClO (λ = 400 nm). (b) Plot of the fluorescence intensity of probe 2 at 528 nm
ex
−
as a function of the concentrations of ClO (λ = 405 nm).
ex
3
.6 Theoretical calculation
In order to further study the optical properties, molecular calculations for probe 1
−
−
and 2 and their oxidation products probe 1-ClO and probe 2-ClO have been
3
7
calculated by the Gaussian 09 program package. The ground state geometries and
electron structures of the complexes were optimized by means of the density
functional theory (DFT) method at the b3lyp/6-31+G (d, p) level and the solvent was
water. As shown in Table S2a, the DFT calculation confirmed that the allowed S0-S1

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electronic transitions with oscillator strength f=0.68634 and 0.68849 were identified
−
as the allowable transitions of the probe 1 and probe 1-ClO , which were composed of
HOMO→LUMO and HOMO→LUMO transitions, respectively. The electronic cloud
of HOMO for the probe 1 was dispersed inside the whole molecular framework and
the LUMO for the probe
1
was locally distributed inside the
6
-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione group (Fig. 5a). However, the
−
electronic cloud of probe 1-ClO both the HOMO and LUMO were dispersed inside
the whole molecular framework. It showed that the fluorogenic process was mainly
mediated by the intramolecular charge transfer (ICT) mechanism. The ICT process
can take place in probe 1, which resulted in weaker fluorescence emissions.
−
Accordingly, in probe 1-ClO , ICT process was prohibited and therefore a stronger
fluorescence was observed.
As shown in Table S2b, the DFT calculation confirmed that the allowed S0-S1
electronic transitions with oscillator strength f=0.0350 and 0.3700 were identified as
−
the allowable transitions of the probe 2 and probe 2-ClO , which were composed of
HOMO→LUMO and HOMO→LUMO, HOMO→LUMO+1 transitions, respectively.
The electronic cloud of HOMO for the probe 2 was locally concentrated inside the
C=C, which was connected with 1, 4-dimethylpyridin-1-ium iodide and 1,
8
-naphthalimide group (Fig. 5b). Besides, the electronic cloud of LUMO for the probe
2
was dispersed inside the whole molecular framework. For the electronic cloud of
−
probe 2-ClO , both the HOMO and LUMO were dispersed inside the whole
molecular framework. It also showed that the fluorogenic process was mainly

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mediated by the intramolecular charge transfer (ICT) mechanism. On the basis of
−
theoretical calculation, the optical properties of probe 1, probe 1-ClO and probe 2,
−
probe 2-ClO have been theoretically revealed.
(a)
(
b)
−
−
Fig. 5. The frontier molecular orbitals of (a) probe 1, probe 1-ClO and (b) probe 2, probe 2-ClO
theoretically predicted at B3LYP/6-31+G (d, p) level.
3
.7. Proposed mechanism
To explore the sensing mechanism, mass spectrometry analyses and NMR
spectroscopic analyses were studied. For probe 1, mass spectrometry analysis of a
−
product obtained from the reaction of probe 1 with ClO in CH OH supported the
3
−
formation of probe 1-ClO compound. A peak at 297.82 corresponding to [Probe
−
+
1
-ClO + H] was clearly observed (Fig. S12a). Further NMR spectroscopic analysis
also provided the evidence that hypochlorite can oxidate oxime structure (-C=N-OH)
of probe 1 for aldehyde group. From Fig. S12b, the peak at 11.87 ppm, which was
assigned to oxime (CH-C=N-OH) of probe 1, with addition of hypochlorite, the peak at
11.87 ppm was gradual disappeared and a new peak at 10.17 was appeared. And the

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peak of hydroxyl (CH-CHO) was displaced from 12.35 ppm to 10.42 ppm. Nuclear
magnetic titration experiment and mass spectrum datum proved that the mechanism of
probe 1 with hypochlorite was hypochlorite can oxidate oxime group into aldehyde
group (Fig. 6a).
Mass spectrometry analysis of a product obtained from the reaction of probe 2 with
−
−
ClO in CH OH supported the formation of probe 2-ClO compound. A peak at
3
−
+
3
50.0748 corresponding to [Probe 2-ClO + H] was clearly observed (Fig. S13a) and
−
+
a peak at 352.0725 was the chlorine isotope peak of [Probe 2-ClO + H] . We tried
our best to isolate the product induced by hypochlorite many times, but always failed.
So we just carried out NMR titration experiments, namely we directly added sodium
hypochlorite solution into DMSO-d including probe 2, as shown in Fig. S13b,
6
1
HNMR showed that the number of aromatic hydrogens obviously decreased after
addition of hypochlorite. It implied that probe 2 was oxidized by hypochlorite,
13
resulting in the pyridine ring leaving. However, after long time scaning, CNMR
could not give any information.
(a)
(b)
−
Fig. 6. The possible mechanism of porbe 1 (a) and probe 2 (b) with ClO .
3
.8. Cellular Imaging
We further investigated the fluorescence imaging of two probes (Fig. 7). The A549

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cells were treated with 4 μM probe 2 in culture media for 30 min at 37 ℃ showed no
light (Fig. 7A). On the contrary, the same cells were treated with 2 μM probe 1 in
culture media for 30 min at 37 ℃ shown distinct green (Fig. 7D). For the control
experiment, the cells were first treated with 5 mM NAC (N-acetyl-L-cysteine) in
culture media for 1 h at 37 ℃ and then incubated with 2 μM probe 1 in culture
media for 30 min at 37 ℃ showed no light (Fig. 7G). The latter two experiments
showed that probe 1 can detect intracellular hypochlorite. And cellular imaging
experiments also indicated that probe 1 has higher sensitivity for hypochlorite than
probe 2.
Fig. 7. Fluorescence and bright field images of A549 cells. (a) Fluorescence image of cells
incubated with probe 2 (4 μM) for 30 min and its bright field image (b), merged image (c). (d)
A549 cells were treated with probe 1 (2 μM) and its bright field image (e), merged image (f). (g)
Fluorescence image of cells pre-treated with NAC (5 mM) for 30 min and then incubated with
probe 1 (2 μM) for 30 min. (h) Bright-field image of g. (i) Merged image of g and h.

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−
3
.9 Detection of ClO in water samples
To take advantage of their good spectroscopic responses of hypochlorite, two
probes were applied to detect the hypochlorite concentration of tap water and river
water. Tap water and river water were obtained from Shanxi University. The analysis
results of two probes were shown in Table 1. From Table 1, the values of recovery
were showed from 99.20 % to 102.00 % in tap water and from 98.60 % to 99.50 % in
river water of probe 1, and the values of recovery were showed from 97.41 % to
9
9.79 % in tap water and from 95.67 % to100.98 % in river water of probe 2. The
experimental results suggested that both probe 1 and 2 were promising fluorescence
probes for the detection of hypochlorite in water samples.
−
Probe
Sample
ClO spiked (M)
Found (M) mean±SD
Recovery (%)
99.20
-6
-6
Tap water 1
Tap water 2
Tap water 3
River water 1
River water 2
River water 3
Tap water 1
Tap water 2
Tap water 3
River water 1
River water 2
River water 3
5×10
8×10
(13.05±0.02)×10
-6
-6
(26.39±0.04)×10
102.00
100.90
99.01
-6
-6
10×10
(33.43±0.01)×10
1
-6
-6
5×10
8×10
(13.21±0.02)×10
-6
-6
(37.16±0.01)×10
99.50
-6
-6
10×10
(32.05±0.03)×10
98.60
-5
-5
-5
-5
-5
-5
-5
13.4×10
26.8×10
33.5×10
13.4×10
26.8×10
33.5×10
(4.95±0.03)×10
97.41
-5
(8.16±0.02)×10
98.62
-5
(10.09±0.05)×10
99.79
2
-5
(4.95±0.04)×10
98.58
-5
(7.96±0.03)×10
100.98
95.67
-5
(9.86±0.06)×10
−
Table 1 The testing results of ClO concentration in tap water and river water samples.
4
. Conclusion
In a word, two novel 1, 8-naphthalimide-based turn-on fluorescent probes were
devised and developed by utilizing the oxidation of hypochlorite. Probe 1 and 2
−
displayed rapid and remarkable turn-on responses to ClO in PBS buffer solution (pH

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7
.4) and PBS buffer solution (pH = 7.4, containing 20% DMSO). Furthermore, the
−
detection limits of probe 1 and 2 for ClO were as low as 2.882 nM and 0.354 μM. In
addition, the detailed signal mechanisms were carried out by MS and NMR
spectroscopic methods. More importantly, fluorescence imaging experiments
−
demonstrated that probe 1 can be more suitable for detecting the endogenous ClO in
living A549 cells. And both two probes had the possibility of potentially applied in
practical applications such as detecting the hypochlorite concentration of tap water
and river water.
Acknowledgments
We thank the National Natural Science Foundation of China (No. 21472118,
2
1672131), the Program for the Top Young and Middle-aged Innovative Talents of
Higher Learning Institutions of Shanxi (2013802), Talents Support Program of Shanxi
Province (2014401), Shanxi province Outstanding Youth Fund (2014021002).

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References
[
1] X. Q. Chen, X. Z. Tian, I. Shin and J. Yoon, Chem. Soc. Rev., 40 (2011)
783-4804.
4
[
[
2] B. D'Autreaux and M. B. Toledano, Nat. Rev. Mol. Cell Biol. 8 (2007) 813-824.
3] K. H. Xu , L. L. Wang , M. M. Qiang , L. Y. Wang , P. Li and B. Tang, Chem.
Commun., 47 (2011) 7386-7388.
[
4] M. Valko, D. Leibfritz, J. Moncol, M. T. D. Cronin, M. Mazur and J. Telser, Int. J.
Biochem. Cell Biol. 39 (2007) 44-84.
[
[
5] M. S. Shim and Y. N. Xia, Angew. Chem. Int. Ed., 52 (2013) 6926-6929.
6] Y. B. Ding, Y. Y. Tang, W. H. Zhu and Y. X. Xie, Chem. Soc. Rev., 44 (2015)
1101-1112.
[
7] X. M. Wang, X. H. Wang, Y. Feng, M. Z. Zhu, H. Yin, Q. X. Guo and X. M. Meng,
Dalton Trans., 44 (2015) 6613-6619.
[
[
[
8] J. Kim and Y. Kim, Analyst, 139 (2014) 2986-2989.
9] S. Goswami and S. Paul, Dalton Trans., 42 (2013) 10097-10101.
10] Y. B. Ding, X. Li, T. Li, W.H. Zhu and Y.S. Xie, J. Org. Chem., 78 (2013)
5
328-5338.
[
[
11] G. P. Li, D. J. Zhu, Q. Liu, L. Xue and H. Jiang, Org. Lett., 15 (2013) 2002-2005.
12] L. Yuan, W. Y. Lin, J. Z. Song and Y. T. Yang, Chem. Commun., 47 (2011)
1
2691-12693.
[
[
13] G. Y. Li, Q. Lin, L. N. Ji and H. Chao, J. Mater. Chem. B, 2 (2014) 7918-7926.
14] Q. A. Best, N. Sattenapally, D. J. Dyer, C. N. Scott and M. E. McCarroll, J. Am.
Chem. Soc., 135 (2013) 13365-13370.

ACCEPTED MANUSCRIPT
-
24 -
[
[
15] H. J. Lee, M. J. Cho and S. K. Chang, Inorg. Chem., 54 (2015) 8644 -8649.
16] B. Chen, Y. B. Ding, X, Li, W. H. Zhu, J. P. Hill, K. Ariga and Y. S. Xie, Chem.
Commun., 49 (2013) 10136-10138.
[
[
17] Y. B. Ding, W. H. Zhu and Y. S. Xie, Development of Ion Chemosensors Based
on Porphyrin Analogues. Chem. Rev., DOI: 10.1021/acs.chemrev.6b00021.
18] Q. Wang, C. Liu, J. J. Chang, Y. Lu, S. He, L. C. Zhao and X. S. Zeng, Dyes and
Pigments, 99 (2013) 733-739.
[
[
19] W. Lin, L. Long, B. Chen and W. Tan, Chem. Eur. J. 15 (2009) 2305-2309.
20] Z. Tang, X. L. Ding, Y. Liu, Z. M. Zhao and B. X. Zhao, RSC Adv., 5 (2015)
9
9664-99668.
[
[
[
[
[
[
21] J. T. Hou, K. Li, J. Yang, K. K. Yu, Y. X. Liao, Y. Z. Ran, Y. H. Liu, X. D. Zhou
and X. Q. Yu, Chem. Commun., 51 (2015) 6781-6784.
22] L. L. Long, D. D. Zhang, X. F. Li, J. F. Zhang, C. Zhang and L. P. Zhou, Anal.
Chim. Acta, 775 (2013) 100-105.
23] J. J. Hu, N. K. Wong, M. Y. Lu, X. M. Chen, S. Ye, A. Q. Zhao, P. Gao, R. Y. Kao,
J. G. Shen and D. Yang, Chem. Sci., 7 (2016) 2094-2099.
24] X. H. Cheng, R. L. Tang, H. Z. Jia, J. Feng, J. G. Qin and Z. Li, ACS Appl. Mater.
Interfaces, 4 (2012) 4387-4392.
25] X. H. Lu, W. Wang, Q. Dong, X. L. Bao, X. F. Lin, W. X. Zhang, X. C. Dong and
W. L. Zhao, Chem. Commun., 51 (2015) 1498-1501.
26] Y. L. Pak, J. Li, K. C. Ko, G. Kim, J. Y. Lee and J. Yoon, Anal. Chem., 88 (2016)
5
476-5481.

ACCEPTED MANUSCRIPT
-
25 -
[
[
[
[
[
[
27] M. M. Salim, E. A. Owens, T. Gao, J. H. Lee, H. Hyun, H. S. Choi and M.
Henary, Analyst, 139 (2014) 4862-4873.
28] S. Y. Lim, K. H. Hong, D. I. Kim, H. Kwon and H. J. Kim, J. Am. Chem. Soc.,
1
36 (2014) 7018-7025.
29] J. F. Li, P. F. Li, F. J. Huo, C. X. Yin, T. Liu, J. B. Chao and Y. B. Zhang, Dyes
and Pigments, 130 (2016) 209-215.
30] W. F. Niu, L. Guo, Y. H. Li, S. M. Shuang, C. Dong and M. S. Wong, Anal.
Chem., 88 (2016) 1908-1914.
31] Y.K. Yue, C.X. Yin, F.J. Huo, J.B. Chao and Y.B. Zhang. Sens. Actuators, B 202
(
2014) 551-556.
32] H. Zhu, J. L. Fan, J. Y. Wang, H. Y. Mu and X. J. Peng, J. Am. Chem. Soc., 136
2014) 12820-12823.
(
[
[
33] W. C. Chen, P. Venkatesan and S. P. Wu, Anal. Chim. Acta, 882 (2015) 68-75.
34] Y. Zhou, J. Y. Li, K. H. Chu, K. Liu, C. Yao and J. Y. Li, Chem. Commun., 48
(2012) 4677-4679.
[
[
35] G. F. Wu, F. Zeng and S. Z. Wu, Anal. Methods, 5 (2013) 5589-5596.
36] L. J. Liang, C. Liu, X. J. Jiao, L. C. Zhao and X. S. Zeng, Chem. Commun., 52
(2016) 7982-7985.
[
37] Y. T. Yang, F. J. Huo, C. X. Yin, A. M. Zheng, J. B. Chao, Y. Q. Li, Z. X. Nie and
R. Martínez-Máñez, D. S. Liu, Biosens. Bioelectron., 47 (2013) 300-306.

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Abstract Graphic
The statement:
In this study, two efficient turn-on fluorescent probes based on 1, 8-Naphthalimide for
hypochlorite have been rationally devised and constructed. The detection limits of
−
probe 1 and 2 for ClO were calculated for 2.882 nM and 0.354 μM. Moreover, the
−
optical properties of two probes and their ClO -addition products were confirmed by
density functional theory calculations. And both two probes had the possibility of
potentially applied in practical applications such as detecting the hypochlorite
concentration of tap water and river water.

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Highlight
1
2
. Two probes based on 1, 8-Naphthalimide had excellent sensitivity and
−
high selectivity for the detection of ClO .
. The recognition mechanisms of the two probes were studied and their
optical properties were confirmed by density functional theory
calculations.
3
. Two probes in practical application was demonstrated