Design, synthesis and application of a dual-functional fluorescent probe for reactive oxygen species and viscosity

Article abstract of DOI:10.1016/j.saa.2020.119059

A fluorescence probe based on cyanine fluorophore was designed and synthesized in this work, which can be used to determine viscosity and reactive oxygen species (e.g., OCl^?, ONOO^?) at different wavelengths. Under a low viscosity med

Full text of DOI:10.1016/j.saa.2020.119059

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119059
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Design, synthesis and application of a dual-functional fluorescent probe
for reactive oxygen species and viscosity
Hongyu Li a,b, Ya Liu a,b, Xiaoyi Li , Xiaohua Li ⁎, Huimin Ma
a
a,
a,b
a
Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
University of Chinese Academy of Sciences, Beijing 100049, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 May 2020
Received in revised form 10 September 2020
Accepted 6 October 2020
Available online 09 October 2020
A fluorescence probe based on cyanine fluorophore was designed and synthesized in this work, which can be
used to determine viscosity and reactive oxygen species (e.g., OCl−, ONOO−) at different wavelengths. Under a
low viscosity medium, the fluorescence quantum yield of the probe is very low; however, with the increase of
the medium viscosity, the probe's emission at 571 nm is enhanced by nearly 25-fold due to the inhibition of in-
tramolecular rotations. On the other hand, the probe shows a rapid and linear fluorescence response at 710 nm to
OCl or ONOO− within 1 min. The different spectral response regions of the probe permit the selective detection
of both viscosity and reactive oxygen species. Furthermore, the probe is demonstrated to be cell permeable and
capable of detecting the viscosity and the total amount of OCl−/ONOO− in living cells with the help of confocal
microscope fluorescence imaging.
−
Keywords:
NIR
Fluorescent probe
Viscosity
Hypochlorous acid (OCl−)
© 2020 Elsevier B.V. All rights reserved.
Peroxynitrite (ONOO−
Cell imaging
)
1. Introduction
To date, various fluorescent probes have been developed for detect-
ing ROS [16–18] and cellular viscosity [19–25], respectively. However,
Cell viscosity is a very important physiological parameter, which is
evidence has suggested that ROS might be associated with the change
of intracellular viscosity [19,26]. Thus, simultaneous detection of these
two parameters is of great importance for studies of various physiolog-
ical processes such as apoptosis. Unfortunately, dual-functional fluores-
cent probes with the capacity of detecting viscosity and ROS are rather
rare [19,26]. Hence, the development of such a dual-functional fluores-
cent probe is very necessary.
related to various diffusion-mediated cellular processes, including the
transmission of substances, the interaction between biological mole-
cules, and the diffusion of metabolites. In general, the cytoplasmic vis-
cosity of normal cells is about 1–2 cP, which however is significantly
enhanced to 140 cP or even higher in abnormal conditions [1–4].
These changes in intracellular viscosity are associated with many dis-
eases [5–11], such as diabetes, hypertension, high cholesterol, arterio-
sclerosis and malignant tumors.
Herein, a cyanine-based dual-functional fluorescent probe (CBRV,
see Scheme 1) is reported for the detection of ROS and viscosity. As
depicted in Scheme 1A, CBRV possesses two small π-conjugations
with two positive charges, thus exhibiting short absorption and emis-
sion wavelengths. In addition, a phenylboronate ester group is
appended to the fluorophore (CRV) for the recognition of ROS (such
Reactive oxygen species (ROS), i.e., hypochlorous acid (OCl−),
−
peroxynitrite (ONOO ), hydrogen peroxide (H
2
O
2
), superoxide anion
•−
radical (O
2
), hydroxyl radical (•OH), nitric oxide (NO) and singlet oxy-
), etc., are a series of substances with high oxidative reactivity,
1
gen ( O
2
−
−
which are also characterized by low concentration and short life span.
as OCl , ONOO
2 2
and H O ) [27–30]; the oxidation of the
−
−
Among these ROS, OCl , produced by the reaction of H
2
O
2
and Cl
phenylboronate ester results in the generation of a larger polymethine
π-conjugation system via electron rearrangement and eventually the
NIR fluorescence off-on response at 710 nm. This NIR fluorescence is
beneficial to eliminating the short wavelength background fluorescence
from bio-samples and thus achieving a sensitive detection. On the other
hand, the typical molecular rotor feature of CBRV is conducive to the de-
tection of viscosity (Scheme 1B). With the increase of environmental
viscosity, the intramolecular rotation of rotatable vinyl bond is re-
stricted, and thus the twisted intramolecular charge transfer (a non-
radiative process) is inhibited [31], resulting in fluorescence
under the catalysis of myeloperoxidase (MPO) [12], has been proved
−
to play an important role in killing multiple pathogens [13]; ONOO ,
generated in the presence of NO and O•
−
[14], possesses strong oxida-
2
tion and nitration ability and has crucial function in cell signaling [15].
Therefore, regulation of ROS production is of great significance for main-
taining cellular immunity as well as many other functions.
⁎
Corresponding author.
E-mail address: lixh@iccas.ac.cn (X. Li).
https://doi.org/10.1016/j.saa.2020.119059
386-1425/© 2020 Elsevier B.V. All rights reserved.
1

H. Li, Y. Liu, X. Li et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119059
Scheme 1. Response mechanism of CBRV to OCl−/ONOO− (A) and viscosity (B).
enhancement of CBRV at 571 nm, which allows independent detection
2.2. Synthesis of CBRV and characterization
of viscosity in a channel different from ROS.
CBRV was synthesized according to the route shown in Scheme S1
through four steps [35]. First, a powder of 1,1,2-trimethyl-1H-benzo
[
e] indole (compound 1, 2.1 g, 10 mmol) and bromoethane (1 g,
2
. Experimental section
10 mmol) in acetonitrile (10 mL) were heated under 100 °C to reflux
for 12 h with stirring. After the reaction was completed, the solvent
was evaporated under reduced pressure. The crude product was puri-
2.1. Materials and instruments
2 2
fied by silica gel chromatograghy (CH Cl /MeOH, 100:4, v/v), obtaining
4
-Hydroxymethylphenylboronic acid pinacol ester was purchased
from Beijing innochem Science&Technology Co., Ltd. 1,1,2-Trimethyl-
H-benzo[e]indole and 4-hydroxy-isophthal-aldehyde were obtained
from Shanghai Aladdin Regent Co., Ltd. 3-(4,5-Dimethyl-2-thiazolyl)-
,5-diphenyl-2H-tetrazolium brominde (MTT), lipopolysaccharide
2 as a grey-green solid.
Next, KI (2 mg, 12 mmol) and chlorotrimethylsilane (24 mL) were
added slowly into 4-hydroxymethylphenylboronic acid pinacol ester
(compound 3, 0.8 g, 3.4 mmol) in anhydrous acetonitrile (30 mL) at
0 °C. After stirring at room temperature for 30 min, 10 mL of ethyl ace-
tate was added, and then washed with saturated sodium thiosulfate to
remove the excess potassium iodide. The organic phase was dried
1
2
(
(
LPS), phorbol-12-myristate-13-acetate (PMA), and N-acetylcystine
NAC) were purchased from Sigma-Aldrich. Murine recombinant
interferon-γ (IFN-γ) was obtained from ProSpec-Tany TechnoGene
Ltd. The cell lines (HeLa and RAW264.7), Dulbecco's modified Eagle's
media (DMEM), and RPMI 1640 media were purchased from KeyGEN
Bio TECH Co., Ltd., Nanjing, China. The preparation and/or concentration
2 4
over anhydrous Na SO , evaporated under reduced pressure and sub-
jected to silica gel chromatography (petroleum ether/ethyl acetate,
100:3, v/v), affording 4 as a white solid.
For compound 6 [30], 4-hydroxyisophthalaldehyde (5, 104 mg,
2 3
0.69 mmol) and K CO (236 mg, 2.38 mmol) in anhydrous acetonitrile
(15 mL) were stirred at 0 °C for 10 min. Then, compound 4 (260 mg,
0.76 mmol) was added and the mixture was stirred at room tempera-
ture overnight. The solvent was removed under reduced pressure to
give 6 as a white solid, which was purified by silica gel chromatography
(petroleum ether/ethyl acetate, 100:30, v/v).
Finally, to get our probe, compound 2 (80 mg, 0.2 mmol) and com-
pound 6 (159 mg, 0.5 mmol) were dissolved in acetic anhydride
(10 mL). The reaction mixture was refluxed for 3 h under 60 °C, and
then the solvent was removed under reduced pressure. The resulting
•
determination of tert-butoxy radical (TBO ) and other reactive oxygen
−
−
species (OCl , ONOO , H
2 2
O , •OH, NO, TBHP) were made according to
the literatures [32–34]. The stock solution (1 mM) of CBRV was pre-
pared by dissolving in methanol. Ultrapure water (over 18 MΩ·cm)
from a Milli-Q reference system (Millipore) was used throughout.
UV–vis absorption spectra were recorded by a TU-1900 spectropho-
tometer (Beijing, China) in 1 cm quartz cells and fluorescence spectra
were determined on a Hitachi F-4600 spectrophotometer in 1 × 1 cm
1
quartz cell with both excitation and emission slit widths of 10 nm. H
and 13C NMR spectra were measured on Bruker Fourier-300 spectrom-
eters. High-resolution electrospray ionization mass spectra (HR-ESI-
MS) were performed on an APEX IVFTMS instrument (Bruker
Daltonics). The incubation was carried out in a shaker water bath
2 2
residue was purified by column chromatography on silica gel (CH Cl /
methanol =100: 4, v/v) to afford CBRV as an orange powder (100 mg,
yield 50.1%). The 1H and C NMR spectra of CBRV are shown in
13
1
(
SKY-100C, Shanghai Sukun Industry & Commerce Company). The ab-
Figs. S1 and S2 in the Supporting Information, respectively. H NMR δ
sorbance for MTT analysis was recorded on a microplate reader (BIO-
TEK Synergy HT, USA) at 490 nm. Confocal fluorescence images were
performed on a FV 1200-IX83 Confocal laser scanning microscope
4
(300 MHz, Methanol-d ): 9.23 (s, 1H), 8.81–8.70 (d, 2H, J = 15.0 Hz),
8.47–8.40 (d, 3H, J = 12.0 Hz), 8.28–8.16 (s, 4H), 8.08–8.05 (s, 3H),
8.02–7.90 (d, 5H, J = 18.0 Hz), 7.84–7.59 (s, 5H), 5.51 (s, 2H),
5.00–4.95 (m, 2H, J = 9 Hz), 4.72–4.68 (m, 2H, J = 6 Hz), 2.18 (s, 6H),
2.08 (s, 6H), 1.73–1.68 (t, 3H, J = 6 Hz), 1.51–1.47 (t, 3H, J = 6 Hz),
(
Olympus, Japan). Image process was accomplished with Olympus soft-
ware (FV10-ASW).
2

H. Li, Y. Liu, X. Li et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119059
3. Results and discussion
1
1
1
1
2
.39 (s, 12H). 13C NMR δ (75 MHz, Methanol-d
):182.9, 182.6,
4
62.8152.2, 147.4, 139.3, 138.8, 137.9, 136.6, 135.0, 134.5, 134.1,
34.0, 131.5, 131.4, 130.0, 128.8, 128.3, 128.3, 127.6, 127.4, 127.3,
27.2, 124.0, 122.9, 114.2, 113.7, 112.3, 110.8, 84.0, 54.3, 43.1, 42.8,
5.2, 25.2, 23.9, 23.6, 13.2, 13.0. HR-ESI-MS: m/z calcd for CBRV
3.1. Spectroscopic response of CBRV to ROS
The spectroscopic properties of CBRV are shown in Fig. 1 and
Table S1. From the absorption and fluorescence spectra of CBRV before
2
+
2+
(
55 2 3
C H59BN O , [M] ); 403.230846, found, 403.230902.
−
−
and after reaction with OCl or ONOO , it is seen that CBRV exhibits a
strong absorption at 460 nm (Fig. 1A) and nearly no fluorescence
2.3. Preparation of ROS
(
Fig. 1B). The reaction of CBRV with ROS produced a new absorption
peak at 590 nm and a strong fluorescence emission at 710 nm, similar
to that of the corresponding reaction product CRV. This supports the oc-
currence of the oxidative reaction, and the product CRV was further
confirmed by mass spectral analyses (Fig. S4).
The effects of pH and reaction time on the reaction system were ex-
plored (Fig. S5A), revealing that CBRV functions well in the range of
pH 6.0–9.0. On the other hand, the fluorescence increase of the reaction
solution reached the maximum within 1 min (Fig. S5B). Thus, 5 min of
the reaction time may be selected for this study.
As mentioned above, the preparation and concentration determina-
tion of ROS were made following the methods reported in literature
32–34,37,38], in which •OH was generated from the Fenton reaction
[
2+
2 2
of Fe (10 μM) and H O (100 μM).
2
.4. General procedure for spectral measurements of ROS and viscosity
CBRV was dissolved in methanol to prepare its stock solution of
mM. Unless otherwise noted, all the spectral measurements were per-
Under the optimized conditions (reaction at pH 7.4 for 5 min), the
1
−
−
linear relationship between CBRV and OCl /ONOO concentration
was investigated. As shown in Fig. 2A and C, the fluorescence intensity
formed in 20 mM phosphate buffer (pH 7.4) according to the following
procedure. A 3 mL of 20 mM phosphate buffer was added to a test tube,
followed by addition of 30 μL CBRV stock solution (1 mM) and appropri-
−
−
increases with increasing OCl or ONOO , and it is found that the fluo-
−
−
rescence enhancement is directly proportional to the concentration of
ate volume of reactant (OCl , ONOO or other ROS and bioactive spe-
cies) solution. After incubation with shaking at room temperature for
−
−
−
OCl and ONOO in the range of 2–10 μM OCl and 2–16 μM
−
2
ONOO , with a linear equation of ΔF = 44.09 × C (μM) + 118 (R =
.99) and ΔF = 37.80 × C (μM) + 4.4 × 10
5
min, the absorption and fluorescence spectra were measured.
To test the effect of viscosity, 50 μL of the stock solution of CBRV was
−10
2
0
93 (R = 0.98), respec-
tively, where ΔF is the difference of fluorescence intensity of CBRV in
mixed with 5 mL of the methanol/glycerol mixture at different volume
proportions in a test tube. The resulting solutions were shaken for 3 h
and then cooled to room temperature. Afterwards, the absorption and
fluorescence spectra of these solutions were measured at room temper-
ature (25 °C).
−
−
the presence and absence of OCl or ONOO . The detection limits
3S/m, in which S is the standard deviation of blank measurements,
(
n = 11, and m is the slope of the linear equation) are determined to
be 128.8 nM and 150.2 nM, respectively.
Next, the selectivity of CBRV was studied for OCl−/ONOO− over
−
other potential ROS, including H
2
O
2
, •OH, NO, O
2
• , TBHP and TBO•. As
−
2
.5. Cytotoxicity assay
shown in Fig. S6, CBRV shows high selectivity to OCl or ONOO− over
other ROS, except that H exhibits a moderate fluorescence response
at higher concentrations, which, however, is a much slower reaction
and lower intensity compared with that of OCl or ONOO under the
same optimal conditions (Fig. S5B). It is also noted that H and •OH
2 2
O
The cytotoxicity of CBRV to HeLa cells was examined by standard
MTT assay [39].
−
−
2 2
O
produced similar signals (Fig. S6), which may be due to that the Fenton
reaction is relatively slow under these conditions, and CBRV was mostly
2.6. Cell culture
2 2
oxidized by H O . Moreover, the response of CBRV to inorganic salts and
RAW 264.7 cells (or HeLa cells) were cultured in RPMI 1640 media
other bioactive substances, such as amino acids, and glucose, were also
examined. As depicted in Fig. S7, the fluorescence of CBRV does not
change significantly with the addition of these species. The above
results.
(
DMEM) supplemented with 10% heat-inactivated new-born calf
serum (fetal calf serum, FBS) in a humidified atmosphere of 95% air
and 5% CO at 37 °C.
2
indicated that CBRV showed relatively high selectivity for OCl−/
−
ONOO , which may be ascribed to the specific oxidation of CBRV by
2
.7. Imaging endogenous ROS in cells
−
−
OCl /ONOO . In order to further investigate the selectivity of CBRV,
the fluorescence of the oxidative product CRV was also examined in
the presence of different ROS, inorganic salts and other bioactive sub-
stances. The results in Figs. S8 and S9 indicated that these interferences
hardly caused fluorescence changes of CRV.
Cells (HeLa or RAW 264.7) were planted in glass bottom dishes,
grown for 24 h and then treated with the stimulator (PMA or LPS/IFN-
γ) for appropriate time. After that the cells were washed and incubated
with 10 μM CBRV for 30 min, and fluorescence imaging was conducted
with a 635 nm excitation source and emissions were collected from 670
to 750 nm. For elimination of ROS, HeLa cells were pretreated with PMA
10 nM) for 6 h, and RAW 264.7 cells were pretreated with LPS
1 μg/mL)/IFN-γ (100 ng/mL) for 15 h; then they were incubated for
h in the presence of NAC (1 mM), and finally washed by serum-free
media and loaded with CBRV for 30 min before imaging.
Further, the cytotoxicity of CBRV toward HeLa cells was studied by
the MTT assay to test whether CBRV can be used for intracellular imag-
ing. As shown in Fig. S10, CBRV at a high concentration of no more than
(
(
1
1
0 μM does not significantly damage the live cells, implying an excellent
biocompatibility of CBRV. Furthermore, the fluorescence confocal imag-
ing results (Fig. S11) suggested that CRV had good cellular permeability.
3.2. Response of CBRV to viscosity
2.8. Imaging endogenous viscosity in HeLa cells
Next, a series of tests on the fluorescence response of CBRV to differ-
HeLa cells were planted in glass bottom dishes, grown for 24 h and
then treated with apoptotic agent (etoposide) for appropriate time.
After that the cells were washed and incubated with 10 μM CBRV for
0 min for fluorescence imaging.
ent viscosities were made by mixing methanol/glycerol at different pro-
portions. From Fig. 3A, it is seen that the emission maximum is at about
571 nm, and as the viscosity gradually increases from 0.59 cP (in pure
methanol) to 945.35 cP (in pure glycerol) [22], the fluorescence is
3
3

H. Li, Y. Liu, X. Li et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119059
(A)
(B)
0
0
0
0
0
0
.5
.4
.3
.2
.1
.0
600
b
d
a
c
450
b
d
c
300
150
a
0
4
00
500
600
700
800
700
750
800
850
Wavelength (nm)
Wavelength (nm)
Fig. 1. (A) Absorption spectra of CBRV (10 μM) in phosphate buffer (pH 7.4) before (a) and after reaction with 10 μM of ONOO (b) or OCl− (c) for 5 min. (B) Fluorescence spectra of CBRV
−
(
10 μM) in phosphate buffer (pH 7.4) before (a) and after reaction with 10 μM of ONOO− (b) or OCl− (c) for 5 min. λex = 485 nm. The absorption and fluorescence spectra of CRV (10 μM)
were given as curve (d) in Panels A and B, respectively.
enhanced largely, accompanied by a slight hypsochromic shift of the
maximum wavelength. Furthermore, a good linearity is obtained be-
tween the fluorescence change and viscosity, which follows the
Forster-Hoffmann equation [40,41] as Log ΔF = x log η + c (where ΔF
stands for the fluorescence change; η is the viscosity of solvent; x is
probe-dependent constant and c is the constant related to CBRV con-
centration and temperature) with the x and c are 0.79 and 0.17 respec-
tively. It should be pointed out that the correlation coefficient of the
linear relationship is as high as 0.99, which indicates that CBRV may
be of capable of monitoring the changes of viscosity.
In addition, pH and polarity of the solvent, which may often affect
fluorescence response, were studied in detail. As shown in Fig. S12A,
the fluorescence of CBRV is not much affected in the range of
pH 6.0–7.4. In addition, the polarity effect of the solvent with different
dielectric constants were also small (Fig. S12B). It should be noted
that dichloromethane caused somewhat fluorescence response, which
Fig. 2. Fluorescence responses of CBRV (10 μM) toward (A) OCl and (C) ONOO− at varied concentrations (0–20 μM) in phosphate buffer (pH 7.4). Linear fitting curves of ΔF against the
−
concentration of (B) OCl− and (D) ONOO−. λex/λem = 485/710 nm.
4

H. Li, Y. Liu, X. Li et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119059
(B)
(A)
3
2
1
400
300
200
100
0
9
45.35 cP
0.59 cP
0
.7
1.4
2.1
2.8
3.5
550
600
650
700
750
Wavelength (nm)
log
Fig. 3. (A) Fluorescence emission spectra of 10 μM CBRV in the mixture of varied ratios of methanol/glycerol (v/v) (from bottom to top): methanol only; 8:2; 7:3; 6:4; 5:5; 4:6; 3:7; 2:8; 1:9
and glycerol only. (B) The linearity between fluorescence change and viscosity. λex/em = 485/571 nm.
however is still smaller than 10% error in the fluorescence signal and
could be neglected. Furthermore, the reversibility of CBRV in response
to viscosity was examined. As shown in Fig. S13, CBRV exhibits good re-
versible fluorescence response to the variation of viscosity between
the other hand, to check the possible influence of CRV, we measured
the fluorescence change of CBRV by introducing CRV. The result
(Fig. S15) showed that there was no significant interference from CRV.
These observations indicated that our probe CBRV may serve as a
dual-functional probe for the assay of ROS and viscosity.
107 cP (methanol/glycerol, v/v = 6:4) and 26.4 cP (methanol/glycerol,
v/v = 6:4). Thus, CBRV is suitable for monitoring the viscosity changes.
Besides, we also tested the response of CRV to viscosity at 571 nm (vis-
cosity channel) and 710 nm (ROS channel). As depicted in Fig. S14, CRV
fluorescence is not sensitive to viscosity at 710 nm, meaning that the
change of viscosity does not interfere with the detection of ROS. On
3
.3. Fluorescence imaging of ROS in cells
CBRV is a lipophilic cation, which implies that it might accumulate in
cell mitochondria. To verify this possibility, a co-localization experiment
a
c
d
e
b
f
8
6
4
2
0
g
a
b
c
d
e
f
Fig. 4. Confocal fluorescence images of HeLa cells under different conditions. (a) Cells only. (b) Cells treated with CBRV (10 μM) for 30 min. (c–e) Cells pretreated with PMA (10 nM) for
c) 2 h, (d) 4 h and (e) 6 h, and then incubated with CBRV (10 μM) for 30 min. (f) Cells pretreated with PMA (10 nM) for 6 h, then incubated with NAC (1 mM) for 1 h, and finally incubated
(
with CBRV (10 μM) for 30 min. The second row shows the corresponding differential interference contrast (DIC) images. λex = 635 nm, λem = 670–750 nm. Scale bar: 10 μm. (g) Relative
pixel intensity of fluorescence images a–f (the pixel intensity from image b is defined as 1.0). The results are presented as mean ± standard deviation (n = 3).
5

H. Li, Y. Liu, X. Li et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119059
was performed. As shown in Fig. S16, the fluorescence from the Mito-
red channel overlaps poorly with that from the CBRV channel, with a
low Pearson's coefficient of 0.54, indicating that CBRV can be distributed
in both cytosol and mitochondria of HeLa cells.
Next, to test whether CBRV could be used to detect the endogenous
ROS change in cells, HeLa and RAW267.4 cells were selected as the can-
cer and macrophage model cells, respectively. HeLa cells were stimu-
lated by PMA (10 nM) for different time, whereas RAW267.4 cells
10 μM etoposide (an apoptotic agent) for different periods of time. As
shown in Fig. 5, the fluorescence intensity (viscosity channel) of HeLa
cells increases with the extension of time, accompanying an obvious
contraction in terms of the contour morphology of cells, suggesting an
increased degree of apoptosis, which may increase the viscosity of
cells. This is consistent with our previous finding [26]. All these results
indicate that CBRV could be applied to monitoring the viscosity change
by fluorescence imaging.
[
42,43] were stimulated by LPS (1 μg/mL) and INF-γ(100 ng/mL) for
1
3
5 h; and then they were incubated with CBRV (10 μM) for another
0 min before subjected to fluorescence measurement. As shown in
4
. Conclusion
Fig. 4, the PMA-stimulated HeLa cells display gradually increased fluo-
rescence with the extension of incubation time. Moreover, this en-
hancement can be greatly inhibited by NAC [44,45], suggesting the
generation of ROS. Interestingly, similar results were observed in the
PMA-stimulated RAW267.4 cells (Fig. S17). These data suggest that
CBRV can be used for monitoring the production of the endogenous
ROS in living cells.
In summary, we have developed CBRV as a new dual-functional
fluorescent probe by conjugating a cyanine skeleton with a benzene bo-
ronic acid ester moiety. The probe exhibits high sensitivity and selectiv-
−
−
ity for OCl /ONOO over other ROS, and good dual responses to both
−
−
viscosity at around 571 nm and OCl /ONOO at the NIR region of
10 nm. In addition, CBRV has been used to monitor the change of
7
total ROS levels as well as the viscosity in living cells. The good analytical
−
performance of CBRV may enable it to be applied to detection of OCl /
−
ONOO or viscosity in more biological samples.
3.4. Fluorescence imaging of viscosity in cells
Next, CBRV was attempted to measure the viscosity change in cells
CRediT authorship contribution statement
during apoptosis. Herein, HeLa cells with relatively large and well-
defined morphology were chosen as the model cells, as well, which
were incubated with CBRV (10 μM) for 30 min and then treated with
Hongyu Li: Methodology, Visualization, Investigation. Ya Liu: Writ-
ing - original draft, testing.
1
2
9
6
3
0
f
a
b
c
d
e
Fig. 5. Confocal fluorescence imaging of HeLa cells under different conditions. (a) Cells only. (b) Cells treated with CBRV (10 μM) for 30 min. Cells firstly treated with 10 μM etoposide for
c) 15 min, (d) 30 min, and (e) 45 min, respectively, and then incubated with CBRV for 30 min before imaging. The second row shows the corresponding DIC images. λex = 488 nm, λem
40–640 nm. Scale bar: 10 μm. (f) Relative pixel intensity of fluorescence images a–e (the pixel intensity from image b is defined as 1.0). The results are present as mean ± standard
deviation (n = 3).
(
=
5
6

H. Li, Y. Liu, X. Li et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 119059
[
[
[
[
19] X. Wang, F.L. Song, X.J. Peng, A versatile fluorescent probe for imaging viscosity and
Xiaoyi Li: Testing and investigation. Xiaohua Li: Review & editing.
Huimin Ma: Supervision, review & editing.
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgment
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.saa.2020.119059.
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