Dual-function fluorescent probe with red emission for sensing viscosity and OCl? and its applications in bioimaging

Article abstract of DOI:10.1016/j.dyepig.2020.108531

The microenvironment in biological systems is associated with pathological of organisms. In this work, HBTC, a red-emitting probe with abilities of responding to OCl^? and environmental viscosity was designed and synthesized to preliminary apply

Full text of DOI:10.1016/j.dyepig.2020.108531

Journal Pre-proof
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Dual-function fluorescent probe with red emission for sensing viscosity and OCl and
its applications in bioimaging
Fanyong Yan, Xiaodong Sun, Yingxia Jiang, Ruijie Wang, Yuyang Zhang, Yali Cui
PII:
S0143-7208(20)30779-8
DOI:
https://doi.org/10.1016/j.dyepig.2020.108531
DYPI 108531
Reference:
To appear in: Dyes and Pigments
Received Date: 28 March 2020
Revised Date: 8 May 2020
Accepted Date: 8 May 2020
Please cite this article as: Yan F, Sun X, Jiang Y, Wang R, Zhang Y, Cui Y, Dual-function fluorescent
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probe with red emission for sensing viscosity and OCl and its applications in bioimaging, Dyes and
Pigments (2020), doi: https://doi.org/10.1016/j.dyepig.2020.108531.
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Fanyong Yan and Xiaodong Sun: Conceptualization, Methodology. Xiaodong Sun, Yingxia Jiang,
Ruijie Wang and Yali Cui: Experiments, Methodology, Software. Xiaodong Sun, Yingxia Jiang,
Yuyang Zhang: Writing-Original draft preparation. Fanyong Yan, Yali Cui: Supervision, Validation,
Writing-Reviewing and Editing.

Dual-function fluorescent probe with red emission for sensing
viscosity and OCl- and its applications in bioimaging
Fanyong Yan § a *, Xiaodong Sun § a, Yingxia Jiang a, Ruijie Wang a, Yuyang Zhang a, Yali Cui b
a
State Key Laboratory of Separation Membranes and Membrane Processes/National Center for
International Joint Research on Separation Membranes, School of Chemistry and Chemical
Engineering, Tianjin Polytechnic University, Tianjin 300387, P.R. China
Department of Nuclear Medicine, Harbin Medical University Cancer Hospital, Harbin,
Heilongjiang, 150081, P.R. China
b
§
Those authors contribute equally to the article
*Corresponding author. Tel: +86 22 83955766. E-mail addresses: yanfanyong@tiangong.edu.cn
(Fanyong Yan).
1

Abstract
The microenvironment in biological systems is associated with pathological of organisms. In this
work, HBTC, a red-emitting probe with abilities of responding to OCl- and environmental viscosity
was designed and synthesized to preliminary apply in cell imaging. HBTC showed weak
fluorescence in the dissolved state due to intramolecular rotation, and it showed about 620 nm red
light emission when molecules are restricted. Through red-to-turquoise ratiometric fluorescence
signal, HBTC showed the capacity to selective detecte OCl-, and the LOD was calculated to be
0.141 μM in OCl- concentration range of 2 – 20 μM. Finally, HBTC was used for preliminary cell
fluorescence imaging. It is expected that HBTC will be used to study the diseases related between
OCl- concentration and viscosity in cells.
Keywords: Fluorescent probe; OCl-; Viscosity; Cell imaging
1
Introduction
Hypochlorous acid (HOCl)/hypochlorite (OCl-) as an essential biological active oxygen (ROS)
plays an important role in the body, relating to many physiological processes such as cell
differentiation, conduction, migration, and immune system.[1-5] OCl- is produced in the organism
by H2O2 and Cl- under the catalysis of myeloperoxidase (MPO), thereby exerting its biological
function.[3, 6, 7] OCl- can play protective function about health in the organism, but abnormal
concentrations of OCl- can cause diseases such as arthritis, rheumatoid disease, tissue damage, lung
injury, kidney disease, cardiovascular disease, neuronal degeneration and even cancer.[8, 9]
Therefore, the detection of OCl- in biological systems is particularly important. On the other hand,
the change in intracellular viscosity is also an important parameter. The interaction between
biomolecules and chemical in cell is concerned with cellular viscosity, therefore, many diseases and
cellular malfunctions are related to it.[10, 11] Apoptosis of the cell is also accompanied by changes
in intracellular viscosity.[12, 13] The cellular apoptosis caused by disease about excess OCl- may be
a condition linked to both parameters, thus it is of great significance that develops methods capable
of detecting both indexes simultaneously in cell. Traditional detection methods such as mass
spectrometry, ion chromatography, electrical analysis, potentiometer, mass spectrometry, and
2

nuclear magnetic methods,[14] these methods are difficult to apply to biological systems, and it is
almost impossible to detect changes in viscosity and OCl- at the same time. Therefore, it is
necessary to find a new detection method.
Fluorescence analysis is an emerging detection method. As a powerful analysis tool, it has the
advantages of flexible application, sensitive signals, easy operation, and economical practicality.[15,
16] In addition, the most important feature compared to traditional detection methods is that it can
enter the organism to perform functions.[17, 18] Due to its own fluorescence emission
characteristics, biological tissues have strong fluorescence interference in the blue to green band
during fluorescence observation.[19-21] This background light has a greater impact on fluorescent
probes in this wavelength range, which is not conducive the process of fluorescence analysis.[22-24]
Another disadvantage of short wavelength light is the damage of high energy light to biological
systems.[25, 26] The feasible solution is the fluorescent probe with a large Stokes shift, thereby
reducing the effect of background light and organism damage.[27] In addition, another advantage of
long-wavelength emission is its strong penetrability, which makes it possible to image deeper
tissues and noninvasive in vivo imaging.[28, 29] Therefore, fluorescent probes with
long-wavelength emission have attracted much attention due to their unique advantages in imaging.
The probe achieves two functions in one molecule and can easily analyze the relationship
between related functions. Qiu et al. synthesized a probe that can sequentially responded Co2+, Hg2+,
and Cu2+, which is due to the affinity of phenolic hydroxyl and imine for divalent cations.[30] Based
on the metabolic process of cysteine (Cys), Yue et al. synthesized a probe with capable of
responding to Cys and SO2 simultaneously and successfully applied it to observe the metabolic
process of Cys in zebrafish.[31] Li et al. synthesized a NIR probe that responds to both viscosity and
hydroxyl radical, which successfully associated it with Ferroptosis.[32] Long-wavelength emission
probes that respond to viscosity and OCl- are rarely reported. In order to study the intrinsic
relationship between them, it is necessary to develop a dual-functional probe with the abilities. For
achieving this purpose, we obtained HBTC by prolonging the conjugated structure of HBT, and it
showed AIE properties and OCl- response capabilities in cells imaging.
3

2 Experimental
2.1 Materials and instruments
Trifluoroacetic Acid (TFA), salicylaldehyde, and acetoacetic ester was purchased from Tianjin
Kemiou Chemical Reagent Co., LTD. Analytical grade 2-aminobenzenethiol was purchased from
Aladdin Reagent Co., LTD. Hexamethylenetetramine (HMTA) was purchased from Tianjin
Guangfu Technology Development Co., LTD. Other solvents and test agents were purchased from
Tianjin Fengchuan chemical reagent technology Co., LTD. All reagents and solvents were used
directly without further purification. Thin layer chromatography (TLC) and column
chromatography (200-300 mesh silica gel) were purchased from Qingdao Ocean Chemical Co.,
LTD. Distilled water was used during all test.
Fluorescent spectra were measured by F-380 fluorescence spectrophotometer. FT-IR spectra were
obtained by TENSOR37 FT-IR (KBr disks). The pH values were carried out on a Mettler Toledo pH
meter. NMR spectra were measured by Bruker Advance III HD 400MHz spectrometer. UV–vis
spectra were obtained by Purkinje General TU-1901 UV–vis spectrometer. Bioimaging was
obtained through Leica SP5 confocal microscopy. HRMS was obtained by Bruker micro TOF-QII
instrument.
2.2 Synthesis of probe HBTC
3-acetyl-2H-chromen-2-one (3-acetylcoumarin). Salicylaldehyde (2 mL, 19.16 mmol) and ethyl
acetoacetate (3.63 mL, 28.74 mmol, 1.5 eq), respectively was added to a three-necked flask with 30
mL of ethanol, two drops of piperidine were added dropwise, heated to reflux, and monitored by
TLC. After the starting point of the system disappeared, the solution was concentrated by rotary
evaporation and recrystallized with ethanol to obtain 2.82 g (y% 78.21%) of white crystals.
3- (3- (3- (benzo [d] thiazol-2-yl) -2-hydroxyphenyl) acryloyl) -2H-chromen-2-one (HBTC). The
synthesis of HBT-CHO accords the pervious reporter.[33] HBT-CHO (0.2 g, 0.78 mmol) and
3-acetylcoumarin (0.44 g, 2.35 mmol, 3 eq) were added to 30 mL of ethanol, two drops of piperidine
were added dropwise, and the reaction was monitored by TLC with reflux. Until the system no
longer changes, the reaction solution was poured into cold water, filter, dry, and purify by column
chromatography (PE / EA = 10/1) to obtain a yellow solid (0.16 g, y% = 45.00%). 1H NMR (400
4

MHz, CDCl3) δ 13.52 (s, 1H), 8.59 (s, 1H), 8.31 (s, 1H), 8.14 (s, 1H), 8.01 (d, J = 6.5 Hz, 1H), 7.92
(d, J = 8.0 Hz, 1H), 7.81 – 7.73 (m, 2H), 7.67 (dd, J = 14.3, 7.7 Hz, 2H), 7.53 (t, J = 7.6 Hz, 1H),
7.49 – 7.36 (m, 2H), 7.35 (d, J = 7.3 Hz, 1H), 7.01 (s, 1H). 13C NMR (101 MHz, DMSO) δ 187.63
(s), 163.65 (s), 158.56 (d, J = 7.8 Hz), 154.42 (s), 151.38 (s), 146.37 (s), 144.41 (s), 134.82 (s),
134.03 (s), 132.28 (s), 130.33 (s), 129.85 (s), 126.55 (s), 126.32 (s), 125.98 (s), 125.21 (s), 124.96
(s), 123.08 (s), 122.31 (s), 122.10 (s), 119.28 (s), 118.53 (s), 117.81 (s), 116.26 (s). HRMS (ESI-,
m/z): Calcd for [M-H+]- 424.0649; found, 424.0657.
Scheme 1. The synthetic route of HBTC.
2.3 Optical measurement
HBTC was dissolve in DMSO to prepare a 0.2 mM mother liquor. Analytes include NaF, NaCl,
NaBr, NaI, NaH2PO4, Na2HPO4, NaNO2, Na2SO3, Na2SO4, Na2S2O3, Na2S2O8, NaS, NaHSO3,
NaAc, KMnO4, NaOCl, H2O2, t-BuOOH, ·OH (1 mM Fe2+ with 100 μM H2O2), ONOO-, O2·- (300
μM xanthine with 2.5 U/mL xanthine oxidase), GSH, Hcy, and Cys.[34, 35] The inorganic salts
were prepared into a mother solution of 0.2 mM by secondary distilled water. The test of AIE
properties was carried out in different ratios of MeOH/Tris solution system.[36] The volume ratio of
MeOH was 0/10 – 10/0, and the final HBTC concentration was 10 μM. Viscosity test was prepared
according to glycerol/MeOH 2/8 – 10/0, and the final HBTC concentration was 20 μM.[37] The rest
of the spectral tests were performed in MeOH/Tris (20/80, v/v, 0.05 mM, pH 7.4) unless otherwise
specified. All spectral tests were performed in a 1 × 1 × 3.5 cm quartz cuvette.
2.4 Cell imaging
According a usual program, MDA-MB-231 cells were cultured in a 5% CO2 saturated water
incubator containing 10% FBS with Penicillin and Streptomycin at 37 °C. HBTC (1 mM, 10 μL)
was added to a 35 mm glass-bottomed petri dish with MDA-MB-231 cells for another 1 h at 37 °C,
then washed by PBS twice. For evaluating the functions of HBTC, the cells were divided into three
groups: blank, Taxol (1 mM, 10 μL), and OCl- (1 mM, 10 μL) were added to the medium for another
30 min, respectively. After washes in PBS twice, fluorescence images were recorded by a
5

fluorescence confocal microscope.
3 Results and discuss
3.1 Fluorescent properties of HBTC
The fluorescent properties of HBTC were first tested. In solid state, HBTC is a yellow powder
and emits red fluorescence under ultraviolet light. As shown in Fig. 1a, the emission spectrum
shows a narrow band at 620 nm, the peak of its excitation spectrum at 406 nm, and the Stokes shift
value reaches about 214 nm. Subsequently, the fluorescence spectra in different MeOH/Tris solvent
systems was recorded. As the proportion of organic solvents continues to decrease, the red
fluorescence of HBTC continues to increase by about 13 times, and the most obvious changes after
the 1:1 solvent system. At the same time, at the edge of the fluorescence spectrum, the Rayleigh
peak resulted from excitation spectrum increases with water content, which shows HBTC
precipitates and agglomerates in water. Prior to this, the solution showed little red fluorescence and
a slight blue fluorescence at 455 nm. These phenomena indicate that HBTC has AIE property,[38,
39] and the enhanced red fluorescence is consistent with the phenomenon of AIE (Fig. 1b). It is the
general understanding about AIE phenomenon that the freely rotating single bond of fluorescent
molecules is limited by the environment, producing enhancement in fluorescence signal. There are
several freely rotating single bonds in HBTC molecules, demonstrating the potentiality of the AIE
properties of the HBTC molecule.
The viscosity of solution is a condition to effectively limit the free-rotating single bond, thereby
achieving AIE process of HBTC. MeOH/glycerol solution of different ratios was used as a tool to
change the viscosity of the solution system. As shown in Fig. 1c, the fluorescence peaks at 455 nm
and 620 nm enhanced as the proportion of glycerol increases, and these two peaks finally make the
color of solution gradually become pale white fluorescence with color coordinates (0.31, 0.25) (Fig.
S5) that is close to white emission. It is strange that the blue fluorescence changes with viscosity and
even more susceptible to viscosity (Fig. 1c). The possible cause of the phenomenon is multiple
freely rotatable single bonds in the structure of HBTC, and their sensitivity to viscosity is different,
which leads to HBTC showing two AIE fluorescence peaks at the same time. It can be speculated
that the single bond connecting the coumarin molecule is more susceptible to the influence of
6

viscosity, so the blue fluorescence may be due to the emission from this part. As shown in Fig. 1d,
The Förster-Hoffmann equation was fitted to the peak of blue fluorescence and viscosity (η, 4.83 –
945 cP, Table S1) to obtain the curve: logF455 = 0.383logη + 1.704 (R2 = 0.9762).[32] In summary,
HBTC has a large Stokes shift, obvious AIE properties, multiple flexible single bonds, and
viscosity-related fluorescence properties.
Fig. 1. Fluorescence properties of HBTC. (a) Optimal excitation and emission wavelengths of HBTC; (b) Changes
in fluorescence spectrum caused by different ratios of MeOH/Tris (insert: physical photos); (c) Changes in
fluorescence spectrum caused by viscosity (insert: physical photos); (d) Förster-Hoffmann equation fitted by
viscosity and fluorescence.
3.1 The detection of HBTC to OCl-
For evaluating specificity of HBTC, the detection of different anions and active species was
carried out. As shown in Fig. 2a, among the 22 species tested, OCl- showed obvious fluorescence
changes, and the fluorescence color of the solution changed from red to turquoise. H2O2 and ·OH
showed a slight fluorescence change, and the remaining species were almost does not affect the
color change of the solution. From the selective experiments, it can be indicated that only OCl- can
respond to HBTC and get obvious fluorescence changes among 22 species. Subsequent interference
7

experiments also obtained the same conclusion. As shown in Fig. 2b, the response of OCl- to HBTC
in the presence of other species has little effect on the fluorescence intensity at 620 nm and 506 nm.
This reveals strong anti-interference ability. The fluorescence at 620 nm decreased and the
fluorescence at 506 nm increased as the concentration of OCl- continued to increase, and a
significant change in fluorescence color was observed (Fig. 2c). The natural logarithm of the
fluorescence ratio I620/I506 shows good linearity in a concentration change of 2 – 20 μM, fitting curve:
ln(I620/I506) = -0.242[OCl-] + 2.851, R2 = 0.9827, and the corresponding LOD for OCl- is calculated
to be 0.141 μM (3σ/k, Fig. 2d).
Fig. 2. The detection of HBTC to OCl-. (a) The fluorescent spectra of HBTC to different species; (b) I620/I506 value
of HBTC in the presence of OCl- with the addition of competitive species; (c) Fluorescent spectra of HBTC in the
presence of different concentrations OCl- (insert: photos of before and after OCl- addition); (d) Fitting curve of
ln(I620/I506) value versus OCl- concentration of (2 − 30 μM).
The details of detecting OCl- need further explanation. As shown in Fig. S6a, HBTC showed a
significant fluorescence change within a few seconds after OCl- was added. The ratio fluorescence
reached the endpoint of detection about 2 min, and then remained stable. The effect of pH on the
detection is shown in Fig. S6b. In the range of pH from 6 to 9, HBTC has a convictive detection
8

effect on OCl-. Fluorescence of HBTC changes under partial acid or alkaline conditions, which
indicates that the solution conditions can affect the fluorescence of HBTC. The structure of HBTC
contains a phenolic hydroxyl group, indicating that it is easily affected by alkaline environment. In
order to prove that alkaline conditions and OCl- have different effects on HBTC, three different
solution conditions were prepared as follows: EtOH/Tris (1/1, v/v; pH 7.4), EtOH/Tris (1/1, v/v; pH
11), and EtOH/Tris (1/1, v/v; pH 11) containing OCl-. As shown in Fig. S6c, HBTC has two
characteristic absorption peaks at 400 nM and 470 nM under neutral conditions. At alkaline
conditions, the absorption peak at 400 nM disappeared, but the absorption peak at 470 nM increased,
and the solution was brown under sunlight. It can be found in the fluorescence spectrum that the
color of the fluorescence changed to blue at pH = 11. However, the color of the fluorescence is
slightly different from that under alkaline conditions, and the fluorescent peak appears around 470
nm. (Fig. S6d). Changes in absorbance and fluorescence indicate that OH- and OCl- have different
effects on HBTC.
3.3 Proposed explanation of detection mechanism
In previous experiments, it can be revealed that both alkaline conditions and OCl- can make
HBTC fluorescence change, and the fluorescent peaks difference is about 30 nm. Therefore, the
structural changes of HBTC under different conditions need to be further proved. The fluorescence
change of HBTC under alkaline conditions can be reasonably inferred: the deprotonation effect of
the phenolic hydroxyl group can cause the ESIPT process to occur, which ultimately brings a
change in color. The mechanism of OCl- response needs to be further discussed. In the reference, the
reaction mechanism was speculated that phenol structure is oxidized to quinone structure by
OCl-.[40, 41] Based on this, a similar mechanism is guessed and verified by 1H NMR to explain the
experiment. It can be found in Fig. 3 that the proton peak on the phenolic hydroxyl group disappears
after the response of HBTC to OCl-, and the proton peak (b) of the hydroxyl counterpoint on the
benzene ring disappears. In addition, the completion of the reaction caused the movement and
deformation of most of the proton peaks, such as the movement of the proton peak (c) on the
conjugated double bond. HRMS also proved this process. As shown in Fig. S7, ion peak emerged at
m/z 440.0613 (Calcd for C25H14NO5S+ 440.0587), representing the formation of p-benzoquinone
derivatives. Therefore, the detection mechanism was preliminarily considered through 1H NMR and
9

HRMS.
Fig. 3. Changes in 1H NMR peaks of HBTC and HBTC + OCl- (d-DMSO).
In summary, three types of HBTC response are reasonably assumed: the redox process with OCl-,
the ESIPT effect under alkaline conditions, and the viscosity-dependent internal rotation restricted
process, which cause changes of the fluorescence (Scheme 2).
O
OCl-
O
O
O
O
O
O
O
N
S
Oxidation
O
O
O
OH-
O
H
N
HO
O
O
N
S
ESIPT
S
O
HO
+
Viscosity
RIR
+
N
S
+
Scheme 1. Three variations of HBTC in different conditions.
4 Application of HBTC in cell imaging
Cell apoptosis will lead to morphological changes and abnormal intracellular indicators, where
changes in viscosity have been reported in many literatures.[12, 13] In order to show whether HBTC
10

can change color with the viscosity change during apoptosis, co-culture staining of HBTC and
breast cancer cells was performed. As shown in Fig. 5, the cancer cells respectively showed
fluorescent signal in the red channel and the blue channel in the case of HBTC alone. After adding
Taxol to promote apoptosis, it is disappointing that the fluorescence signal enhancement is not
obvious. Probable cause is that HBTC is not sensitive to changes in viscosity caused by apoptosis,
and it can also be seen from Fig. 1. Another possible cause is that the watery environment of the cell
may cause HBTC to lose its capability of viscosity response. Therefore, HBTC needs to be further
optimized.
Fig. 5. Intracellular viscosity changes imaging by HBTC. (a) – (c) Photos of HBTC cultured cells in red channel,
blue channel, and after merge; (d) – (f) Photos of Taxol (100 μg/ml) cultured cells in red channel, blue channel,
and after merge.
Another function of HBTC is selective fluorescence response to OCl-. In order to show the
application of HBTC in response to OCl- in cells, exogenous OCl- was used as a stimulant to treat
breast cancer cells cultured in HBTC. As shown in Fig. 6, HBTC mainly showed red fluorescence
under blank conditions. After adding OCl-, the fluorescence in the green channel was significantly
enhanced and the fluorescence after Merge changed from red to yellow-green, which indicates that
HBTC is responded by OCl- with a ratiometric fluorescence signal.
11

Fig. 6. OCl- intracellular imaging by HBTC. (a) – (c) Photos of HBTC cultured cells in red channel, green channel,
and after merge; (d) – (f) Photos of OCl- treated cells in red channel, blue channel, and after merge.
Conclusion
In this work, a red-emitting fluorescent probe with 216 nm Stokes shift was synthesized for
detecting viscosity and OCl-. In the dissolved state, probe HBTC shows weak fluorescence, and the
fluorescent intensity increases with the increase of environmental viscosity or the increase of water
content, which shows the AIE property caused by the restriction of intramolecular rotation (RIR)
effect. On the other hand, HBTC can selectively detect OCl- by red-to-turquoise fluorescence signal
with a LOD of 0.141 μM. At pH 6 – 9, HBTC was able to complete the detection in about 2 min.
Finally, we apply the bifunctional probe HBTC to cell imaging. Viscosity changes caused by
apoptosis did not result in significant fluorescence enhancement, but exogenous OCl- causes a
significant ratiometric fluorescence signal.
Acknowledgement
The work was supported by the National Natural Science Foundation of China (51678409,
51638011, 51578375, 81801808), Tianjin Research Program of Application Foundation and
Advanced Technology (19JCYBJC19800, 18JCYBJC87500, 15ZCZDSF00880), State Key
Laboratory of Separation Membranes and Membrane Processes (Z1-201507), Harbin Medical
University Cancer Hospital Haiyan Foundation (JJZD2018-02), the Program for Innovative
12

Research Team in University of Tianjin (TD13-5042).
13

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Graphic Abstract: HBTC fluorescence changes and cell imaging in three cases.
17

Highlight
1. A red-light emitting probe HBTC with 214 nm Stokes shift was synthesized.
2. Probe HBTC has dual functions response for viscosity and OCl-.
3. Probe HBTC was successfully applied to the detection of OCl- in MDA-MB-231 cells.

Conflict of interest
The authors declared that they have no conflicts of interest to this work.
We declare that we do not have any commercial or associative interest that represents a conflict of
interest in connection with the work submitted