Lysosome-targeted two-photon fluorescent probe for detection of hypobromous acid in vitro and in vivo

Article abstract of DOI:10.1016/j.saa.2018.12.029

It is found that hypobromous acid (HOBr) can affect the activity of type IV collagen. Herein, we synthesized a lysosome-targeted fluorescence probe NA-lyso based on Suzuki coupling reaction with naphthalimide as a fluorescent group. HOBr can oxidize the amino group and methylthio group, which increased the degree of conjugation of the probe, thereby affecting its optical properties. Accordingly, it can establish a method for the specific detection of HOBr. NA-lyso has the properties including fast response, high fluorescence quantum yield (Φ = 59.17%), high selectivity, low cytotoxicity and good membrane-permeability. The probe can locate to lysosome of cells. The potential of the probe as biosensor for HOBr was demonstrated by imaging of exogenous and endogenous HOBr in living cells and in mice. In consequence, NA-lyso is expected to be a powerful tool to detect HOBr in complex biosystem and provides a means of exploring physiological functions associated with HOBr in living organisms.

Full text of DOI:10.1016/j.saa.2018.12.029

Accepted Manuscript
Lysosome-targeted two-photon fluorescent probe for detection of
hypobromous acid in vitro and in vivo
Chen Ma, Minrui Ma, Yida Zhang, Xinyue Zhu, Lin Zhou, Ran
Fang, Xiaoyan Liu, Haixia Zhang
PII:
DOI:
Reference:
S1386-1425(18)31099-0
https://doi.org/10.1016/j.saa.2018.12.029
SAA 16658
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 5 November 2018
Revised date:
Accepted
date:
6 December 2018
15 December 2018
Please cite this article as: Chen Ma, Minrui Ma, Yida Zhang, Xinyue Zhu, Lin Zhou, Ran
Fang, Xiaoyan Liu, Haixia Zhang , Lysosome-targeted two-photon fluorescent probe for
detection of hypobromous acid in vitro and in vivo. Saa (2018), https://doi.org/10.1016/
j.saa.2018.12.029
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
Lysosome-targeted two-photon fluorescent probe for
detection of hypobromous acid in vitro and in vivo
Chen Ma1, Minrui Ma1, Yida Zhang1, Xinyue Zhu1, Lin Zhou1, Ran Fang1, Xiaoyan
Liu1, Haixia Zhang1,*
1College of Chemistry and Chemical Engineering, Lanzhou University
*E-mail: zhanghx@lzu.edu.cn

ACCEPTED MANUSCRIPT
Abstract
It is found that hypobromous acid (HOBr) can affect the activity of type IV
collagen. Herein, we synthesized a lysosome-targeted fluorescence probe NA-lyso
based on Suzuki coupling reaction with naphthalimide as a fluorescent group. HOBr
can oxidize the amino group and methylthio group, which increased the degree of
conjugation of the probe, thereby affecting its optical properties. Accordingly, it can
establish a method for the specific detection of HOBr. NA-lyso has the properties
including fast response, high fluorescence quantum yield (Φ=59.17%), high
selectivity, low cytotoxicity and good membrane-permeability. The probe can locate
to lysosome of cells. The potential of the probe as biosensor for HOBr was
demonstrated by imaging of exogenous and endogenous HOBr in living cells and in
mice. In consequence, NA-lyso is expected to be a powerful tool to detect HOBr in
complex biosystem and provides a means of exploring physiological functions
associated with HOBr in living organisms.
Keywords: Fluorescence probe; Hypobromous acid; Two-photon; Lysosome-targeted

ACCEPTED MANUSCRIPT
Introduction
Reactive oxygen species (ROS) are closely related to many biological processes,
including cell signaling, cell differentiation, cell growth, and apoptosis [1-3]. Further,
they are involved in the pathogenesis of many diseases in organisms [4-6]. HOBr is
an important ROS and its chemical properties and structures are very similar with
HOCl [7]. There were a lot of detailed research on the role of HOCl in human, for
instance, once the body produces an excessive amount of HOCl, it causes various
diseases including rheumatoid arthritis and cancer [8-11]. However, research on the
physiological effects and detection of HOBr are quite rare [12]. Recent researches
have shown that excessive generation and accumulation of HOBr in human body is
toxic to organisms and its damaging influences relate to a wide range of diseases,
including rheumatoid arthritis [13, 14] and inflammatory tissue damage[15-17].
Hudson and his co-workers reported that HOBr plays an irreplaceable role in the
formation of collagen IV in 2014 [18], because HOBr plays a key role in the coupling
reaction of hydroxylysine and methionine, while they are components of collagen IV.
HOBr is generated from the peroxidation of the bromide anion (Br-) with hydrogen
peroxide, the reaction was catalyzed by a heme peroxidase such as myeloperoxidase
(MPO) or eosinophil peroxidase (EPO) [19, 20]. Recently, Tang’s group has
developed two fluorescence probes for detecting HOBr, one is biphenyl probe (BPP),
the other is a mitochondria-targeting probe RhSN-mito, both of probes are capable of
sensitive and selective detection of HOBr [21, 22]. To better understand the

ACCEPTED MANUSCRIPT
physiological and pathological effects of HOBr at the subcellular level, it is necessary
to establish an ultrasensitive method for monitoring the concentration change of
HOBr in different organelles. However, there is no probe for detecting endogenous
HOBr in lysosomes.
Inspired by the above research, we have developed a lysosome-targeted
fluorescent probe NA-lyso for the specific detection of HOBr. The synthetic straegy is
shown in Scheme 1. NA-lyso was obtained by a Suzuki cross-coupling reaction. In
the presence of HOBr, the amino group on the probe and the methylthio group will
undergo a cyclization reaction, and the fluorescence intensity of the cyclic product
(NA-cyc) weakens sharply. The electron clouds orbital distribution of NA-lyso and
NA-cyc by density functional theory were calculated, verifying that the photoinduced
electron transfer (PET) effect results in the fluorescence quenching.
Scheme 1. Synthetic route of probe NA-lyso and proposed reaction between
NA-lyso and HOBr
2. Experimental Section

ACCEPTED MANUSCRIPT
2.1. Materials and instruments
Liquid bromine, dichloromethane (CH2Cl2), ethanol (C2H5OH), methanol
(MeOH) and toluene were purchased from Tianjin Guangfu Chemical Research
Institute (Tianjin, China). 1,1’-bis(diphenylphosphino) ferrocene-palladium (II)
chloride dichloromethane complex, 2-methylthiobenzeneboronic acid, silver nitrate
(AgNO3), 4-(2-aminoethyl)morpholine, 6-Dimethyl sulfoxide (d6-DMSO),
4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES) were purchased from
Energy Chemical Technology (Shanghai, China). N-Acety-L-Cysteine (NAC) was
purchased from Sigma Aldrich China ( Shanghai, China). DEME medium, MTT
(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) was obtained
from Sangon Biotech (Shanghai, China). Lyso-Tracker Red, a commercial lysosomal
targeting reagent was purchased from Beyotime Biotechnology (Shanghai, China),
Ultrapure water was produced from the ALH-6000-U (Aquapro International
Company, USA) purification system. All other chemicals were obtained from
qualified reagent suppliers with analytical reagent grade.
Fluorescence spectra were measured by a RF-5301pc Fluorescence
spectrometer (Shimadzu, Japan) with a Xenon lamp and 1.0-cm quartz cells at the
slits of 5/5 nm. Absorption spectra were measured on a UV-visible spectrophotometer
(TU-1810, China). The fluorescence quantum yields and two-photon absorption cross
section were determined on Fluorescence spectrometer FLSP920 (Edinburgh
Instruments Ltd., U.K). High resolution mass spectra (HRMS) were measured using a

ACCEPTED MANUSCRIPT
APEX II 47e FT-ICR spectrometer with ESI or APCI positive ion mode (Bruker
Daltonics, America). NMR spectra were determined by 400 MHz using a JEOL NMR
1
instruments (JEOL, Japan). All H NMR chemical shifts (δ) are reported relative to
13
residual d6-DMSO (δ = 7.27 ppm), and all C NMR chemical shifts (δ) are reported
relative to d6-DMSO (δ = 40.76 ppm). MTT Assay were performed using the Spark
multimode microplate reader (TECAN, Switzerland). Cell imaging were performed
by Two-photon laser confocal microscopy (LSM 510 META , Zessi , Germany). Mice
imaging were performed with an IVIS Spectrum (Carestream Health, Canada)
2.2 Synthesis of NA-lyso
The synthesis steps were shown in Scheme 1.
Compound 1 was synthesized by previous method in our laboratory [23].
Compound 1 (2 mmol, 0426 mg) was dissolved in 20 mL CH2Cl2, and liquid bromine
(Br2, 2 mmol, 320 mg) was added. The mixture was kept at 40℃ for 12 h, then the
solvent was evaporated under reduced pressure. The crude product was purified by
silica gel chromatography (CH2Cl2: CH3OH = 20:1, v / v) to obtain compound 2 with
yield 85%.
Compound 2 (0.25 mmol, 72.5 mg), 2-methylthiophenylboronic acid (0.75 mmol,
126 mg), tetrabutylammonium bromide (0.025 mmol, 8.10 mg) and
1,1'-(diphenylphosphine) ferrocene-palladium(II) chloride complex ( 0.025 mmol,
20.4 mg) were dissolved in 15 mL toluene. Ethanol (8 mL) and K2CO3 (4 mL, 4 M)
were added to the mixture. The resulting solution was kept at 80℃ and stirring under

ACCEPTED MANUSCRIPT
an Ar gas atmosphere for 24 h and monitored by thin layer chromatography (TLC)
discontinuously. After the reaction was completed, the reaction mixture were cooled
to room temperature, filtered and concentrated. The crude product was purified by
neutral alumina column chromatography (CH2Cl2: CH3OH = 10:1, v / v) to obtain
compound 3, yield 39%.
Continuously, 0.1 mmol (33.5 mg) of compound 3 was dissolved in 10 mL of
ethanol, and 0.1 mmol (13.0 mg) of 4-(2-aminoethyl) morpholine was added in the
solution. The mixture was refluxing at 78 ° C for 6 h. Then the solvent was removed
and the crude product was purified by silica gel chromatography (CH2Cl2:CH3OH =
20:1, v / v). NA-lyso was obtained with a yield of 60%.
NA-lyso: 1H NMR (400 MHz, ) δ = 8.74 (d, J = 6.7 Hz, 1H), 8.43 (d, J = 4.9 Hz, 1H),
7.90 (d, J = 1.9 Hz, 1H), 7.66 (m, 1H), 7.42 (dd, J = 19.3, 7.4 Hz, 2H), 7.25 (m, 2H),
6.69 (s, 2H), 4.12 (s, 2H), 3.50 (s, 4H), 3.28 (m, 6H), 2.34 (d, J = 1.9 Hz, 3H).
13C NMR (100 MHz, ) δ =164.27(s), 163.42(s), 149.58(s), 139.24(s), 135.95(s),
135.76(s), 133.11(s), 131.46(s), 131.05(s), 130.37(s), 129.69(s), 129.54(s), 125.78(s),
125.70(s), 125.03(s), 124.76(s), 122.30(s), 120.52(s), 119.18(s), 108.27(s), 102.93(s),
66.38(s), 56.63(s), 54.09(s), 36.66(s), 14.87(s). HRMS (ESI, m/z) Calcd for
C25H26N3O3S [M+H]+: 448.1689, found: 448.1690.
2.3 Preparation of HOBr and -OBr
Preparation of HOBr. Liquid bromine (100 μL) was dissolved in ultrapure water
(15 mL), then titrated with 0.2 M AgNO3 solution in an ice bath until the brown color

ACCEPTED MANUSCRIPT
disappear. Then the solution was filtered with 0.22 µM filter membrane.
Lambert−Beer’s law was used to calculate the concentration of HOBr (ε260 = 160 L
mol−1 cm−1[21]).
-
Preparation of OBr. Sodium hydroxide (1.20 g) was dissolved in 10 mL
ultrapure water (10 mL), and 0.768 mL Br2 was added to the solution dropwise in an
ice bath. The concentration of -OBr was calculated (ε329 = 332 L mol−1 cm−1[21])
2.4 Determination of fluorescence quantum yield (Φ) and two-photon absorption
cross section (TPACS)
Femtosecond (fs) fluorescence measurement technology was used to determine
fluorescence quantum yield of NA-lyso (1.0 × 10−6 M，HEPES 7.4) with fluorescein
(Φ%: 92% , 2.4×10-5 M in NaOH solution, pH=11) as the reference. The fluorescence
quantum yields are calculated by the equation (1). Eight wavelengths (760, 780, 800,
820, 840, 860, 880 and 900 nm) were utilized as excitation wavelength to measure the
two photon absorption cross section value of NA-lyso, and the value was calculated
with the equation (2).
Φx =Φs(AsF /AxF )(nx /ns )2
(1)
(2)
x
s
s Cs ns Ss
x  
s x Cx nx Sx
Where, Φ is the fluorescence quantum yield, A is the absorbance at the excitation
wavelength, F is the area under the corrected emission curve, n is the refractive index
of solvent, δ is value of the two photon section, C is the concentration, and the S
shows the fluorescence emission intensity of the two photon. Subscript s and x

ACCEPTED MANUSCRIPT
corresponding to the reference and the samples respectively.
2.5 Spectral experiments
The stock solution of NA-lyso (1.0 mM) was prepared in DMSO. Samples were
prepared by placing 20.0 μL of probe (1.0 mM) and an appropriate aliquot of each
analyte solution into a 2.0 mL centrifugal tube, and the volume was fixed finally to
2.0 mL with HEPES (10 mM, pH 7.4 ). The resulting solution were shaken by vortex
at room temperature, fluorescence and UV spectra were recorded. Fluorescence
spectra were recorded at 430/540 nm with the slits 5/5 nm.
2.6 Cytotoxicity Assay
Cell viability was measured by MTT method [24]. Hela cells were seeded in
96-well plates with 200 μL per well at a concentration of 1.0 × 104 cells/well, and
different concentrations of NA-lyso (10, 20, 30, 40, 50 and 100 μM) were added, then
the cells were incubated for 24 h. The control group were treated only with PBS
buffer at pH = 7.4 and incubated under the same conditions. After incubation for 24 h,
the medium were removed from each well, and 100 μL of MTT solution (0.5 mg/mL,
DEME) was added to each well. The cells further were incubated for 4 h at 37 °C.
After that, the medium was removed carefully and the formazan solid was dissolved
by adding 100 μL of DMSO. The UV absorbance of each well plate was read using a
microplate reader. The cell survival ratio (VR) = A/A0 × 100%. Here, A is the
absorbance of the experimental cells, and A0 is the absorbance of the control cells.
Each test was repeated five times, and the data are shown as the mean ± SD.

ACCEPTED MANUSCRIPT
2.7 Imaging of HOBr in HeLa cells
HeLa cells were plated on 6-well plates and allowed to adhere for 24 h, and the
cells were divided into five parallel groups. The first group was as a blank control and
the other groups were incubated with NA-lyso (20 µM). The second group was as a
reagent blank control. All the cells were incubated for 30 min at 37℃. The third was
added Br- (20 µM); the fourth was added HOBr (20 µM); the fifth group was added
Br- (20 µM) and NAC (20 µM). All groups containing 0.1% DMSO as a cosolvent
and then every parallel was cultivated for another 30 min. Before imaging, the cells
were washed with PBS (pH = 7.4) buffer for three times.
For colocalization experiments, one group of cells was incubated with 20 µM
Lyso-Tracker Red for 30 min, another group were incubated with 20 µM NA-lyso for
30 min. Before the imaging experiment, all groups of cells were rinsed with PBS for
three times, and the fluorescence images were acquired through a two-photon laser
confocal microscopy with excitation wavelength as 800 nm, collecting green channel
of 520-560 nm (LSM 510 META , Zessi , Germany).
2.8 Imaging of HOBr in Kunming Mice
Kunming mice (KM, female, 5-6 weeks old) were obtained from Gansu
University of Chinese Medicine. All animal experiments were performed in
accordance with the guidelines issued by The Ethical Committee of Gansu University
of Chinese Medicine.
Kunming mice were divided into four groups, the first group was as control

ACCEPTED MANUSCRIPT
group; the second group was given an Inyraperitoneal (i.p.) injection of saline (200
µL) containing 0.1% DMSO; the third group was given an i.p. injection of 200 μL
NA-lyso; and the last group was given an i.p. injection of NaBr (200 μL, 100 μM),
after 30 min, given i.p. injection of 200 μL NA-lyso. The NA-lyso was prepared in
100 μM in saline containing 0.1% DMSO as cosolvent. After the i.p. injection, each
group was kept for 30 min and then for image.
The mice were imaged as quadruplets, one from each group using an IVIS
Spectrum (Carestream Health, Canada) in fluorescence mode equipped with 420 and
560 nm filters for excitation and emission, respectively. Photographs were taken using
a fixed exposure time.
3. Results and discussion
3.1 Synthesis of probe NA-lyso
The probe was successfully synthesized based on Suzuki coupling reaction
according to Scheme 1 with naphthalimide as a fluorescent group, and the structure
1
of NA-lyso was characterized by H NMR, 13C NMR and HRMS spectra (see
Figure S5-S7 in Supporting Information).
3.2 Spectroscopic property and optical response to HOBr
The fluorescence quantum yield of NA-lyso is 59.17% and its two-photon cross
section under different wavelength excitations were obtained and calculated using
Equation 2. The calculative result is shown in Figure S8. The largest two-photon

ACCEPTED MANUSCRIPT
cross section is obtained at 800 nm excitation, which is 88.8 GM.
NA-lyso has a strong absorption peak at 437 nm (Figure 1a), upon the addition
of HOBr, the absorption of the probe is red-shifted to 451 nm and the fluorescence
intensity at 540 nm is significantly weakened (Figure 1b). This phenomena can be
attributed to the formation of nitrogen-sulfur double bond by the amino group and
methylthio group in the probe under the action of HOBr (Scheme 1). This conjecture
was verified by HRMS, as illustrated in Figure 2, the characteristic peak of the
product NA-cyc (the product of NA-lyso and HOBr) appears in the HRMS spectrum.
Figure 1. UV-Vis (a) and Fluorescence emission spectra (b) of NA-lyso (black) and
the product NA-cyc (red). The concentration of NA-lyso and HOBr were 10 µM and
40 µM respectively. λex = 430 nm.

ACCEPTED MANUSCRIPT
Figure 2. The HRMS of NA-cyc
3.3 Study on the luminescence mechanism of probe
To illustrate the fluorescence "on-off" phenomenon, we calculated the electron
cloud orbital distribution of the probe NA-lyso and the reaction product NA-cyc
through the density functional theory by the Gaussian 09 software [DFT at the
b3lyp/6-311g (d,p) level]. The calculated result is shown in Figure S9, we can get the
information that the electron clouds in the LUMO and HUMO orbits of NA-lyso are
both distributed on the naphthalimide ring, thus there is no photoinduced electron
transfer (PET) effect. For NA-cyc, the electron cloud density in LUMO is mainly
distributed on the naphthalimide ring, however, the electron cloud in HUMO is
mainly distributed on 4-(2-aminoethyl) morpholine, thus cause PET, which leads to
fluorescence quenching of NA-cyc. In addition, the energy differential of NA-lyso
and NA-cyc are 0.3282 and 0.3035 eV respectively, which are basically consistent. It

ACCEPTED MANUSCRIPT
is also confirmed that the PET effect occurs in NA-cyc, leading to the fluorescence
quenching.
3.4 Optimization of measurement conditions
First, the fluorescent intensities of NA-lyso and NA-cyc in HEPES buffers with
different pH ranging from 3.0 to 10 were recorded (Figure 3a). After HOBr was
added, fluorescence intensity were receded dramatically. When the pH is less than 8.0,
the fluorescence of NA-lyso and NA-cyc are relatively stable, but when the pH is
larger than 8.0, the degree of fluorescence response is reduced. The reason is that
-OBr is the main form when the pH is larger than 8 (pKa of HOBr is 8.8 at 25℃[12]),
and the real concentration of HOBr in the system was reduced, which weakened the
response of NA-lyso to HOBr.
The response of NA-lyso to HOBr is so fast that the fluorescence intensity
immediately weakens when added HOBr to the system and remains stable for a long
time. The fluorescence intensities of NA-lyso and NA-cyc show hardly changes
within 60 min (Figure 3b), which indicate the probe has good resistance to
photobleaching and NA-cyc could remain stable.

ACCEPTED MANUSCRIPT
Figure 3. Effect of pH (a) and time (b) on the response of the reaction system
The concentration of NA-lyso and HOBr were 10 µM and 40 µM respectively.
λex = 430 nm; λem = 540 nm.
3.5 Fluorescence responses of NA-lyso to HOBr
The fluorescence responses of NA-lyso to HOBr with different concentration
(0-40 µM) were investigated in HEPES 7.4 (10 mM) at room temperature. Figure 4a
shows that the fluorescence intensity of the reaction product decreased with a
dose-dependent manner over a concentration range of HOBr. As depicted in Figure
4b，there is a good linearity between (F0-F)/F0 and the concentration of HOBr in the
range of 0 to 35 µM, while F0 and F indicate the fluorescence intensity in the
absence and presence of HOBr respectively. The regression equation is (F0-F)/F0=
0.0179[CHOBr(µM)]-0.0333, with a linear coefficient of 0.990. The limit of detection
(3S/σ) was determined to be as low as 33.5 nM, which makes NA-lyso practicable
for monitoring endogenous HOBr.

ACCEPTED MANUSCRIPT
Figure 4. a) Fluorescence intensities of NA-lyso (10 µM) treated with increasing
concentrations of HOBr in HEPES (10 mM, pH 7.4, 0.1% DMSO as a cosolvent).
Silt width: 5/5 nm, λex= 430 nm; b) Linear correlation between (F0-F)/F0 and
concentrations of HOBr in the range of 0-35 µM.
3.6 Specificity of NA-lyso for detecting HOBr
To inspect whether NA-lyso could specifically monitor HOBr in the complicated
intracellular environment, we tested its ability to discriminate between HOBr and
various bioanalytes, such as competing ROS and RNS (Figure 5a). Only HOBr can
reduce the fluorescence intensity of NA-lyso, correspondingly, (F0-F)/F0 has a larger
value. When HOBr coexist with other ROS and RNS (Figure 5b), (F0-F)/F0 shows
almost no change, which indicated NA-lyso could detect HOBr with excellent
selectivity and satisfactory anti-interference performance in complicated system.
Moreover, the probe do not respond with Br- and -OBr, and they have no effect on the
response of probe to HOBr as well.

ACCEPTED MANUSCRIPT
Figure 5. The fluorescence responses of NA-lyso (10 µM) in the presence of various
-
analytes. 1:Blank, 2: HOBr, 3: ·OH ,4: ONOO-,5: t-BuO·,6: O2 ,7: H2O2, 8: NO, 9:
HOCl, 10: t-BuOOH, 11: Br-, 12: -OBr. (a) Fluorescence responses of NA-lyso with
various analytes; (b) fluorescence responses of NA-lyso towards HOBr plus the
corresponding analytes. The concentration of all analytes were 10 µM. λex=430 nm,
λem=540 nm.
3.7 Cell imaging of probe NA-lyso
Inspired by outstanding optical property (Φ=59.17%, TPACS=88.8 GM) of
NA-lyso and its sensitivity towards HOBr, we proceed to investigate the potential
application of the probe in fluorescence imaging in cells. Firstly, an MTT assay was
performed in Hela cells (Figure S10). When the concentration of NA-lyso were 100
µM, there was no significant reduction in cell viability, indicating the probe had
favorable biocompatibility. Subsequently, the colocalization experiment was
conducted by costaining Hela cells with Lyso-Tracker Red (a typical commercially
available lysosomal tracker) and NA-lyso (Figure 6). The fluorescence of NA-lyso
from the costained cells overlaid well with that from Lyso-Tracker Red (Figure 6c),

ACCEPTED MANUSCRIPT
and the Pearson’s coefficient is so high as 0.95 (calculated by Image J), which
demonstrates NA-lyso can locate to the lysosome of Hela cells.
Figure 6. Fluorescence images of lysosome in Hela cells. Hela cells were
incubated with NA-lyso (a, 20 μM, λex = 800 nm, λem = 520−560 nm ) and
Lyso-Tracker Red (b, 10 μM, λex = 600 nm, λem = 620−700 nm ) respectively. (c)
merged image of (a) and (b); (d) bright field image.
Next, we investigated whether the probe can detect HOBr in cells under
physiological conditions. As shown in Figure 7, a strong fluorescence signal was
observed in Hela cells which were treated only with NA-lyso (Figure 7b), which
meant the probe can easily enter into the cells. When the cells were stimulated with
NaBr or HOBr, the fluorescence intensities of cells were declined sharply (Figure 7c
or 7d). The reason of NA-lyso responding to Br- is that Br- changed to HOBr in cells
under the existence of EPO or MPO, but not all the Br- can change to HOBr in a short
time. So the fluorescence intensity in Figure 7c was less darker than in Figure 7d.
Additionally, when the cells were treated with Br- and the scavenger (NAC) of HOBr
in sequence, the fluorescence intensity of the cells became brighter owing to the
decreasing of HOBr concentration (Figure 7e).

ACCEPTED MANUSCRIPT
Through the image J software, we compared the fluorescence intensity from
Figure 7a to 7e, the fluorescence intensity from Figure 7e is slightly stronger than
that from Figure 7b, the reason is that NAC not only reacted with exogenous HOBr,
but also with endogenous HOBr from the cells.
Figure 7. Fluorescence images of Hela cells. (a) blank cells; (b-e) cells were
incubated with NA-lyso (20 µM) for 30 min (b) and subsequently incubated for 30
min with 20 µM NaBr (c) or 20 µM HOBr (d) or 20 µM NaBr plus 20 µM NAC (e).
(f) Normalized fluorescence intensity of cells in panels (a) to (e). λex = 800 nm, λem
=520−560 nm, scale bar = 25 µm.
3.8 Fluorescence imaging of HOBr in mice
The mice that were given an intraperitoneal (i.p.) injection of NA-lyso have
very strong fluorescence (Figure 8c). The mice were i.p. injection with Br- and
NA-lyso (20 µM) (Figure 8d), in which the fluorescence intensity significantly
decreased, indicating that NA-lyso could visualize endogenous HOBr in living mice.
These fluorescence images prove that HOBr can be generated from Br- in mice and

ACCEPTED MANUSCRIPT
confirm NA-lyso is a potential tool for detecting HOBr in vivo.
Figure 8. Fluorescence imaging of HOBr in living mice. a) Mice without any
treatment, b) mice with i.p. injection of saline solution, c) mice with i.p. injection of
NA-lyso (20 µM) for 30 min, d) mice with i.p. injection of Br− (100 µM) for 30 min
and then with NA-lyso (20 µM) for another 30 min.
The properties of probe NA-lyso were compared with the existing probes for
HOBr and the result is shown in Table S1 in Supporting Information. The probe can
target to lysosome and is capable of detection of HOBr, moreover, it can be also used
for cells and mice imaging owing to the longer excitation wavelength.
Conclusion
In summary, we developed a high quantum yield lysosome-targeting two-photo
fluorescence probe NA-lyso for specific monitoring of HOBr. The probe has low
limit of detection (33.5 nM) for HOBr with good sensitivity and selectivity. This

ACCEPTED MANUSCRIPT
probe is capable of targeting to lysosome and monitoring the level of exogenous and
endogenous HOBr in Hela cells under excitation of two-photon wavelength.
Moreover, the probe can also be used to image HOBr in mice. Overall, NA-lyso is
an ideal tool for the further research of detection of HOBr and has potential
application in further exploration of physiological and pathological roles of HOBr in
lysosome. However, it should continue to develop the new probe for accurate
quantification of HOBr in cells.
Acknowledgement
This research was financially supported by the National Natural Science
Foundation of China (No. 21575055).

ACCEPTED MANUSCRIPT
References
[1] X. Chen, X. Tian, I. Shin, J. Yoon. Fluorescent and luminescent probes for
detection of reactive oxygen and nitrogen species. Chem. Soc. Rev. 40 (2011)
4783-4804.
[2] C. Nathan. Specificity of a third kind: reactive oxygen and nitrogen intermediates
in cell signaling. J. Clin. Imvest. 111 (2003) 769.
[3] P. F. Bove, A.V.D. Vliet. Nitric oxide and reactive nitrogen species in airway
epithelial signaling and inflammation. Free Radical Bio.Medi. 41 (2006) 515-527.
[4] S. K. Ko, X. Chen, J. Yoon, I. Shin. Zebrafish as a good vertebrate model for
molecular imaging using fluorescent probes. Chem. Soc. Rev. 40 (2011) 2120-2130.
[5] X. Q. Chen, Y. Zhou, X. J. Peng, J. Y. Yoon. Fluorescent and colorimetric probes
for detection of thiols. Chem. Soc. Rev. 39 (2010) 2120–2135.
[6]K. Kikuchi. Design, synthesis and biological application of chemical probes for
bio-imaging. Chem. Soc. Rev. 39 (2010) 2048-2053.
[7] D. I. P And, M. J. Davies. Kinetic Analysis of the Reactions of Hypobromous Acid
with Protein Components: Implications for Cellular Damage and Use of
3-Bromotyrosine as a Marker of Oxidative Stress. Biochemistry. 43 (2004)
4799-4809.
[8] W. A. Rutala, D. J. Weber. Uses of inorganic hypochlorite (bleach) in health-care
Facilities. Clin. Microbiol. Rev. 10 (1997) 597–610.
[9] Y. W. Yap, M. Whiteman, B. H. Bay, Y. Li, F.-S. Sheu, R. Z. Qi, C. H. Tan, N. S.

ACCEPTED MANUSCRIPT
Cheung, Hypochlorous acid induces apoptosis of cultured cortical neurons through
activation of calpains and rupture of lysosomes, J. Neurochem. 98 (2006) 1597-1609.
[10] B. B. Deng, M. G Ren, X. Q. Kong, K. Zhou, W. Y. Lin. Development of an
enhanced turn-on fluorescent HOCl probe with a large Stokes shift and its use for
imaging HOCl in cells and zebrafish. Sensors and Actuators B 255 (2018) 963–969.
[11] X. Q. Chen, F. Wang, J. Y. Hyun, T. W. Wei, J. Qiang, X. T. Ren, I. Shin, J. Yoon,
Recent progress in the development of fluorescent, luminescent and colorimetric
probes for detection of reactive oxygen and nitrogen species, Chem. Soc. Rev. 45
(2016) 2976–3016.
[12] F .Yu, P. Song, P. Li, B. Wang, K. Han. Development of reversible fluorescence
probes based on redox oxoammonium cation for hypobromous acid detection in living
cells. Chem. Commun. 48 (2012) 7735-7737.
[13] J. J. Hu, N. K. Wong, S. Ye, X Chen, M. Y. Lu, A. Q. Zhao, Y. Guo, A.C. Ma,
A.Y. Leung, J .Shen, D.Yang. Fluorescent Probe HKSOX-1 for Imaging and
Detection of Endogenous Superoxide in Live Cells and In Vivo. J. Am. Chem.
Soc.137 (2015) 6837-6843.
[14] K. Giannakis, T. Andronikos. Membrane automata for modeling biomolecular
processes. Nat. Comput. 16 (2017) 1-13.
[15] A. E. Lane, J. T. Tan, C. L. Hawkins, A. K. Heather, M. J. Davies. The
myeloperoxidase-derived oxidant HOSCN inhibits protein tyrosine phosphatases and
modulates cell signalling via the mitogen-activated protein kinase (MAPK) pathway

ACCEPTED MANUSCRIPT
in macrophages. Free Radical Bio. Medi. 49 (2010) 161-169.
[16] M. J. Ceko, K. Hummitzsch, N. Hatzirodos, W. Bonner, S. A. James, J. K. Kirby,
R. J. Rodgers, H. H. Harris. Distribution and speciation of bromine in mammalian
tissue and fluids by X-ray fluorescence imaging and X-ray absorption spectroscopy.
Metallomics. 7 (2015) 756-765.
[17] J. C. van Dalen, W. M. Whitehouse, C. C. Winterbourn, J. A.Kettle .Thiocyanate
and chloride as competing substrates for myeloperoxidase. Biochem. J. 327(1997)
487-492.
[18] A. S. McCall, C. F. Cummings, G. Bhave, R. Vanacore, B. G.Hudson. Bromine is
an essential trace element for assembly of collagen IV scaffolds in tissue development
and architecture. Cell. 157 (2014) 1380-1392.
[19] W. Wu, Y. Chen, A. D ’ Avignon, S. L. Hazen. 3-Bromotyrosine and
3,5-dibromotyrosine are major products of protein oxidation by eosinophil peroxidase:
potential markers for eosinophil-dependent tissue injury in vivo. Biochemistry.38
(1999) 3538-3548.
[20] B.S. Wang, P. Li, F.B. Yu, J.S. Chen, Z.J. Qu and K.L. Han. A near-infrared
reversible and ratiometric fluorescent probe based on Se-BODIPY for the redox cycle
mediated by hypobromous acid and hydrogen sulfidein living cells Chem. Commun.
49 (2013) 5790—5792.
[21] K. H Xu, D. R. Luan, X. T. Wang, B. Hu, X. J. Liu, F. P. Kong, B. Tang. An
Ultrasensitive Cyclization-Based Fluorescent Probe for Imaging Native HOBr in Live

ACCEPTED MANUSCRIPT
Cells and Zebrafish. Angew. Chem. Int. Ed. 55 (2016) 1-5.
[22] X.J Liu, A.S. Zheng, D.R. Luan, X.T. Wang, F.P. Kong, L.L.Tong, K.H. Xu, B.
Tang. High-Quantum-Yield Mitochondria-Targeting Near-Infrared Fluorescent Probe
for Imaging Native Hypobromous Acid in Living Cells and in Vivo. Anal. Chem.
89(2017) 1787−1792.
[23] C. Ma , F. Y. Zhang , Y. Y. Wang, X. Y. Zhu, X. Y. Liu, C. Y. Zhao, H. X. Zhang.
Synthesis and application of ratio fluorescence probe for chloride. Anal. Bioanal.
Chem. 410 (2018) 6507-6516.
[24] J. Kang, F. J. Huo, Y. B. Zhang , J. B. Chao, T. E. Glass, C.X. Yin. A novel
near-infrared ratiometric fluorescent probe for cyanide and its bioimaging applications.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 209 (2019)
95-99

ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Highlights
1. A lysosome-targeted fluorescence probe NA-lyso for monitoring HOBr was
synthesized.
2. The probe can respond to HOBr with outstanding sensitivity and selectivity.
3. The probe can target in lysosome and monitoring the level of HOBr in Hela cells.
4. The probe can also be used to image HOBr in vivo.