Ratiometric and colorimetric fluorescent probe for hypochlorite monitor and application for bioimaging in living cells, bacteria and zebrafish

Article abstract of DOI:10.1016/j.jhazmat.2020.122029

Hypochlorous acid (HOCl)/hypochlorite (ClO^?) was a biologically important component of reactive oxygen species (ROS) and plays a key role in human immune function systems. HOCl/ClO^? can destroy invasive bacteria and pathogens, and me

Full text of DOI:10.1016/j.jhazmat.2020.122029

Journal Pre-proof
Ratiometric and colorimetric fluorescent probe for hypochlorite monitor
and application for bioimaging in living cells, bacteria and zebrafish
Xiaojun He, Hong Chen, Chuchu Xu, Jinyi Fan, Wei Xu, Yahui Li,
Hui Deng, Jianliang Shen
PII:
S0304-3894(20)30015-7
DOI:
https://doi.org/10.1016/j.jhazmat.2020.122029
HAZMAT 122029
Reference:
To appear in:
Journal of Hazardous Materials
Received Date:
Revised Date:
Accepted Date:
27 October 2019
14 December 2019
4 January 2020
Please cite this article as: He X, Chen H, Xu C, Fan J, Xu W, Li Y, Deng H, Shen J,
Ratiometric and colorimetric fluorescent probe for hypochlorite monitor and application for
bioimaging in living cells, bacteria and zebrafish, Journal of Hazardous Materials (2020),
doi: https://doi.org/10.1016/j.jhazmat.2020.122029
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Ratiometric and colorimetric fluorescent probe for hypochlorite monitor and
application for bioimaging in living cells, bacteria and zebrafish
Xiaojun He,a,§ Hong Chen,c,§ Chuchu Xu,d Jinyi Fan,d Wei Xu,a Yahui Li,b Hui Dengd,* and
Jianliang Shena,b,*
aSchool of Ophthalmology & Optometry, School of Biomedical Engineering, Wenzhou
Medical University, Wenzhou, Zhejiang 325035, China
bWenzhou Institute, University of Chinese Academy of Sciences, Wenzhou, Zhejiang
325001, China
cLuoyang Key Laboratory of Organic Functional Molecules, College of Food and Drug,
Luoyang Normal University, Luoyang, 471934, China
dSchool of Stomatology, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
§These authors contributed equally to this work.
All authors have given approval to the final version of this manuscript.
*Address correspondence to dh0726@163.com (H. Deng); shenjl@wibe.ac.cn (J. Shen)

Graphic abstract
Highlights
 Novelty sensor has employed to sense ClO- with ratiometric and colorimetric.
 The sensing mechanism was explored by theoretical calculation.
 The chemosensors for ClO- successful response in cells, bacteria and zebrafish.
 Visualized fluorescence recovery of ClO- can be realized on filter paper.
Abstract
Hypochlorous acid (HOCl)/hypochlorite (ClO-) was a biologically important component of
reactive oxygen species (ROS) and plays a key role in human immune function systems.
HOCl/ClO- can destroy invasive bacteria and pathogens, and mediate the physiological balance
of the organism with low concentrations, and cause oxidation of the biomolecules such as
proteins, cholesterol and nucleic acid in biological cells, leading to a series of diseases with over
capacity. Therefore, quantifying the content of HOCl/ClO- in organisms are extremely urgent. In
this work, coumarin-salicylic hydrazide Schiff base (CMSH), a ratiometric and colorimetric
fluorescent probe for ClO- detection based on coumarin as the fluorophore unit was rationally

designed and synthesized. The results indicated that CMSH exhibits high selectivity and
sensitivity for ClO- identification. Additionally, the ratios (I470/I532) displayed brilliant ClO--
dependent quick and sensitive performance within 40 s and limitation of 128 nM, respectively.
As well as the color of the solution changes from green to colorless accompanied by the
fluorescence form green turns into blue with addition of ClO-. Totally, CMSH has been
successfully employed as ratiometric sensor to image in living cells, bacteria and zebrafish with
low cytotoxicity and good permeability.
Keywords: Fluorescent probe, Ratiometric and colorimetric, Hypochlorous acid/hypochlorite,
Bioimaging in living cells, bacteria and zebrafish
1. Introduction
Reactive oxygen species (ROS), including superoxide radicals, hydroxyl radicals, hydrogen
peroxide, singlet oxygen and hypochlorous acid (HOCl)/hypochlorite (ClO-), play a crucial role
in the life system.[1-3] HOCl/ClO- was one of the important ROS in the body and plays an
important role in the human immune function system, which was mainly produced by the
reaction of H2O2 and Cl- under catalysis with myeloperoxidase (MPO) in the organism.[4-6] Even
if HOCl/ClO- devotes to the destruction of bacteria in living organisms, and also gives rise to
oxidative stress with excessive, and leading to many diseases such as phlegmonosis,
angiocardiopathy, atherosclerosis, cancer and kidney diseases.[7-11] However, excessive
HOCl/ClO- can also bring about damage of tissue and a range of complication such as
arteriosclerosis, arthritis and cancer, as well as environmental pollution.[12-15] Therefore, it was
important to develop an effective method to monitor the changes of HOCl/ClO- concentration.
Fluorescent probes have the advantages of high sensitivity, real-time monitoring and easy
operation, and have obvious advantages in detecting analytes in environmental and biological
systems.[16, 17] Among them, the organic molecule fluorescent probe has small volume, simple
synthesis, fast reaction time, good selectivity, high sensitivity, and fluorescence as an output
signal, which was convenient for real-time detection.[18, 19] Hence, small molecule probes were

more and more widely used in environmental detection and chemical analysis, and were widely
used in fields such as medicine and biomedicine.[20, 21]
Recently, a number of fluorescent probes capable of specifically identifying ClO- have been
designed and synthesized, including some probes that can be targeted subcellular organelle. Lin
et al.[22] developed a ratiometric type fluorescent probe using dehydration reaction of
hypochlorous acid for the first time, and the molecular charge was increased due to the large
electron pull capability. The transfer process was enhanced, resulting in red shift of the
fluorescence spectrum; Yuan et al.[23] designed two-photon hypochlorite probes with naphthalene
as the fluorophore, which can locate mitochondria and lysosomes separately; Hu and Zeng et
al.[24] based on coumarin and the semi-cyanine dye developed a colorimetric and fluorescent
response hypochlorous acid fluorescent probe for the backbone; Wu and Zeng[25] used pyrene as
the mother to design and synthesize a ratio of hypochlorous acid fluorescent probe. The
introduction of quaternary ammonium salt greatly improved the water solubility of the probe;
Nagano et al.[26] adopted the rhodamine located in the mitochondria was a fluorophore, and a
fluorescent probe selective for high reactive oxygen species was designed. The photoinduced
electron transfer (PET) process in the molecule allows the rhodamine fluorescence to be
quenched. However, to date, most of the constructed ClO- probes were fluorescent enhanced or
quenched. Compared with this single emission wavelength change probe, the ratiometric type
probe has a dual-wavelength emission characteristic, which was less affected by environmental
factors, and has a wide response range, high accuracy, and can realize semi-quantitative analysis,
and has been widely used.[27-29] Moreover, the ratiometric probes reported so far have limited
their application in organisms due to poor water solubility or their inherent toxicity.
Based on the above characteristics, we have developed a ClO- ratiometric and colorimetric type
fluorescent probe with high selectivity, high sensitivity, good water solubility and low toxicity
based on the coumarin parent unit, which can be used for specific rapid detection of aqueous
solutions. The probe uses coumarin as the fluorophore to bind salicylate to make it have the
overall fluorescence red shift for easy detection. Moreover, the probe has obvious changes in
color and fluorescence before and after detection of ClO-, which could be realized as a dual-
function recognition probe with fluorescence and color. It's worth noting that the probe can
detect ClO- in living cells, bacteria and zebrafish, which can effectively achieve imaging results.
2. Experimental section

2.1. Materials and apparatus
All chemicals and solvents were obtained from commercial suppliers with analytical reagents
o
grade. Sodium hypochlorite was purchased from Aladdin stored at 4 C, and all of other
chemicals were purchased from Aladdin and Sigma-Aldrich. MRC-5 cells were provided by
ATCC (Manassas, VA) and zebrafish were got from the Animal Ethics Committee of Wenzhou
Medical University. Absorption spectrum and emission spectrum were recorded on UV-2600
form SHIMADZU and RF-45301 from SHIMADZU, respectively. NMR spectra were obtained
on a Bruker Ascend 400 spectrometer in DMSO-d6. LCMS spectra were recorded on the waters
zq 2000 spectrometer with HPLC (waters 2695). Cells and zebrafish fluorescent images were
acquired on confocal microscopy with Nikon-A1.
2.2. Synthesis of CMSH
Accurately weigh (250 mg, 1.0 mmol) of 7-(diethylamino)-2-oxo-2H-methylene-3-carbaldehyde
and dissolve in 15 mL of absolute ethanol. To the above solution was added (150 mg, 1.0 mmol)
of 2-hydroxybenzohydrazide and mixed well. After stirring the reaction for 16-18 hours at room
temperature, the mixture after the reaction was filtered through a Buchner funnel, and the solid
precipitate on the cake was washed with ethanol several times to obtain 330 mg of probe CMSH,
the solid powder of which was orange.[30] Yield: 84%.; 1H NMR (400 MHz, DMSO) δ 11.90 (s,
2H), 11.85 (s, 1H), 8.43 (s, 1H), 8.31(d, J = 7.6 Hz, 1H), 7.82 (d, J = 8.9 Hz, 1H), 7.36 (t, J = 7.6
Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 6.70 (d, J = 7.5 Hz, 1H), 6.68 (d, J = 7.6 Hz, 1H), 6.50 (s, 1H),
3.47 (q, J = 6.7 Hz, 4H), 1.14 (t, J = 6.7 Hz, 6H); 13C NMR (150 MHz, DMSO) δ 165.35, 161.28,
159.99, 157.12, 152.02, 144.01, 139.50, 131.49, 128.67, 119.32, 117.81, 115.94, 112.69, 110.27,
108.54, 96.87, 45.04, 13.10; LC-MS (ESI+) m/z [M+1]+: Calcd for C21H22N3O4, 379.15, found,
380.00.
2.3. Spectrum Measurements
The stock solution of CMSH was prepared in EtOH with the concentration of 1 mM, which
prepared working solution with EtOH/H2O (v/v, 1:19) system in 10 μM and 5 μM for absorption
spectrum and emission spectrum tests, respectively. The detecting ions (ClO-) and other various
-
-
2-
2-
interferential analytes stock solutions [ClO-, O2 , Cl-, NO2 , AcO-, CO3 , 1O2, NO, ONOO-, SO4
2-
, •OH, S2O3 , Cys, GSH and H2O2] were prepared at 1 mM in deionized water. The work system
solution contained CMSH, EtOH/H2O (1:19) and 16 equiv. of each analytes.

2.4. Cytotoxicity Assays
The toxicological evaluation of CMSH to MRC-5 cells were employed by traditional methods
MTT assays.[31] MRC-5 cells were cultured and seeded in 96-well plates with 2 × 104 cells/mL,
o
and after treatment by various concentrations of CMSH from 0 µM to 80 µM at 37 C for 24h.
After that 10 µL MTT (5 mg/mL) was added to each well, and incubated with 4 h under the same
condition, and was added 150 µL DMSO to dissolve precipitation after the supernatants were
aspirated. To record the absorbance at 570 nm by microplate reader. The cell viability (%) =
(ODs-ODb) / (ODc-ODb) × 100 %, there s-sample, b-blank and c-control.
2.5. Cell Culture and Imaging
Dulbecco's Modified Eagle Medium (DMEM, Hyclone) used for MRC-5 cells cultured, and 10%
fetal calf serum (FBS, Sijiqing), penicillin (100 U / ml, Hyclone) and streptomycin sulfate (100
o
U / ml, Hyclone) were added in above medium, and cultured at 37 C of 5% CO2 and 95% air
atmosphere condition.[32-34] MRC-5 cells were seeded in new confocal dish processing for the
night prior to imaging experiments. After that the cells were pretreated with 5 μM free CMSH
for 20 min, and then incubated with 20 μM, 40 μM and 80 μM of ClO- for 20 min. Cellular
imaging was performed after washing the cells thrice with PBS. Fluorescent imaging was taken
in green/blue channels on confocal microscopy with Nikon-A1.
2.6. Fluorescence Imaging in Zebrafish
The 4-day old zebrafish were cultured and transferred to 24-well plate, and 5 μM of free CMSH
was added for 20 minutes and washed with PBS, and then 20 μM, 40 μM and 80 μM of ClO-
were added respectively, which were incubated with 20 minutes further. The zebrafish were
washed and transferred to a new confocal dish for narcosis (3-Aminobenzoic acid ethyl ester
methanesulfonate, MS222) and imaging.
2.7. Quantum calculations
All calculations were done in the Gaussian 09 package.[35] Firstly, the density functional
theory(DFT) method was used to optimize the geometry of the ground state structure before and
after the reaction of CMSH with ClO- at the B3LYP/6-31G* level, and the natural bond orbital
(NBD) analysis was performed on the optimized model. Based on the optimized configuration of
the ground state, the TD-DFT method was applied to calculate the excited state at the B3LYP/6-
311++G** level. Then, the excited state geometry was optimized at the B3LYP/6-31G* level

and the excited state was calculated. In order to investigate the influence of calculation method
and solvation effect on the calculation results, we use the continuous polarization medium model
(PCM) to complete the geometric structure optimization and the excited state calculation with
ethanol and water as the medium. The results show that the geometry optimization after solvent
addition was completed. There was no significant difference between the calculation results of
the energy levels. Therefore, the calculation results and discussion in this paper do not include
the solvent effect.
2.8. Live subject statement
All animal operations (fluorescence imaging in zebrafish) were performed in accordance with the
guidelines for laboratory animal care and use at Wenzhou Medical University and approved by
the Animal Ethics Committee of Wenzhou Medical University.
3. Results and discussion
3.1 Design and synthesis
Diethylamino-coumarin dye was used as the building basics to assemble fluorescent probe
CMSH with wonderful photophysical property, such as high fluorescence quantum yield, which
were an important indicator of the application of probes in the field of biology. Whereas,
absorption and emission spectra of diethylamino-coumarin were restricted at 400 nm -500 nm,
which was not only lacks tissue penetration, but also was strongly interfered by the background
of its own fluorescence. To push the emission with red shift, we were employed diethylamino-
coumarin aldehyde as one building basic, and then modified with salicylhydrazide to form water
solubility CMSH with specific spectral properties (Scheme 1). The structure of CMSH was
1
13
confirmed by H and C NMR and MS as shown in Fig.S1-S3. Details of the synthesis steps,
structural analysis, etc. were provided in the experimental section and supplementary
information. According to existing reports, hypochlorite (ClO-)/hypochlorous acid (HOCl) have
strong affinity to double bonds. It was foreseeable that when the C=N bond was destroyed with
oxygenization by ClO-, the large π conjugate of CMSH will be destroyed, which was acted as a
colorimetric and ratiometric chemosensor respond to ClO-. The coumarin–salicylhydrazide
sensor displayed emission maximum at 532 nm (Fig. S5). Consequently, with increase of ClO-,
the color of probe solution system from green turn into colourless. Meanwhile, original green

fluorescence from CMSH will gradually fade, and the blue fluorescence from coumarin
aldehyde building block will rise. It could be seen that CMSH will reveal dual colorimetric and
proportional fluorescence responses to ClO-.
3.2 Ratiometric and colorimetric response of CMSH towards ClO-
The spectroscopic analysis with CMSH to the monitoring of ClO- was investigated, which were
carried out in EtOH/H2O system at ambient temperature. The probe CMSH solution system
displayed green color with green fluorescence under the excitation wavelength at 390 nm. With
addition of ClO- (16 equiv.), the green color of CMSH instantly turn into colourless, which could
be distinguished ClO- use colorimetric with the naked eye, and the fluorescence from green turn
into blue (Fig. 1 inset). To examine the response of CMSH to ClO-, the spectra titrations were
performed. As shown in Fig. 1, CMSH shows a conspicuous absorption signal at 456 nm and the
expected emission signal at 532 nm. With the increase of ClO- concentration, the fluorescence
intensity of CMSH at 532 nm regularly attenuated, and a newly blue-shifted emission signal at
470 nm appeared. At the same time, the symbolic absorption signal shifts from 456 nm to 348
nm, indicating that the whole π conjugate of CMSH were destroyed with addition of ClO- to 16
equivalents. In addition, the newly formed emission signal reached a ratio of (F470/F532) at 9.2-
times, indicating that CMSH can be used as a ratiometric chemosensor for ClO-. It is noteworthy
that proportional fluorescent probes require a significant proportional signal change at two
wavelengths because the dynamic range of the proportional probe are proportionally controlled.
Linear correction was performed between the absorbance ratio (A348/A456) and ClO-concentration
(0-80 μM) of the corresponding coefficients, and the correction coefficient was higher (R2 =
0.9728) (Fig. 2c), indicating that CMSH can be used for quantitative detection of ClO-
concentration. Additionally, a good linear relationship (R2 = 0.9716) was obtained by plotting
the relative fluorescence intensity ratio value (F470/F532) versus ClO- concentration at a ClO-
concentration of 0 to 80 μM (Fig. 2d). In addition, CMSH could be sensed to low concentrations
of ClO- with a detection limit of 128 nM, which was faceoff with previously published ClO-
proportional fluorescent probes (Table S1). The results indicate that CMSH could be detected
ClO- with good advantages of sensitivity.
3.3 Selectivity of CMSH toward ClO-

Selectivity was one of the crucial indicators for measuring fluorescent probe applications. To
evaluate the selectivity for ClO-, 5 µM CMSH was treated to various biologically relevant ROS,
including various analytes (80 μM). In Fig. 2a, only with addition of 80 µM ClO- led to an
obvious color and fluorescence variation of CMSH, which could be perceived with the naked
-
-
2-
2-
2-
eye. However, other analytes (O2 , Cl-, NO2 , AcO-, CO3 , 1O2, NO, ONOO-, SO4 , •OH, S2O3 ,
Cys, GSH and H2O2) only given rise to a negligible ratiometric sensing of CMSH. To further
insight into the selectivity of CMSH for ClO- over other analytes, we compared the fluorescence
ratios (F470/F532) differences of CMSH for other interferent. As shown in Fig. 2b, this probe
responds to ClO- significantly strong with the fluorescence over than other analytes. The probe
exhibited an apparent chromatic aberration from the color of solution and fluorescence sensing to
ClO- on the strength of oxidization on cleavage of C=N group in CMSH, which generated a blue
shift emissive diethylamino-coumarin aldehyde. Under the visible light and 365nm UV lamp, the
green color of CMSH solution changed to colorless only in the presence of ClO- with the
fluorescence changes from green to blue, which was well discriminated by ‘naked eye’ and
fluorescence from CMSH in presence of other analytes as shown in Fig. 2c. These results
display that CMSH could be recognition ClO- with good selectivity over other biological
analytes, which have potential applications to detect ClO- in various biological environment.
3.4 Time and pH dependent response of CMSH toward ClO-
Time dependent fluorescence sensing ClO- with CMSH was investigated. As shown in Fig. 3a,
with increasing of ClO- (16 equiv.), the fluorescence intensity (at 470 nm) increased rapidly and
arrived in equilibration within 40 s, which illustrated that CMSH could be employed as a "fast
sensing" chemosensor for ClO- and has a potential application to realize for visualized ClO- in
living systems with real-time detection. Furthermore, to evaluate the feasibility of CMSH as a
multifunctional sensor for ClO- in different environment conditions, pH-dependent fluorescence
sensing to ClO- was exposed in EtOH/H2O (v/v = 1/19) with various pH values. In Fig. 3b, the
fluorescence intensity ratios (F470/F532) of CMSH was unchanged as motionless as a statue from
pH 2.0 to 12.0. With present of ClO- (16 equiv.), the fluorescence intensity of CMSH maintain
significantly at the pH values undulating from 2.0 to 8.0, which was similar to the pKa of ClO-
(pKa = 7.53).[36, 37] Hence, CMSH can used to detect ClO- at physiological pH values.
3.5 Proposed sensing mechanism

To confirm the response mechanism of CMSH for detecting ClO-, the reaction product of
CMSH with ClO- was were characterized by LC-MS. The probe CMSH structure has a very
good conjugated system and exhibits strong green fluorescence (532 nm). With addition of
sodium hypochlorite, the original conjugated system of the probe was destroyed due to the
oxidation of hypochlorite (Fig. 4a). This results in a decrease in fluorescence at Em=532 nm and
an increase in fluorescence at Em=470 nm. In order to verify the above conjecture, mass
spectrometry was performed on the probe CMSH solution to which sodium hypochlorite
solution was added. Mass spectrometry showed that the signal peak of m/z=246.0 appeared in
the reaction solution, and the mass spectrum signal peak of the probe CMSH was [M+H] =
380.0 (Fig. S4). This indicates that the probe CMSH was oxidized in the action of hypochlorous
acid with the C=N bond was broken, and the original product coumarin aldehyde molecule [M] =
245.1 was formed. The above results indicate that the probe CMSH was oxidized under the
action of hypochlorous acid to form coumarin aldehyde, and the original conjugated system was
destroyed, thereby generating a change in the fluorescent signal. To further estimate the optical
behavior of the probes CMSH and ClO- before and after the oxidation reaction, we calculated
the optimal geometry of the probe from the B3LYP/6-31G(d) level using density functional
theory (DFT) based on the Gaussian 09 program. As shown in Fig. 4b, the highest occupied orbit
(HOMO) of the probe CMSH was mainly distributed on the salicylhydrazone moiety, and the
lowest unoccupied orbital (LUMO) was distributed on the coumarin skeleton. However, when
CMSH was oxidized by ClO- to form coumarin aldehyde molecule, the HOMO electron cloud of
the coumarin aldehyde molecule was uniformly distributed throughout the structure, and the
LUMO was biased toward the other end of the molecular skeleton. In addition, after reacting
with ClO-, the HOMO-LUMO level difference of the probe CMSH was 0.11558 eV lower than
the energy level difference of the probe CMSH (0.13416 eV), which causes the Stokes shift to
decrease, so that the maximum emission wavelength becomes shorter.
3.6 Cell imaging
Encouraged by the merits results of spectroscopy displayed above, to investigate the potential
application of CMSH for detection ClO- in living cells, cell bioimaging were executed in MRC-5
cell lines. Cell cytotoxicity of CMSH was performed by MTT assay, and the results displayed
that about 80% of cells still remained alive even though 80 µM CMSH was normalized for 24 h
(Fig. S6), revealing that CMSH has low cytotoxicity and could be further used for cell imaging

experiments. Subsequently, to demonstrate the capability of this probe to sense exogenous ClO-
in living cells, we performed imaging assay with control group and treatment group as powerful
evidence. As shown in Fig. 5, MRC-5 cells presented a clear cell profile with green fluorescence
in green channel and non-fluorescence in blue channel after incubation with 5 μM CMSH for 20
min. Additionally, the MRC-5 cells were incubation with 5 μM CMSH, and further treatment
with 20 μM, 40 μM and 80 μM ClO- for 20 min respectively, which the green fluorescence was
faded away and blue fluorescence increasingly prominent. As the concentration of ClO-
increased, the blue fluorescence from MRC-5 cells became much stronger and the original green
fluorescence gradually faded away. The ratio images (Fblue/Fgreen) (Fig.5d, 5h, 5l and 5q) were
recorded by mediating the blue channel images (Fig. 5b, 5f, 5j and 5o) with the green channel
images (Fig. 5c, 5g, 5k and 5p), which was prominent movement for blue channel after the
addition of ClO-. Cells treated with coumarin (5 μM) as a control group, and found that the cells
imaged blue fluorescence, and the cells still imaged blue fluorescence after treatment with 80 μM
ClO- (Fig. S7). Cell imaging results revealed that CMSH has good cell permeability and could be
act as a ratiometric fluorescence imaging of ClO- in living cells.
3.7 Bacteria imaging
Reactive oxygen species (ROS) was closely related to the aerobic metabolism and the formation
of enzyme in bacteria, and the enzyme-catalyzed reaction was mainly carried out on the cell
membrane. Therefore, the production of ROS was focused on the membrane of bacteria.[38-40]
Recently, It has been reported that ROS has a direct relationship with the growth of bacteria, and
it has great significance to explore the content of ROS in bacteria for the development of new
antibacterial drugs, clinical medicine, and food safety. Hence, fluorescence monitor of ROS
(ClO-) in bacteria would be useful to get insight into this antimicrobial mechanism. However,
there was little research on imaging in bacteria at present, which was required for the sensor to
have powerful fluorescence intensity, stability and permeability. To explore its application
further, CMSH were employed to sense ClO- in E. coli via bioimaging using fluorescence
imaging. As illustrated in Fig. 6, the E. coli stained by 5 μM CMSH alone showed an obvious
fluorescence in the green channel in E. coli and no fluorescence the blue channel. With the
addition of 20 μM, 40 μM and 80 μM ClO- for 20 min respectively, the fluorescence of E. coli in

the green fluorescence was faded away and blue fluorescence increasingly with prominent, and
the “rhabditiform” shape of E. coli could be clearly visualized from the fluorescent pattern in the
zoomed area. The ratiometric images (Fblue/Fgreen) (Fig.6d, 6h, 6l and 6q) were recorded by
mediating the blue channel images (Fig. 6b, 6f, 6j and 6o) with the green channel images (Fig.
6c, 6g, 6k and 6p), which was prominent movement for blue channel after the addition of ClO-.
E. coli treated with coumarin (5 μM) as a control group, and found that E. coli imaged blue
fluorescence, and E. coli still imaged blue fluorescence after treatment with 80 μM ClO- (Fig. S7).
These results proved that CMSH could be applied in tracing ClO- in E. coli, which would be a
potential tool to study the biotransformation of ClO- in bacteria.
3.8 Zebrafish imaging
In normal metabolic processes, organs of an organism could spontaneously produce various
ROS. So far, the majority of reported studies demonstrated the possibility of fluorescence
imaging of ROS either by single fluorescent channel. However, bioimaging ROS by fluorescent
probes in normal organisms with multi-fluorescent channel was still limited by the sensitivity
and stability of the probe and its ability to pass various biological barriers. Numerous biological
processes were produced in the purtenance of living animals, causing ROS (ClO-) to self-
generate there. Therefore, to investigate the potential biological application of CMSH
responding to ClO- in a living organism with green and blue fluorescent channel, four-day-old
zebrafishes, a popular vertebrate model, were chosen as our research model system. In Fig.7, it
showed that when the zebrafish were cultured in embryo medium, and incubated with free probe
CMSH (5 µM) for 20 min to allow CMSH to permeate into the whole tissues of zebrafish, they
exhibited a visible green fluorescence in their abdomen area, this results indicated that the probe
was able to penetrate in tissues of zebrafish with emitting fluorescence. There was almost no
fluorescence found in the blue channel when the zebrafish were pretreated with CMSH. In
addition, the zebrafish pretreated with CMSH and washed thrice with PBS, and further
incubated with 20 μM, 40 μM and 80 μM ClO- for 20 min respectively, displayed a remarkable
changed that the green fluorescence was faded away and blue fluorescence was increasingly
prominent in the head and abdomen of zebrafish. The ratiometric images (Fblue/Fgreen) (Fig.7d, 7h,
7l and 7q) were recorded by mediating the blue channel images (Fig. 7b, 7f, 7j and 7o) with the
green channel images (Fig. 7c, 7g, 7k and 7p), which was prominent movement for blue channel
after the addition of ClO-. Zebrafish treated with coumarin (5 μM) as a control group, and found

that zebrafish imaged blue fluorescence, and zebrafish still imaged blue fluorescence after
treatment with 80 μM ClO- (Fig. S7). Thus, these results convincingly indicated that CMSH
possessed high tissue penetration capacity and could be realized the visualization of ClO- in
zebrafish with ratiometric fluorescence.
3.9 Application of paper-based probe
As mentioned above, CMSH exhibited a noticeable colour change from green to colorless and a
significant fluorescence change from green to blue in the presence of ClO-. Thus, we tentatively
prepare a colorimetric solution and test paper model for visual detection of ClO-. CMSH (5 µM)
was loaded into a vial, and then different concentrations of ClO- (0 µM, 10 µM, 15 µM, 20 µM,
25 µM, 30 µM, 35 µM, 40 µM, 45 µM, 50 µM, 55 µM, 60 µM, 65 µM, 70 µM, 75 µM and 80
µM) were added and incubated with CMSH (5 µM) for 60s. As shown in Fig. 8, the color of
CMSH solution in vial turned into colorless gradually with the increase of ClO-, which was
recognition with the naked eye (Fig. 8a). Meanwhile, the fluorescence of CMSH solution (5 µM)
changed from green fluorescence to blue fluorescence under 365 nm UV lamp (Fig. 8b). For
practical applications, it is necessary to develop a sensitive, simple, hand-held and cheap paper
sensor.[41-43] Herein, CMSH act as a ClO--sensing paper sensor, which could be realized
visualizing by fluorescence detection of ClO-. Firstly, the filter paper was labelled by EtOH/H2O
solution of CMSH (5 µM) with ‘WMU’ pattern, followed by air-drying. And then dropping
concentrations of ClO- (80 µM) on the paper, the sensing signal was recorded under both visible
light and UV lamp (365 nm). As shown in Fig. 8e and 8f, with addition of ClO-, the color of the
test paper gradually changed from green to blue under UV lamp (365 nm). Both color and
fluorescence of the paper changed under ClO- with rapid and conspicuous. These results showed
that CMSH test paper can quickly and conveniently detect ClO- in an aqueous medium without
any spectroscopic instrumentation.
4. Conclusion
In conclusion, we have rationally designed and synthesized CMSH as a novel colorimetric and
ratiometric chemosensor for ClO- via the oxidation of fracture the hydrazine into the aldehyde.
CMSH functions as a HClO-specific fluorescent indicator that features a fast and ultrasensitive
response. The detection limit is as low as 128 nM, which is the highest sensitivity achieved to
date. Notably, this reaction of the oxidation of fracture the hydrazine into the aldehyde has been

used in fluorescent for ClO- monitor with rarely reported in previously. Significantly, CMSH
can be applied to image ClO- in MRC-5 cells and bacteria for the first time and monitor the
concentration-dependent with ratiometric fluorescence. Additionally, the outstanding sensing
property and good biocompatibility of CMSH in zebrafish revealed that CMSH could be
successfully applied for ClO- detection in the organization. Such work on developing
colorimetric and ratiometric chemosensors based on ClO- is ongoing.
Author contribution：
X.H., H.C. and J.S. conceived and designed the experiments; X.H., H.C. and H.D. carried out experiments; H.D. analyzed
experimental results; C.X., J.F. and W.X. contributed reagents/materials/analysis tools, and assisted with bioimaging experiments
including cells and zebrafish imaging; Y.L. and H.D. performed and analyzed density functional theory calculation; X.H. and J.S.
wrote and modified the manuscript.
Declaration of interests
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
The authors declare no competing financial interest.
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China
(31800833 and 21977081), Zhejiang Provincial Natural Science of Foundation of China
(Z19H180001), the Wenzhou Medical University and Wenzhou Institute of Biomaterials &
Engineering (WIBEZD2017001-03).

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Figure/caption:
Scheme 1 (a) Design strategy and (b) synthetic route for probe CMSH.

Fig. 1 (a) UV-vis absorption (10 µM) and (b) fluorescence spectra (5 µM) changes of CMSH in EtOH/H2O
solution (v/v = 1/19) upon addition of increasing amount of ClO- (0 – 80 µM). Each spectrum was recorded
after 3 min. Inset: (a) The color and (b) fluorescence images of CMSH in the absence/presence of ClO- with
365 nm lamp. Linear relationships between (c) UV-vis absorbance ratios (A348/A456) and (d) fluorescence
intensity ratios (F470/F532) of CMSH versus concentrations of HOCl in EtOH/H2O solution. R was the
correlation coefficient of linear.

Fig.2 Changes of fluorescence spectra (a), ratio of fluorescence intensity of F470/F532 (b), and the fluorescence
colour photographs (c) of CMSH (5 μM) with various biologically relevant species (80 μM) in EtOH-H2O
-
-
2-
1
2-
2-
(v/v= 1/19): blank, ClO-, O2 , Cl-, NO2 , AcO-, CO3 , O2, NO, ONOO-, SO4 , •OH, S2O3 , Cys, GSH and
H2O2 with visible and 365 nm lamp.

Fig. 3 (a) Time-depend fluorescence responses of 5 μM CMSH towards 80 µM ClO- in EtOH-H2O solution.
(b) Ratiometric fluorescence changes of 5 μM CMSH in the absence and presence of 80 μM ClO- in EtOH-
H2O solution with different pH values. Each spectrum was recorded after 3 min for 3 times.

Fig. 4 (a) Proposed mechanism for CMSH sensing of ClO-. (b) HOMO-LUMO energy levels and the
interfacial plots of the molecular orbitals for CMSH with and without addition of ClO-.

Fig. 5 Relative confocal fluorescence images of MRC-5 cells under different conditions with CMSH. MRC-5
cells treated with 5 μM CMSH (a-d), then further incubated with 20 μM ClO- (e-h), 40 μM ClO- (i-l) and 80
μM ClO- for 10 min (m-q). Fluorescence images from left to right: blue channel, green channel and ratiometric
images (Fblue/Fgreen). Scale bar: 100 μm.

Fig.6 Confocal fluorescence images of E. coli. Fluorescent image of E. coli stained with CMSH (5 µM) for 20
min (a-d). E. coli were incubated with CMSH for 20 min, further incubated with 20 μM ClO- (e-h), 40 μM
ClO- (i-l) and 80 μM ClO- for 10 min (m-q). Insert: local zoom of the photograph.

Fig.7 Confocal fluorescence images of zebrafish. Fluorescent image of zebrafish stained with CMSH (5 µM)
for 20 min (a-d). Zebrafish were incubated with CMSH for 20 min, further incubated with 20 μM ClO- (e-h),
40 μM ClO- (i-l) and 80 μM ClO- for 10 min (m-q).

Fig. 8 CMSH (5 µM) as a reagent kit for determination of ClO-. (a) The color and (b) fluorescence images of
CMSH reagent (5 µM) in the presence of different concentrations of ClO-. Photograph of (c, d) color and (e, f)
fluorescence of probe CMSH (5 µM) test paper in the absent and presence of (80 µM) of ClO- under a hand-
held UV lamp (365 nm).

Supporting Information
Ratiometric and colorimetric fluorescent probe for hypochlorite monitor and
application for bioimaging in living cells, bacteria and zebrafish
Xiaojun He,a,§ Hong Chen,c,§ Chuchu Xu,d Jinyi Fan,d Wei Xu,a Yahui Li,b Hui Dengd,* and
Jianliang Shena,b,*
aSchool of Ophthalmology & Optometry, School of Biomedical Engineering, Wenzhou Medical
University, Wenzhou, Zhejiang 325035, China
bWenzhou Institute, University of Chinese Academy of Sciences, Wenzhou, Zhejiang 325001, China
cLuoyang Key Laboratory of Organic Functional Molecules, College of Food and Drug, Luoyang Normal
University, Luoyang, 471934, China
dSchool of Stomatology, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China

Fig. S1 1H NMR spectra of CMSH in DMSO-d6.

Fig. S2 13C NMR spectra of CMSH in DMSO-d6.