The near-infrared fluorescent probes based on phenoxazine for the rapid detection of hypochlorous acid

Article abstract of DOI:10.1016/j.dyepig.2020.108404

Hypochlorous acid (HOCl) is tightly related with a series of diseases, and plays an important part in biological processes. Herein, we designed and synthesized two “turn-on” near-infrared (NIR) probes (BC-2 and BC-3) based on phenoxazine to sense and imag

Full text of DOI:10.1016/j.dyepig.2020.108404

Journal Pre-proof
The near-infrared fluorescent probes based on phenoxazine for the rapid detection of
hypochlorous acid
Wubin Zheng, Jiajia Yang, Yang Shen, Yusi Yao, Guanglei Lv, Shiyou Hao, Chunxia
Li
PII:
S0143-7208(19)33050-5
DOI:
https://doi.org/10.1016/j.dyepig.2020.108404
DYPI 108404
Reference:
To appear in: Dyes and Pigments
Received Date: 30 December 2019
Revised Date: 15 March 2020
Accepted Date: 26 March 2020
Please cite this article as: Zheng W, Yang J, Shen Y, Yao Y, Lv G, Hao S, Li C, The near-infrared
fluorescent probes based on phenoxazine for the rapid detection of hypochlorous acid, Dyes and
Pigments (2020), doi: https://doi.org/10.1016/j.dyepig.2020.108404.
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Author statement
Wubin Zheng: Investigation, Data Curation, Writing - Original Draft
Jiajia Yang: Investigation
Yang Shen: Data Curation
Yusi Yao: Validation
Guanglei Lv: Conceptualization, Writing - Review & Editing, Supervision,
Shiyou Hao: Project administration
Chunxia Li: Writing - Review & Editing, Supervision

Graphical abstract

The near-infrared fluorescent probes based on phenoxazine for the rapid
detection of hypochlorous acid
Wubin Zheng, Jiajia Yang, Yang Shen, Yusi Yao, Guanglei Lv*, Shiyou Hao* and Chunxia
Li*
Key Laboratory of the Ministry of Education for Advanced Catalysis Materials, Zhejiang Normal
University, Jinhua 321004, Zhejiang, P. R. China.
* Corresponding Authors. E-mail: guanglei@zjnu.edu.cn; sky54@zjnu.cn; cxli@zjnu.edu.cn.
Abstract: Hypochlorous acid (HOCl) is tightly related with a series of diseases, and plays an
important part in biological processes. Herein, we designed and synthesized two “turn-on”
near-infrared (NIR) probes (BC-2 and BC-3) based on phenoxazine to sense and image HOCl
both in vitro and in vivo. In the presence of HOCl, the fluorophore phenoxazine was released and
the fluorescence intensity significantly increased. The probes show excellent selectivity, high
sensitivity, and rapid response towards HOCl. Moreover, the probe BC-3 is capable of rapidly
detecting endogenous HOCl in living cells and in the inflammation area from the mouse models
of arthritis.
Keywords: fluorescent probes; near-infrared (NIR); hypochlorous acid; reactive oxygen species;
phenoxazine
1

1. Introduction
Reactive oxygen species (ROS), as one of endogenous metabolites, are of great importance for
physiological processes, while excessive ROS are closely correlated with a wide range of various
diseases, including inflammation, cancer and neurodegenerative diseases [1-6]. Hypochlorous
acid (HOCl), generating from the reaction between hydrogen peroxide and chloride ions via
myeloperoxidase (MPO)-mediated in vivo [7,8], is a member of the highly active ROS. It is
considered to be an important bactericidal oxidant in the immune system against pathogenic
microorganisms including germs, viruses and so on [9-11]. However, excessive production of
HOCl can oxidize phospholipids, proteins and deoxyribonucleic acid (DNA), leading to cell and
tissue damage [12-15]. Meanwhile, many diseases are closely related to the overproduction of
HOCl, such as atherosclerosis, rheumatoid arthritis and so on [16-19]. Therefore, it is greatly
urgent to develop highly selective and sensitive fluorescent probes for the rapid detection of
HOCl.
Owing to fluorescent probes enabling real-time and in situ detecting HOCl in biological
organisms [20-23], a wide range of fluorescent probes, on the basis of a series of fluorophores
including BODIPY [24-27], naphthalimide [28,29], rhodamine [30,31], coumarin [32] and so on,
have been reported. Although numerous fluorescent probes are capable of detecting HOCl in
vitro or in cells [33-45], few probes can be employed in living system mainly owing to the
limitation of emission wavelength. Therefore, it is urgently required to develop NIR fluorescent
probes to detect HOCl in living organisms because they possess deeper penetration depth and
little autofluorescence interference [46-48]. It is well established that the construction of
“turn-on” NIR fluorescent probes is an important strategy for detecting HOCl because of
significant fluorescence increase after response with high signal-to-noise ratio. Yi developed a
2

novel “turn-on” mechanism for specifically detecting HOCl, taking advantage of remarkable
fluorescence change between the reduced form and oxidized form of methylene blue (MB)
[49,50]. It is well known that phenoxazine possesses similar photophysical properties to
methylene blue (Scheme 1), its maximum fluorescence emission lying in the NIR area (roughly
680 nm) and its reduced form of phenoxazine (RO1) emits little fluorescence. Herein, RO1 and
its derivatives are ideal scaffolds for the design of turn-on fluorescent probes.
Taking these into consideration, we designed and synthesized two fluorescent probes BC-2
and BC-3 based on RO1 (Scheme 1) for the specific detection of HOCl both in vitro and in living
organisms. The two probes BC-2 and BC-3 released fluorophore phenoxazine (Scheme 1) in the
presence of HOCl, accompanying with significant fluorescence intensity increase in NIR
emission and remarkable colour change from colourless to blue. It is worth noting that the probe
BC-3 exhibited high selectivity, sensitivity and rapid response towards HOCl both in cells and in
mouse models of arthritis.
2. Experimental section
2.1. Materials and general methods
The stock solutions (1 mM) of probes was prepared in ethanol (HPLC grade), and further
diluted with PBS (sodium phosphate buffer, pH = 7.4) to from a final concentration (10 μM).
Materials involved in the preparation of active species, including t-BuOOH (tert-butyl
hydroperoxide), 5% NaOCl solution, 30% H2O2 solution, nitroferricyanide (III) dehydrate, KO2,
3-morpholinosydnonimine hydrochloride, 2, 2′-azobis(2-amidinopropane)dihydrochloride and
ferrous sulfate (hydroxyl radicals was produced by the Fenton reaction), were used to prepare
ROS or reactive nitrogen species (RNS) in ultrapure water. Unless otherwise noted, other
3

experimental materials (including metal salts, amino acids, and solvents and so on) were obtained
from companies. All of the reagents were analytical grade in the optical spectroscopic studies.
The fluorescence spectra of two probes towards HOCl in solution was measured on a F-4600
FL spectrophotometer, and the emission wavelength ranges was collected from 640 to 680 nm,
λex=620 nm. The absorption spectra of probes in solution were performed on UH5300 UV-Vis
1
13
spectrophotometer, ranging from 550 to 750 nm. H NMR (400 MHz) and C NMR (100 MHz)
were measured on Bruker AV-400 or AV-600 NMR spectrometer. High-resolution mass
spectrometry (HRMS) analysis was carried out using a TOF MS instrument. CLSM was recorded
on Olympus FV1000 Confocal Laser Scanning Microscope.
2.2. Synthesis details of probes
2.2.1. Synthesis of BC
Phenoxazine (1.0 g, 0.7 mmol, 25%) and Na2CO3 (297 mg, 2.8 mmol) were dissolved in 8.0
mL water and then dichloromethane (DCM, 4.0 mL) was added. Above mixture was stirred at 40
°C under nitrogen atmosphere, the sodium dithionite (487 mg, 2.8 mmol) in 10.0 mL water was
slowly injected into the reaction system directly using a syringe device. After that, the mixture
was stirred for 1 h until the solution became yellow. The mixture was transferred into a dried
flask with three necks and then Na2CO3 (297 mg, 2.8 mmol) was added. Lastly, triphosgene (125
mg, 0.42 mmol) in 5 mL DCM was added dropwise under low temperature. The reaction was
o
carried out under a nitrogen atmosphere at 40 C for 3 h. The reaction mixture was poured into
ice/water and extracted with DCM-water system. After that, the combined organic solvent was
dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The target
product was obtained via column chromatography (BC, white solid, yield 66.8%; ethyl acetate:
4

petroleum ether = 1:20). 1H NMR (400 MHz, CDCl3) δ = 7.38 (d, J = 9.6 Hz, 2H), 6.41-6.36 (m,
4H), 3.34 (q, J = 7.2 Hz, 8H), 1.16 (t, J = 7.2 Hz, 12H). HR-MS (ESI, m/z): calculated for
C21H27N3O2Cl [M+H]+, 388.1786, found 388.1787.
2.2.2. Synthesis of BC-2
Compound BC (50 mg, 0.13 mmol), Na2CO3 (55.4 mg, 0.52 mmol), and 4-isopropylaniline
(70.3 mg, 0.52 mmol) dissolved in 5 mL DCM. The mixture was stirred at 40 oC under nitrogen
atmosphere until the reaction was complete through TLC analysis. The reaction mixture was
cooled to room temperature, then the target product was obtained via column chromatography
1
(BC-2, light blue solid, yield 64.8%; ethyl acetate: petroleum ether=1:5). H NMR (400 MHz,
CDCl3) δ =7.39 (d, J = 9.6 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 7.23 (s, 1H), 7.15 (d, J = 8.4 Hz, 2H),
6.46-6.41 (m, 4H), 3.36 (q, J = 7.0 Hz, 8H), 2.93-2.81 (m, 1H), 1.26-1.16 (m, 18H). 13C NMR (100
MHz, CDCl3) δ = 153.3, 152.5, 146.8, 143.9, 136.2, 126.8, 125.0, 119.7, 117.6, 106.7, 100.0, 44.7,
33.5, 24.1, 12.6. HR-MS (ESI, m/z): calculated for C30H39N4O2 [M+H]+, 487.3068, found
487.3076.
2.2.3. Synthesis of BC-3
The synthesis method of BC-3 referred to compound BC-2. The target product BC-3 was
obtained via column chromatography (BC-3, white solid, yield 65.3%; ethyl acetate: petroleum
ether = 1:3). 1H NMR (400 MHz, CDCl3) δ = 7.37 (d, J = 9.5 Hz, 2H), 7.32 (d, J = 9.0 Hz, 2H),
7.15 (s, 1H), 6.82 (d, J = 9.0 Hz, 2H), 6.44-6.42 (m,4H), 3.77 (s, 3H), 3.34 (q, J = 7.0 Hz, 8H), 1.16
13
(t, J = 7.0 Hz, 12H). C NMR (100 MHz, CDCl3) δ = 155.8, 153.6, 152.5, 146.8, 131.6, 125.1,
121.7, 117.6, 114.0, 106.7, 100.0, 55.5, 44.6, 12.6. HR-MS (ESI, m/z): calculated for C28H35N4O3
[M+H]+, 475.2704, found 475.2735.
5

2.3. Determination of limit of detection (LOD)
The determination of LOD using fluorescence titration based on currently widely used method
[51]. The fluorescence spectra of free probe were measured eleven times and its standard
deviation (σ) was obtained. Next, the fluorescence intensities at 669 nm were plotted as the
increasing concentrations of HOCl and the slope (k) was obtained, and the LOD was calculated
using the following equation: LOD = 3σ/k
2.4. Cell culture and cytotoxicity assay
The HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin sulphate, and then the
HeLa cells were cultivated for 24 h at 37 oC in air incubator (5% CO2). The cytotoxicity of BC-3
was investigated in HeLa cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. HeLa cells were seeded in a 96-well micro-assay culture plate at a density
of 104 cells per well and then grew for 24 h in air incubator (5% CO2) at 37 oC. These cells were
further incubated upon addition of different concentrations of BC-3 (0, 5, 10, 20, and 30 μM) for
o
3 and 6 h at 37 C in air incubator (5% CO2). The wells with cell culture media only were
employed as the blank. Then, cell culture medium was removed and cells were washed three
times with PBS. The MTT (20 μL, 5 mg/mL) solution in PBS was added to each well and the
cells were incubated at 37 oC under 5% CO2 for 4 h. The excess MTT solution was drained from
per well, and then 100 μL of DMSO (dimethyl sulfoxide) was added. Microplate reader was used
to measure absorbance at 490 nm. All sample concentrations were tested in parallel for 5 groups.
2.5. Confocal fluorescence imaging
6

The response of probe BC-3 toward the endogenous HOCl was observed in HL-60 cells
(highly expressing endogenous HOCl cells). The HL-60 cells in good condition were cultured in
Roswell Park Memorial Institute (RPMI 1640) supplemented with 10% fetal bovine serum
(FBS), and 1% penicillin and 1% streptomycin sulphate at 37 °C under 5% CO2 for 24 h. The
o
HL-60 cells were further incubated with 10 μM BC-3 for 12 h at 37 C in air incubator (5%
CO2). As a control experiment, 4-aminobenzoic acid hydrazide (ABAH, a MPO inhibitor) was
also added, which could decrease the level of endogenous HOCl in HL-60 cells. The HL-60 cells
were pretreated with ABAH (200 μM) for 1 h and further incubated with BC-3 (10 μM) and
ABAH (200 μM) for another 12 h. CLSM imaging performance of BC-3 toward HOCl in HL-60
cells was performed on a Olympus FV1000 confocal laser scanning microscope. The
fluorescence channel was recorded at λem = 700 ± 50 nm, λex = 633 nm.
2.6. The detection of HOCl in the mouse model of rheumatoid arthritis
The ability of BC-3 to detect endogenous HOCl was demonstrated in Rheumatoid Arthritis
mice. The model of rheumatoid arthritis was produced by injecting λ-carrageenan (inflammatory
initiator, 5 mg/mL in PBS buffer). The right ankle joint from a 6 to 8-week old mouse was
injected by 100 μL λ-carrageenan solution. As a control experiment, the left ankle joint from the
same mouse was injected with 100 μL physiological saline. And then the probe BC-3 (100 μL, 1
mM) was injected into the both ankles after 4 hours. The fluorescence signal was gathered on
Bruker In-Vivo Xtreme 4MP imaging system, λem = 715 ± 60 nm.
3. Results and discussion
3.1. The response ability of probes towards HOCl
7

We first evaluated the ability of BC-2 and BC-3 to detect HOCl by spectroscopy in PBS
solution (pH = 7.4). The intensity of fluorescence and absorption considerably rose with the
increase of HOCl concentrations (Fig. 1 and Fig. S1). Furthermore, the limit of detection (LOD)
of probes were tested using fluorescence titration, and the LOD values of BC-2 and BC-3 were
as much low as 20.4 nM and 11.1 nM, respectively (Fig. 1b and Fig. S1b). More importantly, the
two probes exhibited fast response to HOCl within less than 50s (BC-2, Fig. S1d) and 30s (BC-3,
Fig. 1d), along with remarkable fluorescence increase. As shown in Fig. S2，upon the addition
of 25 μM HOCl, the fluorescence of BC-2 (38-fold, quantum yield ΦF = 5.82 %) and BC-3
(114-fold, ΦF = 6.23 %) dramatically increased at the wavelength of 669 nm. In addition to
fluorescence change, the colour of solutions containing BC-2 or BC-3 turns to obvious blue from
colourless after the addition of HOCl, which can clearly be observed by the naked eye (Fig. S3).
These results demonstrated that the probes exhibited not only high sensitivity but also fast
response towards HOCl.
3.2. The selectivity of probes towards HOCl
Compared with BC-2, BC-3 displayed faster response and lower LOD towards HOCl (Fig. 1
and Fig. S1). Therefore, we took BC-3 as an example to perform the subsequent investigation.
The selectivity of the BC-3 towards HOCl was studied using fluorescence spectrum in PBS (pH
= 7.4). Firstly, we investigated the selectivity of the probe in the presence of HOCl and other
−
ROS/RNS, including H2O2, t-BuOOH, NO, ROO., O2 , .OH, t-BuOO. and ONOO−. In the
presence of 10 μM HOCl, the fluorescence enhancement of BC-3 was greater than that of other
100 μM ROS/RNS (Fig. 2a). Furthermore, the reactivity of BC-3 towards some common anions
and cations was also investigated. The fluorescence intensity of BC-3 showed little change when
a wide range of anions and cations were added, such as CH3COO−, CO32−, SO42−, Cl−, F−, I−,
8

−
+
NO2 , S2O42−, Fe3+, Ni2+, Mg2+, Fe2+, Al3+, Ca2+, K+, Cu2+, NH4 (Fig. 2b and c). We also tested
the interference of amino acids towards the detection of HOCl. Therefore, a series of amino
acids, such as Pro, Ser, Ala, Gln, Val, Thr, lle, Gly, Tyr, Met, Trp, Phe, Cys, Glu, and Lys, were
employed to perform this experiment. As expected, the fluorescence intensity did not increase
even if the concentrations of amino acids rose to 100 μM (Fig. 2d). Moreover, BC-2 also
exhibited high selectivity for HOCl among various species under the same conditions (Fig. S4).
It is worth noting that the existence of HOCl can induce the colour change from colourless to
remarkable blue (Fig. S5–S8), which enables the detection of HOCl by means of the naked eye.
In addition, both probes also showed good selectivity towards HOCl even in acidic solution
(pH=5) and basic solution (pH=9) (Fig. S9 and S10). Taken all together, both of the probes
exhibited high selectivity towards HOCl and little interference against various substances.
3.3. The anti-interference ability of probes
In order to guarantee the effective application of probes in vivo, the interference of several
cellular reductants (including N-acetylcysteine (NAC), glutathione (GSH), glucose and aldehyde)
for the detection of HOCl was studied (Fig. 3). With the increase of concentrations of NAC and
GSH (0-25 μM), the fluorescence intensity of BC-3 gradually decreased. This mainly because
NAC and GSH all contain the active thiol group (-SH) which is prone to being attacked by
oxidizing reagents. This means NAC and GSH can easily react with HOCl. Therefore, the
presence of NAC or GSH could consume HOCl to some extent, leading to the fluorescence
intensity decrease [49,52]. Nevertheless, even if the addition of higher concentrations of NAC or
GSH (25 μM), HOCl (10 μM) still triggered BC-3 to emit obvious fluorescence (> 5.7-fold and >
5.4-fold in the presence of NAC and GSH compared to the probe itself, respectively). More
importantly, the fluorescence intensity of the probe BC-3 showed little changes under the high
9

concentration of glucose and aldehyde. In addition, the probe BC-2 also exhibited
anti-interference ability (Fig. S11). Then, we explored the stability of the luminous nucleus
phenoxazine in different buffer systems with the addition of a large excess of HOCl (10 μM
phenoxazine in the presence of 50 μM HOCl) for one hour (Fig. S12). The results showed that
the fluorescence intensity of phenoxazine rarely changed, which indicated that the fluorophore
phenoxazine could be stable enough for the detection of HOCl in complex physiological
environment. Taken together, BC-2 and BC-3 showed superior anti-interference ability towards a
wide range of cellular species, indicating both of probes possessed great potential in the efficient
detection of HOCl in a wide variety of biological environments.
3.4. Mechanism exploration
On the basis of previous literature [50] and our results, we proposed a possible recognition
mechanism (Scheme 2). Initially, hypochlorous acid attacks the electron-deficient carbon atom
from amide carbonyl group, resulting in the cleavage of the amide to release the reduced
phenoxazine. Finally, the fluorophore phenoxazine is generated via the rapid oxidation of RO1.
As expected, both fluorescence and absorption spectra characteristics of the probe are consistent
with phenoxazine after reaction with HOCl (Fig. S13). Moreover, HRMS and HPLC analysis
further confirmed the formation of phenoxazine (Fig. S14 and S15).
3.5. The detection of endogenous HOCl in cellular level
The desirable selectivity and rapid response properties of BC-3 for HOCl promoted us to
explore its utility in in vivo detection of HOCl. Firstly, the biocompatibility of the probe was
examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
The result showed that the probe exhibited low cytotoxicity (Fig. S16). Even after 6 hours of
incubation with 30 μM BC-3, more than 95% of the HeLa cells still survived. To evaluate the
10

capability of BC-3 for intracellular HOCl detection, human promyelocytic leukemia (HL-60)
cells with abundant MPO for endogenic generation of HOCl, were chosen as the cell model in
this study. Then, fluorescent cell images were obtained using confocal laser scanning microscopy
(CLSM) after HL-60 cells were incubated with 10 μM BC-3 for 12 h at 37 . The fluorescence
signal was not observed in the absence of probe BC-3 in HL-60 cells (Fig. 4a-c). However, the
apparent fluorescence signal was observed in the red channel in the presence of probe BC-3 (Fig.
4d-f), indicating the probe showed good cell membrane penetrability and imaging ability. As a
control experiment, 4-aminobenzoic acid hydrazide (ABAH, an MPO inhibitor) was added,
which could decrease the level of endogenous HOCl in HL-60 cells. As expected, the
fluorescence signal of BC-3 was significantly suppressed after the addition of ABAH (Fig. 4g-i).
Taken all together, BC-3 can be used to detect endogenous HOCl in living cells.
3.6. The detection of HOCl in the mouse model of rheumatoid arthritis
Inspired by the above results, we studied whether BC-3 was able to detect endogenous HOCl
in the mouse model of rheumatoid arthritis (RA). The model of rheumatoid arthritis was
produced via the injection of λ-carrageenan (inflammatory initiator, 5 mg/mL in PBS buffer) into
the right ankle. As the control group, physiological saline was injected in left ankle joint without
λ-carrageenan. After four hours, the same amount of BC-3 (100 μL, 1 mM) was injected into
both of ankles of the mouse. The differences between two ankles were observed immediately
after BC-3 injection. It is clear to find that only the obvious fluorescence emission was recorded
in the paw area from right ankle (Fig. 5). However, negligible fluorescence was observed for the
11

control group. These results indicated that the probe BC-3 exhibited high capability for the
detection of endogenous HOCl in vivo.
4. Conclusion
In summary, two new fluorescent probes (BC-2 and BC-3) are designed and synthesized based
on phenoxazine for the detection of HOCl both in vitro and in vivo. In the presence of HOCl, the
probes can rapidly detect HOCl using both fluorescence and absorption changes in vitro.
Furthermore, the probes exhibited excellent sensitivity and selectivity for HOCl over other
ROS/RNS. More importantly, the probe BC-3 was able to detect the endogenous HOCl level in
living cells. Moreover, BC-3 could specifically respond to HOCl in mouse model of rheumatoid
arthritis. The further study on the applications of phenoxazine derivatives is ongoing in our
laboratory.
Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgments
This research was funded by the National Natural Science Foundation of China (51572258,
51872263 and 21876158), Zhejiang Provincial Natural Science Foundation of China
(LZ19E020001 and LQ19B050003), Zhejiang Provincial Ten Thousands Talents Program
(2018R52005), and the Key Construction Project (2017XM022) of Zhejiang Normal University.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at XXXX
12

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19

Scheme 1 The synthetic route of probes BC-2 and BC-3. The reaction conditions: (a)
Na2CO3, Na2S2O4; (b) Na2CO3, triphosgene; (c) Na2CO3; (d) HOCl.
Scheme 2 The recognition mechanism of probes towards HOCl.

a
b
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
10
0
µM
0
2
4
6
8
µ
µ
µ
µ
µ
M
M
M
M
M
y=15.58+42.95x
R2
0.996
=
HOCl
10 µM
µ
M
640 660 680 700 720 740 760 780 800
0
2
4
6
8
10
Wavelength (nm)
HOCl (µM)
1.0
D
c
10 µM
1.0
0.8
0.6
0.4
0.2
0.0
0.8 30
µ
M
0
5
µM
µM
0.6
10
15
20
25
30
µ
µ
µ
µ
µ
M
HClO
M
M
M
M
0.4
0
0.2
30 s
µ
M
HOCl
0.0
0
20 40 60 80 100 120 140
560
600
640
680
720
Time (s)
Wavelength (nm)
Fig. 1 (a) Fluorescence spectra of 10 μM BC-3 (in PBS, pH=7.4) in the presence of
different concentrations of HOCl (0, 2, 4, 6, 8, 10 μM); (b) The linear relationship
between the fluorescence intensity at 669 nm and the concentration of HOCl (0–10
μM); (c) Absorption spectra of BC-3 (10 μM) in the presence of different
concentrations of HOCl (0–30 μM); (d) The fluorescence intensity of BC-3 (10 μM)
varied with time in the presence of 10 μM HOCl. The excitation and emission
wavelength (λex/λem) are 620/669 nm, respectively.
a
b
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
A
B
C
D
E
F
G
H
I
A
B
C
D
E
F
G
H
I
ROS/RNS
Anions
d
1.0
0.8
0.6
0.4
0.2
0.0
c
1.0
0.8
0.6
0.4
0.2
0.0
A B C D E F G H I J K L M N O P
A
B
C D E F G H
I
J
Amino acids
Cations

Fig. 2 The fluorescence intensity of BC-3 at 669 nm after the addition of other species
in PBS. (a) The selectivity of BC-3 in the presence of different ROS/RNS (from A to
− .
J: H2O2, t-BuOOH, NO, ROO., O2 , OH, t-BuOO., ONOO− and HOCl) with
2−
concentrations of 100 μM; (b) 100 μM various anions (from A to I: CH3COO−, CO3 ,
2−
−
2−
SO4 , Cl−, F−, I−, NO2 , S2O4 and HOCl) and (c) cations (from A to J: Fe3+, Ni2+,
+
Mg2+, Fe2+, Al3+, Ca2+, K+, Cu2+, NH4 and HOCl) were added; (d) The fluorescence
intensity of BC-3 in the presence of a series of amino acids (100 μM, from A to P:
Pro, Ser, Ala, Gln, Val, Thr, lle, Gly, Tyr, Met, Trp, Phe, Cys, Glu, Lys and HOCl).
The excitation and emission wavelength (λex/λem) are 620/669 nm, respectively.
a
b
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
25
µM
BC-3
10 µM
15 µM
25 µM
BC-3
10
µM
15
µM
20
µM
20 µM
10
µM HOCl + NAC
10 µM HOCl + GSH
c
1.0 d
1.0
0.8
0.6
0.4
0.2
0.0
0.8
0.6
0.4
0.2
0.0
0.1 mM 0.25 mM 0.5 mM 1.0 mM
0.0 mM
0.1 mM 0.25 mM 0.5 mM 1.0 mM
0.0 mM
10 µM HOCl + Glucose
10 µM HOCl + HCHO
Fig. 3 The interference of several cellular reductants for the detection of BC-3 (10
μM) was studied in the present of HOCl, including (a) NAC; (b) GSH; (c) HCHO; (d)
Glucose. The excitation and emission wavelength (λex/λem) are 620/669 nm,
respectively.

Fig. 4 Fluorescence images of BC-3 for endogenous HOCl. (a-c) Fluorescence
images of HL-60 cells alone. (d-f) The HL-60 cells were cultured with probe BC-3
(10 μM) for 12 h. (g-i) The HL-60 cells were pretreated with ABAH (200 μM) for 1 h
and further cultivated with BC-3 (10 μM) and ABAH (200 μM) for another 12 h.
Confocal fluorescence imaging from left to right: bright field, red channel (700 ± 50
nm, λex = 633 nm) and merged images.
Fig. 5 Fluorescence imaging of BC-3 in the mouse model of rheumatoid arthritis, (a)
brightfield; (b) rainbow channel; (c) the merged image of a and b. The fluorescence
signal was collected at λem = 715 ± 60 nm under the excitation wavelength 610 nm.

Highlights
ò We designed and synthesized “turn-on” near-infrared (NIR) probes (BC-2 and
BC-3) based on phenoxazine for the detection of HOCl.
ò The probes show excellent selectivity, high sensitivity, and rapid response
towards HOCl.
ò The probe BC-3 is capable of rapidly detecting endogenous HOCl in living cells
and in the inflammation area from the mouse models of arthritis.

Declaration of interests
The authors declare no competing financial interests.