A turn-on fluorescent probe for hypochlorous acid based on HOCl-promoted removal of the C = N bond in BODIPY-hydrazone

Article abstract of DOI:10.1039/c5nj01083g

A boron dipyrromethene-based fluorescent probe, BODH, has been successfully developed for hypochlorous acid detection based on the HOCl-promoted oxidative removal of the C = N bond in response to the amount of HOCl. The reaction is accompanied by a 6-fold

Full text of DOI:10.1039/c5nj01083g

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A boron dipyrromethene-based fluorescent probe for sensing HOCl was developed .

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A turn-on fluorescent probe for hypochlorous acid based on HOCl-promoted
removal of the C=N bond in BODIPY-hydrazone
a
a
a,
WeiꢀChieh Chen , Parthiban Venkatesan , ShuꢀPao Wu *
a
Department of Applied Chemistry, National Chiao Tung University, Hsinchu,
Taiwan 300, ROC. Eꢀmail: spwu@mail.nctu.edu.tw. Tel.: +886ꢀ3ꢀ5712121ꢀext56506;
Fax: +886ꢀ3ꢀ5723764
*
Corresponding author.
1

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Abstract
A boron dipyrrometheneꢀbased fluorescent probe, BODH, has been successfully
developed for hypochlorous acid detection based on HOClꢀpromoted oxidative
removal of the C=N bond in response to the amount of HOCl. The reaction is
accompanied by a 6ꢀfold increase in the fluorescent quantum yield (from 0.05 to 0.29).
The fluorescence intensity of the reaction between HOCl and BODH shows a good
linearity in the HOCl concentration range of 1 to 25 ꢁM with a low detection limit of
2
05 nM (S/N = 3). Confocal fluorescence microscopy imaging using RAW264.7 cells
shows that the new probe BODH can be used as an effective fluorescent probe for
detecting HOCl in living cells.
Keywords: Hypochlorous acid; Fluorescent probe; Boron dipyrromethene;
Bioimaging.
2

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1
. Introduction
Hypochlorous acid (HOCl) is a reactive oxygen species (ROS) produced as a
1
ꢀ4
defense mechanism in the immune system. Endogenous hypochlorous acid is
formed from the reaction of chloride ion with hydrogen peroxide catalyzed by the
enzyme myeloperoxidase (MPO) in leukocytes including macrophages, monocytes,
5
,6
and neutrophils. However, overproduction of HOCl causes several human diseases
7
ꢀ9
such as arthritis, cancer and neurodegeneration. Due to its biological importance,
the need to design highly sensitive and selective probes for HOCl has become a
significant research issue. Fluorescent probes that can directly indicate the change in
concentration of HOCl would be useful for examining the dynamic spreading of
HOCl in living cells.
HOCl has shown high oxidation properties, which can be used to design a probe
for HOCl. Several functional groups, such as pꢀmethoxyphenol, oxime, selenide,
10ꢀ33
telluride and thiol, have been found to be HOClꢀreactive moieties.
To construct a
HOCl fluorescent probe, a HOClꢀreactive moiety is connected to an organic
fluorophore so that it can control the fluorescence of the fluorophore. In particular, the
isomerization of the C=N group is known to undergo nonꢀradiative decay in the
11
excited state; this can be used as a signal alteration for fluorescence turnꢀon sensing.
In the absence of HOCl, the fluorescence of the fluorophore is quenched due to imine
3

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isomerization; however, in the presence of HOCl, the imine isomerization is blocked
by HOCl oxidation and the fluorescence of the fluorophore is reestablished. To
facilitate the practical application of HOCl probes in cell imaging, development of
these probes has focused on selectivity and sensitivity. In this paper, we have reported
a highly sensitive and selective fluorescent probe for HOCl based on the
HOClꢀpromoted oxidation of imine.
In this work, a BODIPYꢀbased fluorescent probe BODH, was designed for HOCl
detection based on HOClꢀpromoted removal of the C=N bond. BODH displays weak
fluorescence with a quantum yield of Φ = 0.05 due to imine (C=N) isomerization,
which has been known to exhibit nonꢀradiactive decay in the excited state. The strong
fluorescence of BODIPY is restored after the imine bond is oxidized to aldehyde
(RꢀCHO) by HOCl. This new probe exhibits high selectivity and sensitivity towards
HOCl over other ROS and reactive nitrogen species (RNS) in aqueous solutions. Most
importantly, BODH shows good cellꢀmembrane permeability and can be successfully
used to obtain images of endogenous HOCl in living cells.
2
2
. Experimental
.1Materials and Instrumentation
All reagents were obtained from commercial sources and used as received without
further purification. UV/Vis spectra were recorded on an Agilent 8453 UV/Vis
4

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spectrometer. Fluorescence spectra measurements were performed on a Hitachi
Fꢀ7000 fluorescence spectrophotometer. NMR spectra were obtained on a Bruker
DRXꢀ300 and Agilent Unity INOVAꢀ500 NMR spectrometer. Fluorescent images
were taken on a Leica TCS SP5 X AOBS Confocal Fluorescence Microscope.
2
.2 Preparation of ROS and RNS
●
2 2 2 2 3
Various ROS and RNS including HOCl, OH, H O , O , NO
−, NO −, NO, ONOO−,
1
−
O
2
and tꢀBuOOH were prepared according to the following methods. HOCl was
ꢀ
prepared from NaOCl; the concentration of hypochlorite (OCl ) was determined by
ꢀ1
ꢀ1
using an extinction coefficient of 350 M cm (292 nm) at pH 9.0. Hydroxyl radical
●
(
4 2 4 2 2
OH) was generated by Fenton reaction on mixing Fe(NH ) (SO ) ‧6H O with 10
●
equivalents of H
O
2 2
; the concentration of OH was estimated from the concentration
2
+
2 2
of Fe . The concentration of the commercially available stock H O solution was
1
estimated by optical absorbance at 240 nm. Singlet oxygen ( O
2
) was generated by the
34
−
addition of NaOCl and H
2
O
2
according to the literature. The source of NO
2
and
−
NO
3
was from NaNO
2
and NaNO
3
. Nitric oxide (NO) was generated from sodium
−
nitroferricyanide(III) dihydrate. Peroxynitrite (ONOO ) was prepared as the reported
35
method; the concentration of peroxynitrite was estimated by using an extinction
ꢀ1
ꢀ1
2 2
coefficient of 1670 M cm (302 nm). Superoxide (O .
−) is prepared from KO
tꢀBuOOH was obtained commercially from Alfa Aesar.
5

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2
.3 Synthesis of BODH
Hydrazine (99%, 172.12 ꢁL, 2.8 mmol) in MeOH was added to a solution of
BODIPYꢀaldehyde (100 mg, 0.28 mmol) in THF/MeOH (5 mL / 30 mL). The reaction
mixture was stirred for 12 h at room temperature. The solvent was removed under
reduced pressure and the solid was dissolved in acetone (30 mL) and then further
stirred for 12 h. The solvent was removed under reduced pressure and the solid was
2 2
taken up into a mixture of water and CH Cl . The mixture was extracted four times
4
with EtOAc. The combined organic fractions were dried over MgSO , and then the
solvent was removed under reduced pressure. The crude material was purified by
column chromatography (hexane: CH
Cl
2 2
= 1:1) to give the compound as a dark
1
purple solid. Yield: 60.5 mg (53 %). m.p. 187.3 °C. H NMR (300 MHz, CDCl
3
): δ
8
3
1
1
.43 (s, 1H), 7.53ꢀ7.51 (m, 3H), 7.32ꢀ7.28 (m, 2H), 6.08 (s, 1H), 2.85 (s, 3H), 2.61 (s,
1
3
3
H), 2.11 (s, 6H), 1.59 (s, 3H), 1.40 (s, 3H). C NMR (125 MHz, CDCl ): δ 167.8,
58.1, 155.8, 152.7, 145.0, 142.4, 141.5, 134.7, 132.5, 130.6, 129.2, 129.1, 127.9,
+
23.9, 122.5, 29.6, 25.2, 18.6, 14.6, 14.4, 14.2, 12.2. HRMS (EI ): calcd. for
+
C
23
H
25BF
2
N
4
[M] 406.1240; found 406.1247.
2
.4 Cell Culture for RAW264.7 Macrophages
6

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The cell line RAW264.7 was provided by the Food Industry Research and
Development Institute (Taiwan). RAW264.7 cells were cultured in Dulbecco’s
modied Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS)
at 37 °C under an atmosphere of 5% CO
coverslips and allowed to adhere for 24 h.
2
. Cells were plated on 18 mm glass
2
.5 Cytotoxicity assay
The methyl thiazolyl tetrazolium (MTT) assay was used to measure the cytotoxicity
of BODH in RAW264.7 cells. RAW264.7 cells were seeded into a 96ꢀwell
cellꢀculture plate. Various concentrations (5, 10, 15, 20, 25 ꢁM) of BODH were added
2
to the wells. The cells were incubated at 37℃ under 5% CO for 24 h. 10 ꢁL MTT (5
。
2
mg/mL) was added to each well and incubated at 37 C under 5% CO for 4 h. Remove
the MTT solution and yellow precipitates (formazan) observed in plates were
dissolved in 200 ꢁL DMSO and 25 ꢁL Sorenson’s glycine buffer (0.1 M glycine and
0
.1 M NaCl). Multiskan GO microplate reader was used to measure the absorbance at
70 nm for each well. The viability of cells was calculated according to the following
5
equation:
Cell viability (%) = (mean of absorbance value of treatment group) / (mean of
absorbance value of control group).
7

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2
.6 Fluorescence Imaging of Exogenous BODH in Living Cells
Experiments to assess the sensing ability of BODH for exogenous NaOCl were
performed in 0.1 M phosphateꢀbuffered saline (PBS) with NaOCl (10 ꢁM). Treat the
cells with 2 ꢁL of 10 mM BODH (final concentration: 10 ꢁM) dissolved in DMSO
and incubated for 30 min at 37 °C. The treated cells were washed with 0.1 M PBS (2
mL × 3) to remove remaining BODH. DMEM (2 mL) was added to the cell culture,
which was then treated with 10 mM solution of NaOCl (2 ꢁL; final concentration: 10
◦
ꢁ
M) dissolved in sterilized 0.1 M PBS (pH 7.4). The samples were incubated at 37 C
for 10 min. The culture medium was removed, and the treated cells were washed with
.1 M PBS (2 mL × 3). Paraformaldehyde solution (1 mL, 4%, in PBS) was added to
0
the treated cell and keep in room temperature 20 min for cell fixation. The
Paraformaldehyde solution was removed and the treated cells were washed with 0.1
M PBS (2 mL × 3) and DI water (2 mL× 3). The cell specimen was made before
observation. Confocal fluorescence imaging of cells was performed with a Leica TCS
SP5 X AOBS Confocal Fluorescence Microscope (Germany), and a 63x
oilꢀimmersion objective lens was used. The cells were excited with a white light laser
at 470 nm, and emission was collected at 495ꢀ535 nm.
2
.7 Fluorescence Imaging of PMA-Induced BODH Production in Living Cells
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RAW264.7 cells were treated with PMA (25 ng/mL) and BODH in culture medium
for 2 h. The culture medium was removed, and the treated cells were washed with 0.1
M PBS (2 mL × 3) before observation. Fluorescence imaging was performed with a
Leica TCS SP5 X AOBS Confocal Fluorescence Microscope. The cells were excited
with a white light laser at 480 nm, and emission was collected at 495ꢀ535 nm.
3
3
. Results and discussion
.1 Synthesis of the probe BODH
The synthesis of the probe BODH is outlined in Scheme 1. BODIPYꢀaldehyde
was obtained from the formylation reaction of BODIPY with VilsmeierꢀHaack
reagent (DMF / POCl
3
). In the next step, the BODIPYꢀaldehyde was further treated
1
with hydrazine, followed by acetone. The structure of BODH was confirmed using H
13
NMR, C NMR and high resolution EI MS. The probe BODH was pink and had an
absorption band centered at 535 nm, which was redꢀshifted by 50 nm from the typical
BODIPY absorption band at 480 nm, due to longer conjugated double bonds. The
probe BODH exhibited weak fluorescence (Φ = 0.05) compared to BODIPY
(Φ = 0.6–0.9) due to imine (C=N) isomerization, which exhibits nonꢀradiactive decay
in the excited state.
9

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3
.2 Fluorescent response of BODH with HOCl
The sensing ability of the probe BODH was tested for various ROS and RNS,
●
1
ꢀ
ꢀ
ꢀ
ꢀ
2 2 2 2 3 2
including NaOCl, OH, H O , O , NO , NO , NO, ONOO , O , and tꢀBuOOH, in a
phosphateꢀbuffered saline (PBS) solution. It was found that strong green fluorescence
emission only occurred for the addition of HOCl to the BODH solution; other ROS
and RNS produced no change in fluorescence (Fig. 1). Quantitative fluorescence
spectra of BODH were recorded in the presence of several ROS and RNS, but HOCl
was the only reactive species to cause an obvious fluorescence enhancement (Fig. 2).
The reaction of BODH with HOCl was fast; addition of NaOCl(aq) to the solution of
the probe BODH within 3 min caused a significant change in fluorescence intensity
(see Figure S5 in the supporting information). During NaOCl titration with BODH,
the absorbance at 512 nm decreased and a new band at 492 nm appeared (Fig. 3). The
oxidized product, BODIPY-Al, has a shorter absorption wavelength centered at
4
92 nm, which is blueꢀshifted by 20 nm from the absorption band of BODH at 512
nm (Scheme 2). This is due to shorter double bond conjugation in the oxidized
product, BODIPY-Al. This absorption change was also observed as color change
from pink to red (Fig. 1). In addition, upon gradual addition of HOCl to a solution of
BODH, an emission band centered at 508 nm was formed (Fig. 3). The emission
intensity reached its maximum after the addition of four equivalents of NaOCl.
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BODH displays weak fluorescence with a quantum yield of Φ = 0.05 due to imine
C=N) isomerization, which has been known to exhibit nonꢀradiactive decay in the
(
excited state. The strong fluorescence of BODIPY is restored after the oxidation of
imine to aldehyde (RꢀCHO) by HOCl (Scheme 2). The quantum yield of the oxidized
BODH was Φ = 0.29, which is 6ꢀfold higher than that of BODH, at Φ = 0.05. The
1
13
structure of oxidized BODH was confirmed by H NMR, C NMR, HPLC, and MS
1
spectrometry. H NMR spectra of BODH showed that the proton (H
a
, imine) signal at
δ = 8.41 ppm disappeared as NaOCl was added and a new peak at δ = 10.03 ppm was
formed (Fig. 4). This indicates that the oxidization of BODH by NaOCl happens on
the imine group. According to the MS and NMR spectra of oxidized BODH, the
imine group was oxidized to be an aldehyde group (CHO). Notably, there was a good
linear correlation between the fluorescence intensity and the concentration of NaOCl
(0 – 20 ꢀM). Furthermore, it was found that BODH had a detection limit of 0.205 ꢁM
(see Figure S7 in the supporting information), which makes it sufficiently sensitive
for HOCl detection in living systems.
A pHꢀdependence experiment was conducted with BODH, to investigate a suitable
pH range for HOCl sensing. Fig. 5 shows that the emission intensities of BODH are
very low at a pH range of 4.5 – 10. After addition of NaOCl, the emission intensity at
5
08 nm became significantly higher at a pH range of 5.0 – 10.0, which means that the
11

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probe could be used in physiological conditions. The emission enhancement is much
more pronounced under basic conditions (pH > 7) than under acidic conditions (pH <
ꢀ
7
), implying that hypochlorite (ClO ) plays a key role in the sensing process.
.3 Imaging live cells
3
The potential of the probe BODH for imaging HOCl in living cells was
investigated. Murine RAW264.7 macrophages were used as a model because
macrophages are known to generate ROS and RNS in the immune system. First, an
MTT assay with a RAW264.7 cell line was used to determine the cytotoxicity of
HBP. In Fig. 6, the cellular viability was estimated to be greater than 80% after 24 h,
which indicates that BODH (<30 ꢁM) has low cytotoxicity. Furthermore, the images
of cells were obtained using a confocal fluorescence microscope. When RAW264.7
cells were incubated with BODH (10 ꢁM), no fluorescence was observed (Fig. 7a).
After the treatment of NaOCl, bright green fluorescence was observed in the
RAW264.7 cells (Figure 7b). An overlay of fluorescence and brightꢀfield images
shows that the fluorescence signals are localized in the intracellular area, indicating a
subcellular distribution of HOCl and good cellꢀmembrane permeability of BODH.
Finally, BODH was used to detect PMAꢀinduced endogenous HOCl production
in RAW264.7 cells. Phorbol myristate acetate (PMA) is known to activate the
3
6
generation of ROS and RNS in macrophage cells, including HOCl. After stimulation
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with PMA (25 ng/mL) for 2 h in the presence of BODH, strong green fluorescence
was observed in RAW264.7 cells (Fig. 8). These results demonstrated that BODH is
able to visualize PMAꢀinduced endogenous HOCl production in the macrophages.
When MPO inhibitor (4ꢀaminibenzohydrazide) was added along with PMA to RAW
2
64.7 cells, fluorescence enhancement was not observed. This clearly indicates that
HOCl only caused fluorescence enhancement while remaining reactive oxygen
species caused negligible fluorescence enhancement. In order to localize where the
TM
probe BODH can be used to detect endogenous HOCl production, HiLyte Fluor
5
94 acid (cytosol stain) and DAPI (nuclear stain) were used. Fig. 8 shows that the
TM
staining by the probe BODH is identical to that by HiLyte Fluor 594 acid. In the
nuclear region, there is no BODH staining. These observations indicate that the probe
BODH can be used to detect endogenous HOCl production in the cytosol.
4
. Conclusion
In summary, we have developed a BODIPYꢀbased green fluorescent probe
BODH that exhibits a highly selective and sensitive response to HOCl over other
reactive species. This system utilizes the HOClꢀpromoted oxidation and removal of
the C=N bond to respond to the amount of HOCl. BODH is rapidly oxidized by
HOCl to be BODIPYꢀAl with an emission enhancement. Confocal fluorescence
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microscopy imaging using RAW264.7 cells showed that the probe BODH could be
used to evaluate the important role of HOCl in biological systems.
Acknowledgements
We gratefully acknowledge the financial support of Ministry of Science and
Technology (Taiwan, MOST 103ꢀ2113ꢀMꢀ009ꢀ005) and National Chiao Tung
University.
Appendix A. Supplementary data
1
13
H NMR, C NMR and mass spectra of BODH, the reaction profile of BODH with
NaOCl, calibration curve of the reaction BODH with NaOCl.
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2Z. Yang, M. Wang, M. She, B. Yin, Y. Huang, P. Liu, J. Li, S. Zhang, Sens.
Actuat. B Chem. 2014, 202, 656–662
33P. Venkatesan, S. Wu, Analyst, 2015, 140, 1349ꢀ1355.
34X. Li, G. Zhang, H. Ma, D. Zhang, J. Li and D. Zhu, J. Am. Chem. Soc., 2004,
1
26, 11543ꢀ11548.
5J. W. Reed, H. H. Ho and W. L. Jolly, J. Am. Chem. Soc., 1974, 96
248ꢀ1249.
6Y.ꢀC. Yang, H.ꢀH. Lu, W.ꢀT. Wang and I. Liau, Anal. Chem., 2011, 83
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3
3
,
,
1
8
16

New Journal of Chemistry
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1
. N H
2 4
N N
O
DMF,POCl3
ClCH CH Cl
2.acetone
N
N
N
N
H
N
N
2
2
B
B
B
F
F
F
F
F
F
BODIPY-Al
BODH
BODIPY
Scheme 1. Synthesis of the probe BODH
17

Page 19 of 28
New Journal of Chemistry
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DOI: 10.1039/C5NJ01083G
O
N N
HOCl
N
N
H
N
N
B
B
F
F
F
F
BODIPY-Al
BODH
Scheme 2. The reaction of the probe BODH with HOCl
18

New Journal of Chemistry
Page 20 of 28
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DOI: 10.1039/C5NJ01083G
Fig. 1. (a) Color and (b) fluorescence photographs of BODH (5 ꢁM) in a PBS
bufferꢀMeOH (v/v = 70/30, 70 mM PBS, pH7.4) solution. BODH was excited by UV
irradiation (λex: 365 nm).
19

Page 21 of 28
New Journal of Chemistry
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DOI: 10.1039/C5NJ01083G
0.4
0.3
0.2
0.1
0.0
BODH + ROS and RNS
BODH+NaOCl
400
500
600
700
Wavelength (nm)
2
1
1
000
500
000
BODH+NaOCl
500
BODH + others ROS & RNS
0
5
00
550
600
650
Wavelength (nm)
Fig. 2. (a) Absorption spectrum (b) Fluorescence spectra of BODH (5 ꢁM) upon
adding various ROS & RNS (25 ꢁM) in a H OꢀMeOH (v/v = 70/30, 70 mM PBS,
2
pH7.4) solution. The excitation wavelength was 470 nm.
20

New Journal of Chemistry
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DOI: 10.1039/C5NJ01083G
1
1
1
.4
.2
.0
0.3
0.2
0.1
0.0
5
12 nm
4
92 nm
0.8
.6
0
0
1
2
3
4
5
Equivalents of NaOCl
400
500
600
700
Wavelength(nm)
5
08 nm 2000
2
1
1
000
500
000
1
500
000
1
500
0
1
2
3
4
5
6
Equivelents of HOCl
5
00
0
500
550
600
650
700
Wavelength (nm)
Fig. 3. UVꢀvis (top) and fluorescence (bottom) changes of BODH (5 ꢁM) in the
presence of various equivalents of NaOCl in a H OꢀMeOH (v/v = 70/30, 70 mM PBS,
2
pH7.4) solution. The excitation wavelength was 470 nm.
21

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DOI: 10.1039/C5NJ01083G
1
Fig. 4 H NMR (500 MHz) spectra of BDP+HOCl in CDCl .
3
22

New Journal of Chemistry
BODH + HOCl
BODH
Page 24 of 28
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DOI: 10.1039/C5NJ01083G
2
1
1
000
500
000
500
0
4
5
6
7
8
9
10
pH
Fig. 5. Fluorescence response (508 nm) of free BODH (5 mM) (
of NaOCl (25 mM, 5eq) ( ) in a H OꢀMeOH (v/v = 70/30, 70 mM buffer) solution
as a function of different pH values. The excitation wavelength was 470 nm.
▓) and after addition
●
2
23

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New Journal of Chemistry
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DOI: 10.1039/C5NJ01083G
1
00
8
6
4
2
0
0
0
0
0
0
5
10
15
20
25
-
6
[
BODH], 10 M
Fig. 6. MTT assay of RAW 264.7 cells were treated in the presence of BODH (0 – 25
M) incubated for 24 hours.
ꢁ
24

New Journal of Chemistry
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DOI: 10.1039/C5NJ01083G
Bright field
Fluorescence
(b)
Overlay
(
a)
(c)
BODH
(
d)
(e)
(f)
BODH
+
NaOCl
Fig. 7. Confocal fluorescence images of RAW264.7 cells. (a) (d) Bright field image;
b) (e) fluorescence image; and (c) (f)merged image. (aꢀc) The cells incubated with
(
BODH (10 μM) for 30 min. (dꢀf) Subsequent treatment of the cells with NaOCl (10
μM) for 10 min. Excitation wavelength = 470 nm, scan range = 495ꢀ535 nm
25

Page 27 of 28
New Journal of Chemistry
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DOI: 10.1039/C5NJ01083G
TM
BODH
HiLyte Fluor 594 acid
DAPI
Overlay
(
a)
e)
(b)
(c)
(d)
BODH
+
PMA
BODH
+
(
(f)
(g)
(h)
4
-ABH
+
PMA
Fig. 8. Detection of PMAꢀinduced hypochlorite production in RAW264.7 cells. (aꢀd)
The cells treated with stimulant PMA (25 ng/mL) for 2 h in the presence of BODH
(10 µM). (eꢀf) MPO inhibitor (100 µM), (4ꢀABH) was coꢀincubated during PMA
stimulation, and incubated with of BODH (10 µM) for 1 hour at 37ºC. The cell was
TM
colabeled with (b, f) HiLyte Fluor 594 acid, (c)(g) DAPI, (e, h) merge image of (aꢀc)
and (eꢀg). The excitation wavelength for (a, e), (b, f) and (c, g) were 470 nm, 594 nm
and 405 nm, respectively; the emission scanning range were 495ꢀ535 nm, 637ꢀ737 nm
and 430ꢀ550 nm, respectively.
26

New Journal of Chemistry
Page 28 of 28
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DOI: 10.1039/C5NJ01083G
Figure and Scheme captions
1. Scheme 1. Synthesis of the probe BODH
2. Scheme 2. The reaction of the probe BODH with HOCl
3. Fig. 1. (a) Color and (b) fluorescence photographs of BODH (5 ꢁM) in a PBS
bufferꢀMeOH (v/v = 70/30, 70 mM PBS, pH7.4) solution. BODH was excited by UV
irradiation (λ : 365 nm).
ex
4
.Fig. 2. (a) Absorption spectrum (b) Fluorescence spectra of BODH (5 ꢁM) upon
adding various ROS & RNS (25 ꢁM) in a H OꢀMeOH (v/v = 70/30, 70 mM PBS,
2
pH7.4) solution. The excitation wavelength was 470 nm.
5
. Fig. 3. UVꢀvis (top) and fluorescence (bottom) changes of BODH (5 ꢁM) in the
presence of various equivalents of NaOCl in a H OꢀMeOH (v/v = 70/30, 70 mM PBS,
2
pH7.4) solution. The excitation wavelength was 470 nm.
1
6
. Fig. 4 H NMR (500 MHz) spectra of BDP+HOCl in CDCl .
3
7
. Fig. 5. Fluorescence response (508 nm) of free BODH (5 mM) (
▓) and after
2
addition of NaOCl (25 mM, 5eq) (●) in a H OꢀMeOH (v/v = 70/30, 70 mM buffer)
solution as a function of different pH values. The excitation wavelength was 470 nm.
8
2
9
. Fig. 6. MTT assay of RAW 264.7 cells were treated in the presence of BODH (0 –
5 ꢁM) incubated for 24 hours.
. Fig. 7. Confocal fluorescence images of RAW264.7 cells. (a) (d) Bright field image;
(
b) (e) fluorescence image; and (c) (f)merged image. (aꢀc) The cells incubated with
BODH (10 M) for 30 min. (dꢀf) Subsequent treatment of the cells with NaOCl (10
M) for 10 min. Excitation wavelength = 470 nm, scan range = 495ꢀ535 nm
0. Fig. 8. Detection of PMAꢀinduced hypochlorite production in RAW264.7 cells.
aꢀd) The cells treated with stimulant PMA (25 ng/mL) for 2 h in the presence of
μ
μ
1
(
BODH (10 µM). (eꢀf) MPO inhibitor (100 µM), (4ꢀABH) was coꢀincubated during
PMA stimulation, and incubated with of BODH (10 µM) for 1 hour at 37ºC. The cell
TM
was colabeled with (b, f) HiLyte Fluor 594 acid, (c)(g) DAPI, (e, h) merge image of
(aꢀc) and (eꢀg). The excitation wavelength for (a, e), (b, f) and (c, g) were 470 nm, 594
nm and 405 nm, respectively; the emission scanning range were 495ꢀ535 nm, 637ꢀ737
nm and 430ꢀ550 nm, respectively.
27