A novel fluorescent probe with extremely low background fluorescence for sensing hypochlorite in zebrafish

Article abstract of DOI:10.1016/j.ab.2020.113795

Development of an efficient fluorescent probe for sensing hypochlorite in water samples and biological samples is highly demanded. However, the currently reported fluorescent probes for hypochlorite frequently suffered from the problem of high background fluorescence. Herein, based on the combined effect of two different fluorescence quenching groups, we rationally developed a novel fluorescent probe for hypochlorite with extremely low background fluorescence. Notably, due to the doubly quenching groups, the probe could even keep low background fluorescence in a solution with high viscosity. Furthermore, the probe displayed highly sensitive and selective response to hypochlorite, with the detection limits calculated to be 10.5 nM. Practical application demonstrated that the probe was able to quantitatively detect hypochlorite in various water samples with good recovery. Significantly, the probe showed extremely low background fluorescence in living cells and was capable of detecting minor variation of endogenous hypochlorite in RAW 264.7 cells. Moreover, the fluorescence imaging different concentration of hypochlorite in zebrafish has been successfully conducted. The probe developed herein will be widely used as a reliable tool to accurately monitor the variation of hypochlorite in living organism.

Full text of DOI:10.1016/j.ab.2020.113795

Journal Pre-proof
A Novel Fluorescent Probe with Extremely Low Background Fluorescence for
Sensing Hypochlorite in Zebrafish
Zhixiang Han, Lianghuan Dong, Fan Sun, Lingliang Long, Shu Jiang, Xiaoting Dai,
Min Zhang
PII:
S0003-2697(20)30327-4
https://doi.org/10.1016/j.ab.2020.113795
YABIO 113795
DOI:
Reference:
To appear in: Analytical Biochemistry
Received Date: 16 February 2020
Revised Date: 4 May 2020
Accepted Date: 20 May 2020
Please cite this article as: Z. Han, L. Dong, F. Sun, L. Long, S. Jiang, X. Dai, M. Zhang, A Novel
Fluorescent Probe with Extremely Low Background Fluorescence for Sensing Hypochlorite in Zebrafish,
Analytical Biochemistry, https://doi.org/10.1016/j.ab.2020.113795.
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A Novel Fluorescent Probe with Extremely Low Background Fluorescence for
Sensing Hypochlorite in Zebrafish
,a
a
a
,b
a
Zhixiang Han,* Lianghuan Dong, Fan Sun, Lingliang Long,* Shu Jiang, Xiaoting
a
b
Dai, and Min Zhang
a
School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang,
Jiangsu 212013 (P. R. China).
b
School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, Jiangsu
2
*
12013 (P. R. China).
Corresponding author:
Fax: +86-511-88790955
Email: zhixianghan69@126.com; longlingliang@ujs.edu.cn.
Abstract
Development of an efficient fluorescent probe for sensing hypochlorite in water samples
and biological samples is highly demanded. However, the currently reported fluorescent
probes for hypochlorite frequently suffered from the problem of high background
fluorescence. Herein, based on the combined effect of two different fluorescence
quenching groups, we rationally developed a novel fluorescent probe for hypochlorite
with extremely low background fluorescence. Notably, due to the doubly quenching
groups, the probe could even keep low background fluorescence in a solution with high
viscosity. Furthermore, the probe displayed highly sensitive and selective response to
hypochlorite, with the detection limits calculated to be 10.5 nM. Practical application
1

demonstrated that the probe was able to quantitatively detect hypochlorite in various
water samples with good recovery. Significantly, the probe showed extremely low
background fluorescence in living cells and was capable of detecting minor variation of
endogenous hypochlorite in RAW 264.7 cells. Moreover, the fluorescence imaging
different concentration of hypochlorite in zebrafish has been successfully conducted. The
probe developed herein will be widely used as a reliable tool to accurately monitor the
variation of hypochlorite in living organism.
Keywords: Fluorescent probes, Doubly-quenching groups, Low background detection,
Hypochlorite, Zebrafish.
1
. Introduction
Hypochlorite (ClO ), a common reactive oxygen species (ROS), has been extensively
-
used in our daily lives as disinfectant and household bleaching agent [1]. Generally, it is
4
used in a concentration range of 10 -10 μM. Nevertheless, exposure to high levels of
-
ClO residue in water may cause eye/nose irritation and stomach discomfort, and thus
-
pose severe adverse effect to human health. On the other hand, ClO can be endogenously
-
produced in living organism by the reaction of H O and Cl under the catalysis of
2
2
myeloperoxidase (MPO). It plays significant roles in human immune system and
-
maintaining intracellular redox homeostasis. However, imbalance of ClO concentration
in human body often causes oxidative stress related diseases, such as cardiovascular
diseases [2], lung injury [3], rheumatoid arthritis [4], and other diseases [5]. Therefore,
-
construction of an efficient method for detection of ClO in water samples and biological
samples is urgently required.
-
Recently, several strategies for detection of ClO have been developed, including
2

spectrophotometry [6], colorimetric assays [7], chemiluminescence method [8],
electrochemical analysis [9], fluorescent probe, and so on. Compared with other
strategies, the fluorescent probe has gained especially attention due to its superior
properties of high selectivity and sensitivity, facile operation, and capability for detecting
analyte of interest in biological system in non-invasive way. So far, many fluorescent
-
probes have been devised for detection of ClO based on its strong oxidation reactivity to
acylhydrazines [10], chalcogenides [11-13], hydrazones [14], oximes [15],
p-methoxyphenols [16], ether [17], and C=C bonds [18]. However, despite the fabulous
developments, the currently reported fluorescent probes often suffered from the problem
of high background fluorescence from the probes themselves. For practical application,
the residual background fluorescence signal tends to decrease the detection sensitivity,
and even cause false positive results. To get an accurate and reliable detection, the
-
fluorescent probe for ClO with extremely low background fluorescence is highly
desirable.
-
In our previous work, we developed compound 1b as fluorescent probe for ClO
(Scheme 1) [19]. For probe 1b, the free rotating N-N group was considered to be
fluorescence quenching group for 7-diethylamino-coumarin fluorophore. Upon treatment
-
with ClO , the N-N group can be selectively removed and thus the fluorescence of
7
-diethylamino-coumarin fluorophore was recovered. It is known that the free rotation of
a specific group was tightly associated with viscosity of surrounding medium [20]. The
free rotation of N-N bond in 1b might be significantly inhibited in a solution with high
viscosity. Therefore, probe 1b possibly exhibits high background fluorescence in living
cells, as the viscosity is varied in distinct regions of the living cells [21].
3

Herein, on the basis of compound 1b, we would like to design compound LH-1 as a
-
novel fluorescent probe for ClO with extremely low background fluorescence (Scheme
1
). In compound LH-1, the coumarin 6H (blue color) was used as fluorophore. Compared
with 7-diethylamino-coumarin (red color in 1b), the coumarin 6H with twist-blocked and
stronger electron-donating julolidine group exhibits higher quantum yield, better
biocompatibility, as well as less background fluorescence when suppressed by a
fluorescence quenching group [22-23]. Thus, the coumarin 6H in probe LH-1 may help
to reduce the background fluorescence. Importantly, apart from the free rotating N-N
group as fluorescence quenching strategy, the 2,4-dinitrobenzene group in LH-1 can be
used as the second type of fluorescence quenching group, owing to the
2
,4-dinitrobenzene group frequently resulted in fluorescence quenching of a fluorophore
via donor-excited photoinduced electron transfer (d-PET) mechanism [24]. Consequently,
probe LH-1 was expected to exhibit extremely low background fluorescence by the
combined effect of two different types of fluorescence quenching groups. In addition, as
the d-PET based fluorescence quenching group will not be influenced by medium
viscosity, probe LH-1 would even display low background fluorescence in a solution
with high viscosity, which is favourable for the probe applying in complex biological
environment.
4

Scheme 1. (a) The molecular structures of compound LH-1, LH-2, and 1b. The FQG
-
represents fluorescence quenching group. (b) The sensing reaction of LH-1 with ClO .
2
2
. Experimental
.1. Materials and instruments
Unless otherwise noted, all the chemical and biological reagents were purchased from
commercialized
companies
and
used
without
further
purification.
8
-hydroxyjulolidine-9-aldehyde (compound 4) was synthesized according to the literature
procedure [25]. Twice-distilled water was prepared by UPU ultrapure water machine
UPC-1-5, was used throughout all experiments. TLC analyses were performed on silica
gel plates and column chromatography was conducted over silica gel (mesh 100-200),
both of which were obtained from the Qingdao Ocean Chemicals.
1
13
H-NMR and C-NMR spectra were recorded on a Varian INOVA-400 spectrometer
operating at 400 MHz and 100 MHz respectively. In NMR, the chemical shift (δ) is the
corresponding resonances signal that was recorded in ppm with tetramethylsilane (TMS)
as reference. The chemical shift mutiplicities were given in following format: singlet (s),
5

doublet (d), triplet (t), multiplet (m), double doublet (dd). Mass spectra were recorded on
an LXQ Spectrometer (Thermo Scientific) operating on ESI. HRMS was recorded on a
SolanX 70 FT-MS spectrometer. Absorption spectra were obtained on a SHIMADZU
UV-2600 spectrometer. Fluorescence emission spectra were measured on a Thermo
Scientific Lumina fluorescence spectrophotometer with 10 nm excitation and emission
slit widths. The pH measurements were performed with a Swiss Mettler Toledo Five Easy
Plus FE28 pH meter. Fluorescence imaging experiments were performed on an inverted
fluorescence microscopy (Carl Zeiss Microscopy GmbH 37081 Gottingen, GERMANY)
and a stereofluorescence microscope (SZX2-ILLT, Olympus Corporation, JAPAN). HeLa
and macrophage cells (RAW264.7) were cultured in a 5% CO / 95% air incubator
2
(Sanyo, MCO-18AICUVL-PC, JAPAN). Zebrafish were incubated in illumination
incubator (MGC-450BP-2, CHINA) at 28.5 °C. The cytotoxicity of the probe was
measured on a Synergy|H5 microplate reader.
2
2
.2. Synthetic procedure
The synthetic routes were outlined in Scheme S1.
.2.1 Synthesis of compound 3 (coumarin 343)
A mixture of compound 4 (1.12 g, 5.18 mmol), Meldrum's acid (0.74 g, 5.18 mmol),
piperidine (0.044 g, 0.52 mmol), and acetic acid (two drops) in 8 mL of ethanol was
stirred at room temperature for 30 minutes. Then the reaction mixture heated to reflux for
3
hours. After cooling to room temperature, 100 mL of ice water was added in the
mixture. The resulting precipitate was washed with ethanol, dried and purified by column
chromatography on silica gel (CH Cl : CH OH : CH COOH = 50 : 1: 0.1) to afford
2
2
3
3
1
yellow product 3 (0.93 g, yield 63%). H NMR (400 MHz, CDCl ) δ (ppm): 12.497 (s,
3
6

1
2
H), 8.506 (s, 1H), 7.022 (s, 1H), 3.412 (q, J = 4.8 Hz, 4H), 2.903 (t, J = 6.4 Hz, 2H),
1
3
.806 (t, J = 6.2 Hz, 2H), 2.013 (t, J = 5.6 Hz, 4H); C NMR (100 MHz, DMSO-d ) δ
6
(ppm): 165.074,161.081, 153.103, 149.621, 149.178, 127.940, 120.005, 107.714, 105.438,
1
2
2
05.182, 50.113, 49.580, 27.209, 20.920, 19.954. HRMS: m/z: calcd for C H NO :
16 16 4
+
86.10793 [M+H] , Found: 286.10775.
.2.2 Synthesis of compound 2
Oxalyl chloride (0.6 mL, 7.1 mmol) and DMF (cat.) were added to
a solution of compound 3 (100 mg, 0.35 mmol) in dried CH Cl (10 mL). The resulting
2
2
solution was stirred at room temperature for 24 hours. After the solvent being removed
under reduced pressure, the obtained residue was dried under high vacuum for 2-3 hours
and used directly for next step.
2
.2.3. Synthesis of compound LH-1
Compound 2 was dissolved in dried CH Cl (7 mL) and treated with a solution of the 2,
2
2
4
-dinitrophenylhydrazine (70 mg, 0.35 mmol) in dried CH Cl (5 mL) and triethylamine
2 2
(1 mL, 7.2 mmol). The solution was stirred at room temperature for 48 hours. After the
solvent removed under reduced pressure, the residue was purified by column
chromatography on silica gel (CH Cl ). The product was isolated as a yellow powder ( 84
2
2
1
mg, yield 51.6 %). H NMR (400 MHz, CDCl ) δ (ppm): 10.721 (s, 1H), 9.725 (s, 1H),
3
9
1
2
1
1
.163 (d, J = 2.4 Hz, 1H), 8.594 (s, 1H), 8.297 (dd, J = 9.2Hz, J =2.4Hz, 1H), 7.057 (s,
1 2
H), 5.322 (s, 1H), 3.412 (m, 4H), 2.931 (t, J = 6.4 Hz, 2H), 2.811 (t, J = 6 Hz, 2H),
1
3
.026 (m, 4H); C NMR (100 MHz, CDCl ) δ (ppm): 163.725,163.046, 153.131,
3
49.397, 149.298, 148.754, 130.960, 130.199, 127.552, 123.602, 120.382, 114.937,
08.282, 105.780, 105.677, 53.434, 50.438, 50.006, 27.435, 20.960, 20.013; MS (ESI):
7

+
+
m/z: calcd for C22
H
20
N O
5 7
: 466.14 [M+H] , C22
66.34 and 488.41, respectively.
.2.4 Synthesis of compound LH-2
The synthesis procedure is similar as compound LH-1, and the crude product was
purified by column chromatography on silica gel (CH Cl : EtOAc = 1 : 7). The product
19 5 7
H N O Na 488.12 [M+Na] . Found:
4
2
2
2
1
was isolated as a yellow powder (66 mg, yield 50.4 %). H NMR (400 MHz,CDCl ) δ
3
(ppm): 10.396 (s, 1H), 8.592(s, 1H), 7.249 (q, J =7.6 Hz, 2H), 7.022 (s, 1H), 6.956 (d, J
=
7.6 Hz , 2H), 6.907(t, J = 7.4 Hz,1H), 5.072(br, 1H), 3.369 (m, 4H), 2.925 (t, J = 6.4
1
3
Hz, 2H), 2.790 (t, J = 6.2 Hz, 2H), 2.004 (m, 4H); C NMR (100 MHz, CDCl
3
) δ (ppm):
1
1
63.79, 162.70, 152.81, 148.57, 148.30, 129.11, 127.18, 121.01, 119.85, 113.86, 108.21,
07.46, 105.72, 50.29, 49.88, 27.45, 21.08, 20.15, 20.08. HRMS: m/z: calcd for
+
C H NO : 376.16612 [M+H] , Found: 376.16579.
16
16
4
-
2
.3. Determination of ClO in Water Sample
The crude water samples from the Yu Dai River, Jing Lake and tap water in Jiangsu
University were passed through a microfiltration membrane before use. Aliquots of the
-
water samples were then spiked with different concentrations of ClO (0, 5, 10, 20 μM).
The results were reported as the mean ± standard deviation of triplicate experiments.
2
.4. Cytotoxicity Study
RAW 264.7 cells or HeLa cells were seeded in each well of 96-well plates at a density
5
of 1×10 cells/well and incubated for 24 h for cell attachment. The stock solution of
LH-1 dissolved in DMF (10 mM) was diluted with a cell culture medium containing 10%
fetal bovine serum (FBS) to have a final concentration of 0, 10, 20, 30, 40 and 50 μM.
Existing culture medium was replaced with 100 µL of the fresh one containing LH-1, and
8

the
cells
were
incubated
for
12
h.
Then
20
μL
of
MTT
-
1
(
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (5 mg mL ,
o
HEPES) was added to each well. After 4 h in 37 C, the remaining MTT solution was
removed, 100 μL dimethyl sulfoxide (DMSO) was added to each well to dissolve the
formazan crystals. Absorbance was measured at 490 nm on a Synergy|H5 microplate
reader.
-
2
.5. Image of exogenous ClO in HeLa cells
HeLa cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotics
2
(penicillin/streptomycin, 100 U/mL) at 37 °C in a 5% CO / 95% air incubator. The cells
4
-1
were seeded on 24-well plates in the culture medium (4×10 cells mL ) and incubated at
the incubator for 24h. Firstly, HeLa cells were incubated with 10 μM LH-1 or LH-2 in
culture media for 30 min at 37 °C, and washed with PBS three times. Then, HeLa cells in
another five groups were pretreated with 10 μM LH-1 in culture media for 30 min,
washed by PBS buffer three times, and subsequently incubated with different
-
concentrations of ClO (0, 5, 10, 15, 30 μM) for another 30 min at 37 °C, and washed
with PBS three times, respectively. All of the cells above were imaged by inverted
fluorescence microscopy with a 20× objective lens.
-
2
.6. Image of endogenous ClO in RAW264.7 cells
RAW264.7 cells were cultured in DMEM supplemented with 10% FBS and 1%
o
antibiotics (penicillin/streptomycin, 100 U/mL) at 37 C in a 5% CO / 95% air incubator.
2
4
-1
The cells were seeded on 24-well plates in the culture medium (6×10 cells mL ) and
-
incubated at the incubator for 24h. The cell experiments for imaging of endogenous ClO
can be divided into three groups. The first group is that RAW 264.7 macrophage cells
9

were incubated with 10 μM LH-1 for 30 min. In the second group, RAW 264.7
macrophage cells were incubated with 4-aminobenzoic acid hydrazide (ABAH, 200 μM)
for 30 min and then incubated with 10 μM LH-1 for 30 min. In the third group, RAW
2
64.7 macrophage cells were stimulated with Lipopolysaccharide (LPS, 20 μg/mL) for 12
h and then incubated with 10 μM LH-1 for 30 min. All of the cells above were washed by
PBS buffer three times before imaging and imaged by inverted fluorescence microscopy
with a 20× objective lens.
-
2
.7. Image of exogenous ClO in zebrafish
The 3-day-old zebrafish were incubated with LH-1 (10 μM) for 30 min in illumination
incubator, and then washed with zebrafish embryo culture water (0.1% NaCl, 0.003%
KCl, 0.004% CaCl ·H O, 0.008% MgSO ) to remove the remaining probe and imaged by
2
2
4
stereofluorescence microscope. Meanwhile, the zebrafish of other two groups were
-
incubated with LH-1 (10 μM) for 30 minutes followed by ClO (5 or 10 μM) for 30 min,
and washed with culture water. Then the fluorescence imaging of zebrafish was carried
out. All the experiments were strictly performed according to ethics guidelines of
Regulations for the Administration of Affairs Concerning Experimental Animals
published by the Bureau of Legislative Affairs of the State Council of the People’s
Republic of China.
3
. Results and discussion
3.1 Synthesis and fluorescence properties
Probe LH-1 was readily prepared by condensation of coumarin 343 with
,4-dinitrophenylhydrazine. The control compound LH-2 was also synthesized in a
2
1
0

similar procedure. Compared with LH-1, two nitro groups were absent in the
molecular structure of LH-2 (Scheme 1). All the compounds were well
1
13
characterized by H NMR spectra, C NMR spectra and Ms spectrum. To test
whether the fluorescence of coumarin 6H in LH-1 can be completely suppressed
by the two combined fluorescence quenching groups, the optical properties of
LH-1 and LH-2 were investigated in 10 mM potassium phosphate buffer (pH 7.4,
containing 20% CH
3
CN as co-solvent). As shown in Figure 1, the fluorescence of
Φf=0.052) was significantly suppressed.
both LH-1 Φf=0.0024) and LH-2
(
(
Apparently, the fluorescence of coumarin 6H fluorophore in LH-1 and LH-2 could
be quenched by the rotation of N-N bond. In addition, compared with that of
LH-2, almost no residual background fluorescence was observed in LH-1 (Figure
1
inset). This was apparently attributed to the additional d-PET process from the
excited coumarin 6H fluorophore to the electron deficient 2,4-dinitrobenzene
moiety in LH-1. To verify this d-PET process, time-dependent density functional
theory (TD-DFT) calculations at the B3LYP/6-31G* level was carried out. As seen
in Figure 2, the electron in the excited coumarin 6H moiety was able to transfer to
the LUMO of 2,4-dinitrobenzene moiety. Thus, the d-PET process can be
proceeded in LH-1. In contrast, no d-PET was occurred in compound LH-2
(
Figure S1). Thus, the d-PET process in LH-1 indeed could further suppress the
background fluorescence from coumarin 6H. The background fluorescence of
LH-1 and LH-2 in a solution with different viscosity was also examined (Figure 3).
Clearly, LH-2 showed noticeable fluorescence enhancement with the solution
viscosity increased, suggesting that the free rotation of N-N bond in LH-2 was
11

progressively inhibited. Nonetheless, no perceptible variation of background
fluorescence was observed for LH-1 when the solution viscosity was increased.
This is probably due to the high viscosity gives negligible effect on the d-PET
process in LH-1, albeit the free rotation of N-N bond in LH-1 was also inhibited.
Therefore, by the combined effect of two fluorescence quenching groups, LH-1
displayed extremely low residual background fluorescence, even in a solution with
high viscosity, in good agreement with our design.
Figure 1. The fluorescence spectra of LH-1 and LH-2 in 10 mM potassium phosphate
buffer (pH 7.4, containing 20% CH CN as co-solvent). For comparison, the fluorescence
3
spectra of coumarin 343 was also displayed.
1
2

Figure 2. Frontier molecular orbital energy illustrations show the relative energetic
dispositions of the orbitals of probe LH-1.
Figure 3. Fluorescence intensity at 488 nm of LH-1 and LH-2 (10 μM) in glycerol /
acetonitrile solution as a function of increasing viscosity (λex = 415 nm).
-
3
.2 Optical response of LH-1 to ClO
-
The optical response of LH-1 to ClO was then inspected. As design, probe LH-1 itself
1
3

displayed no observable background fluorescence (Figure 4a). However, with the
-
addition of increasing amount of ClO , dramatic fluorescence enhancement was detected
at 488 nm (Φ ꢀ=ꢀ0.55), with the fluorescence color changed from dark to bluish green
f
-
(
Figure 4a, inset). Notably, when 40 μM ClO was introduced, the magnitude of
fluorescence enhancement at 488 nm reached 739-fold. The drastic fluorescence
enhancement implies that all of fluorescence quenching groups were removed upon
-
treatment with ClO . In addition, the fluorescence intensity of LH-1 at 488 nm showed
-
linear relationship to ClO in the concentration range of 0 to 20 μM (Figure 4b),
-
indicating that LH-1 can be potentially used for quantitatively detecting ClO . Moreover,
-
LH-1 displayed very sensitive response to ClO , and the detection limits was estimated to
-
be 10.5 nM, which is favourable for detecting minor variation of intracellular ClO .
-
Recently reported fluorescent probes for ClO were summarized in Table S1. The results
-
clearly denoted that LH-1 showed superior fluorescence sensing properties to ClO .
Figure 4. (a) Fluorescence spectra (λ = 415 nm) of LH-1 (10 μM) in the presence of
ex
-
various concentrations of ClO (0-40 μM) in 10 mM potassium phosphate buffer (pH 7.4,
containing 20% CH CN as co-solvent). Inset: photographs of 10 μM LH-1 without and
3
1
4

-
with ClO (40 μM) under a hand-hold 365 nm UV light. (b) The linear relationship of
-
fluorescence intensity at 488 nm of LH-1 (10 μM) to various amount of ClO (0 to 20
μM).
-
The UV/Vis absorption spectra of LH-1 also showed a significant response to ClO
-
(
Figure S2). In the absence of ClO , LH-1 displayed an intense absorption centered at 459
-
nm. After addition of ClO , the absorption was blue-shifted to 424 nm. The blue-shifted
absorption suggested that the electron withdrawing fluorescence quenching groups were
removed. To investigate the sensing reaction mechanism, the sensing reaction product
was isolated and used for characterization. According to the high-resolution mass
1
spectrometry (HRMS) (Figure S3), H NMR (Figure S4), absorption spectra (Figure S5),
and fluorescence emission spectra (Figure S6), the reaction product was unambiguously
proved to be coumarin 343 (Scheme 1b), consistent with the sensing reaction mechanism
of previous work [19].
-
The selectivity of LH-1 toward ClO were evaluated. As displayed in Figure 5, addition
1
of 40ꢀμM other potential interfering species including H
2
O
2
, •OH, O
2
, and NO caused no
visible enhancement of fluorescent intensity at 488 nm. A prominent fluorescence
-
enhancement was only triggered after the addition of 40ꢀμM ClO . Furthermore, the
visual fluorescence response of LH-1 to various species (Figure 5, inset) showed that
-
probe LH-1 can be served as “naked-eye’’ indicator for ClO . Also, the time-dependent
fluorescence intensity changes of LH-1 (10 μM) upon introducing different concentration
-
of ClO (0, 10, 20, 30, 40 μM) were monitored (Figure 6). The fluorescence intensities at
-
4
88 nm of LH-1 can level out after incubation with ClO for 20 minutes. Thus, probe
-
LH-1 can be employed for rapid detection of ClO . The fluorescence responses of LH-1
1
5

-
3
to ClO in solution with different CH CN volume fraction were also studied (Figure S7).
Probe LH-1 itself exhibit no background fluorescence in solution with various CH CN
3
-
volume fraction. After addition of ClO , the largest fluorescence intensity enhancement
was observed in a solution with 20% CH CN. Thereby, all the experiments for the optical
3
-
response of LH-1 to ClO were carried out in 10 mM potassium phosphate buffer
containing 20% CH CN.
3
Figure 5. Fluorescence intensity responses (λ =415 nm, λ = 488 nm) of LH-1 (10 μM)
ex
em
in the presence of 40 μM various species in 10 mM potassium phosphate buffer (pH 7.4,
containing 20% CH CN as co-solvent). Inset: visual fluorescence color changes of LH-1
3
-
(
10 μM) in the presence of 40 μM various species (from left to right: Blank, ClO , H O ,
2
2
1
•
2
OH, O , NO).
1
6

Figure 6. Time-dependent fluorescence intensity of LH-1 (10 μM) after incubating with
-
various concentration (0, 10, 20, 30, 40 μM) of ClO in 10 mM potassium phosphate
buffer (pH 7.4, containing 20% CH
3
CN as co-solvent) at room temperature (λex=415 nm,
λem=488 nm).
-
Moreover, the effect of pH value on the fluorescence response of LH-1 to ClO
-
was determined (Figure S8). In the absence of ClO , almost no background
fluorescence was observed in LH-1 solution in a wide pH range of 2-11. Thus, the
pH value of the solution showed an ignorable effect on the background
fluorescence in LH-1, probably because of the combined effect of two types of
-
fluorescence quenching groups. Upon introducing 40 μM ClO , remarkable
fluorescence enhancement was observed when the pH value of the solution was
-
above 6.0. Consequently, LH-1 could be used to detect ClO under physiological
pH conditions.
1
7

-
3
.3 Determination of ClO in water samples
-
-
ClO was widely used in our daily life, and the high-level residue of ClO in water may
-
lead to hazard to human health. Thus, probe LH-1 was applied for determining ClO in
various water samples by means of standard addition method. The water samples were
obtained from Yu Dai river, Jing Lake and tap water. No significant fluorescence
enhancement was observed when LH-1 was directly treated with the water samples
-
(
Table S2). When different concentrations of ClO (5, 10 and 20 μM) were added to the
-
water samples, probe LH-1 could detect ClO in these water samples with good recovery.
-
These data led us to conclude that LH-1 can accurately quantify ClO levels in various
water samples.
-
3
.4 Fluorescence imaging of exogenous and endogenous ClO in living cells
-
Inspired by the good performance of LH-1 to ClO in aqueous solution, probe LH-1
-
was further utilized for detecting exogenous and endogenous ClO in living cells. Firstly,
the background fluorescence in living cells was investigated. Obviously, probe LH-1
showed no discernible background fluorescence in HeLa cells (Figure 7a). Thus, LH-1
-
can be used as a reliable fluorescent probe for detecting ClO in living cells. By contrast,
the control compound LH-2 still displayed weak background fluorescence (Figure 7b),
probably because the relative high viscosity in living cells inhibits the free rotation of
N-N group in LH-2.
1
8

Figure 7. (a, b) The fluorescence image of LH-1 and LH-2 in HeLa cells, respectively;
c, d) merged image of (a) and (b) with their corresponding bright field images,
(
respectively; (e) quantification of mean fluorescence intensity in (a) and (b)
correspondingly. Scale bar: 20 μm.
-
Then, the fluorescence imaging of LH-1 to exogenous ClO in HeLa cells was carried
out. The cells were pre-stained with LH-1 (10 μM), and then incubated with different
-
concentration of ClO (0 μM, 5 μM, 10 μM, 15 μM, and 30 μM). As shown in Figure 8,
the fluorescence intensities of the cells were gradually enhanced with the addition of
-
increasing amount of ClO . The results indicated that probe LH-1 had the capability to
-
sense different concentration of exogenous ClO in living cells.
-
Figure 8. Fluorescence images for ClO detection in HeLa cells using probe LH-1. (a-e)
1
9

image of HeLa cells incubated with LH-1 (10 μM) for 30 min, and then treating with 0, 5,
-
1
0, 15, and 30 μM ClO , respectively. (f-j) merged image of (a-e) with their
corresponding bright field image. (k) quantification of mean fluorescence intensity in (a-e)
correspondingly. Scale bar: 20 μm.
As probe LH-1 displayed extremely low background fluorescence as well as
-
highly sensitive response to ClO , we anticipated that LH-1 could be applied for
-
imaging very trace amount of endogenous ClO in living cells. Thus, the feasibility
-
of LH-1 for imaging endogenous ClO was investigated in RAW 264.7 cells. After
incubating with LH-1 for 30 min, the RAW 264.7 cells showed weak intracellular
fluorescence (Figure 9a). However, when the cells were pre-treated with
4-amino-benzoicacid hydrazide (ABAH) (well-known MPO inhibitors [26]) for 30
min, and then further incubated with LH-1 for 30 min, no fluorescence was
observed (Figure 9b). Thus, the weak fluorescence in Figure 9a was apparently
-
originated from basal endogenous ClO . Accordingly, LH-1 was sensitive enough
-
to detect the basal endogenous ClO . It is known that RAW264.7 cells may
-
produce more concentration of endogenous ClO by lipopolysaccharide (LPS)
stimulation [27]. Subsequently, RAW 264.7 cells were incubated with LPS (20
μg/mL) for 12 h, and then stained with LH-1 for 30 min. Notable fluorescence
enhancement was observed (Figure 9c). Therefore, probe LH-1 was able to detect
-
the minor variation of endogenous ClO in living RAW 264.7 cells. The
cytotoxicity of LH-1 was studied by MTT assays. The results demonstrated that
probe LH-1 showed low cellular cytotoxicity (Figure S9).
2
0

-
Figure 9. Fluorescence images for detection of endogeous ClO in RAW 264.7 cells
using LH-1. (a) RAW 264.7 cells were incubated with LH-1 (10 μM) for 30 min; (b)
RAW 264.7 cells were pre-treated with ABAH (200 μM) for 30 min, and then incubated
with LH-1 (10 μM) for 30 min; (c) RAW 264.7 cells were stimulated with LPS (20
μg/mL) for 12 h and then incubated with LH-1 for 30 min. (d-f) merged image of a-c
with their corresponding bright field image. (g) Quantification of mean fluorescence
intensity in (a-c) correspondingly. Scale bar: 20 μm.
-
3
.5 Fluorescence imaging of ClO in zebrafish
Encouraged by the successful fluorescence imaging in living cells, we further explored
-
the ability of LH-1 for detection of ClO in vivo. In the control group, the 3-day-old
zebrafish was stained with 10 μM LH-1 for 30 min. Almost no fluorescence can be seen
in the zebrafish (Figure 10b). However, when the LH-1 stained zebrafish was further
-
incubated with ClO (5 μM) for 30 min, a weak green fluorescence was observed (Figure
1
0d). Moreover, the green fluorescence become stronger after the LH-1 stained zebrafish
-
incubated with 10 μM ClO for 30 min (Figure 10f). Therefore, probe LH-1 has the
-
potential capacity to monitor ClO in vivo.
2
1

-
Figure 10. Fluorescence imaging of ClO in zebrafish: (a–b) the zebrafish was stained
with LH-1 (10ꢀμM) for 30ꢀmin; (c–d) the zebrafish was stained with LH-1 (10ꢀμM) for
-
3
0ꢀmin, and then incubated with ClO (5ꢀμM) for 30ꢀmin; (e–f) the zebrafish stained with
-
LH-1 (10ꢀμM) for 30ꢀmin, and then incubated with ClO (10ꢀμM) for 30ꢀmin; (a, c, e) the
bright-fields; (b, d, f) the green fluorescence channels. Scale bar: 500 μm.
4
. Conclusions
In summary, we have rationally developed a novel fluorescent probe, LH-1, for
-
detection of ClO in water samples and biological samples. In probe LH-1, the
fluorescence of coumarin 6H fluorophore not only can be quenched by the rotation
of N-N group, but also can be quenched by the 2,4-dinitrobenzenze group via
d-PET mechanism. Thus, probe LH-1 displayed extremely low background
fluorescence. Moreover, by the combined effect of two fluorescence quenching
groups, the probe exhibited low background fluorescence even in a solution with
high viscosity. The optical experiments indicated that probe LH-1 displayed
-
dramatical fluorescence enhancement response to ClO with high sensitivity and
selectivity, and the detection limits were determined to be 10.5 nM. In addition, the
2
2

-
fluorescence enhancement showed good linear relationship to ClO in the
concentration range of 0 to 20 μM. Probe LH-1 has been applied for quantitatively
-
detecting ClO in various water samples. Importantly, the fluorescence imaging
experiments demonstrated that LH-1 was successfully used for imaging the minor
-
variation of ClO in living cells and zebrafish. The probe illustrated herein will be
-
widely employed to reliably monitor the variation of ClO in living system.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(
Grants 201105038, 51878317, 21874059), the Priority Academic Program
Development of Jiangsu Higher Education Institutions, and the Jiangsu Collaborative
Innovation Center of Technology and Material of Water Treatment.
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[
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[
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7

Highlights
ò
ò
ò
The fluorescent probe displayed extremely low background fluorescence by a novel
doubly-quenching strategy.
The fluorescent probe showed highly sensitive and selective response to hypochlorite even
with no perturbation by viscosity.
The fluorescent probe has been applied for fluorescence imaging of exogenous and
endogenous hypochlorite in living cells and zebrafish.