A novel endoplasmic reticulum-targeted ratiometric fluorescent probe based on FRET for the detection of SO2 derivatives

Article abstract of DOI:10.1016/j.dyepig.2021.109180

A novel FRET-based fluorescence probe, NBIS, constructed by benzoindole-based hemicyanine as an acceptor and naphthalimide derivatives as a donor was developed for the detecting of sulfur dioxide (SO₂) derivatives. Probe NBIS exhibited high energy transfer efficiency (89%) and showed high sensitivity (LOD: 16.2 nM) toward HSO₃^?/SO₃^2?. Probe NBIS could target the endoplasmic reticulum (ER) and was applied to image exogenous and endogenous HSO₃^?/SO₃^2? in living cells.

Full text of DOI:10.1016/j.dyepig.2021.109180

Dyes and Pigments 188 (2021) 109180
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: http://www.elsevier.com/locate/dyepig
A novel endoplasmic reticulum-targeted ratiometric fluorescent probe
based on FRET for the detection of SO2 derivatives
Zhang-Yi Lia, Xiao-Ling Cuib, Ye-Hao Yana, Qiao-Ling Chea, Jun-Ying Miaob,
,
,**
Bao-Xiang Zhaoa *, Zhao-Min Linc
a Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan, 250100, PR China
b Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Science, Shandong University, Qingdao, 266237, PR China
c Institute of Medical Science, The Second Hospital of Shandong University, Jinan, 250033, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A novel FRET-based fluorescence probe, NBIS, constructed by benzoindole-based hemicyanine as an acceptor
and naphthalimide derivatives as a donor was developed for the detecting of sulfur dioxide (SO2) derivatives.
Probe NBIS exhibited high energy transfer efficiency (89%) and showed high sensitivity (LOD: 16.2 nM) toward
HSOꢀ3 /SO23ꢀ . Probe NBIS could target the endoplasmic reticulum (ER) and was applied to image exogenous and
endogenous HSOꢀ3 /SO23ꢀ in living cells.
Ratiometric fluorescence probe
FRET
SO2 derivatives
Cell image
ER-Targeted
1. Introduction
advantages such as low cost, rapidity, high selectivity and sensitivity,
non-invasive detection, good bio-compatibility as well as real-time
It is known that continuous exposure to air pollution, in which sulfur
dioxide (SO2) is a main composition, might enhance chance of suffering
cardiovascular and lung diseases. On the other hand, SO2 derivatives are
among the most widely utilized preservatives in the food and beverages
industries, such as winemaking, and one of their main functions is to
inhibit microorganisms. Under physiological conditions, SO2 can
dissolve in water and form its derivatives, bisulfite and sulfite [1,2].
There are many analytical methods for the assay of SO2 preserving
agents including titrimetric, spectrometric, chromatographic, electro-
chemical (voltammetric, amperometric, potentiometric) [3]. To date,
the endogenous nature and possible physiological roles of SO2 has
gained attention. There is mounting evidence that SO2 can be generated
during normal cellular metabolism and may possibly function as a
signaling molecule in normal physiology. For example, a complete
endogenous SO2 pathway was detected in the cardiovascular system and
found to play an important role in cardiovascular physiology and
pathophysiology [4]. Endogenous SO2 could regulate hippocampal
neuron apoptosis in developing epileptic rats [5]. Therefore, it is
important to develop methods for detecting exogenous and endogenous
SO2 (HSOꢀ3 /SO23ꢀ ). Compared with traditional detection methods,
fluorescent probe assay is more practical because of excellent
detectability. In the last decade, fluorescent probes for the detection of
SO2 derivatives have been developed rapidly. Many fluorescent probes
based on different sensing mechanisms including Michael addition,
deprotection reaction, nucleophilic reaction with aldehyde have been
designed and used to the detection of SO2 derivatives [6–8]. Fluorescent
probes typically fall into two categories, those which rely upon a single
emission signal and those which employ a ratiometric. Because ratio-
metric fluorescence probes that allow measurement of the fluorescence
emission intensities at two different wavelengths can eliminate inter-
ference from, for example, environmental effects, and the concentration
of the probe by self-calibration, ratiometric fluorescence probes are
highly desirable. So far, a number of ratiometric fluorescence probes
based on different response mechanism, such as the intramolecular
charge transfer (ICT), photoinduced electron transfer (PET),
excited-state intramolecular proton transfer (ESIPT), through bond en-
ergy transfer (TBET) and fluorescence resonance energy transfer (FRET)
have been reported to sense a wide variety of analytes, such as pH
[9–11], HSOꢀ3 [12–19], peroxynitrite [20], thiol [21–23], hypochlorite
[24,25], Fe3+ [26], Ca2+ [27], β-Galactosidase [28], hydrogen sulfide
(H2S) [29,30], Among them, FRET-based ratiometric fluorescence
probes are very popular [31–37]. However, some deficiencies require
* Corresponding author.
** Corresponding author.
E-mail addresses: bxzhao@sdu.edu.cn (B.-X. Zhao), linzhaomin@sdu.edu.cn (Z.-M. Lin).
https://doi.org/10.1016/j.dyepig.2021.109180
Received 12 December 2020; Received in revised form 21 January 2021; Accepted 21 January 2021
Available online 30 January 2021
0143-7208/© 2021 Elsevier Ltd. All rights reserved.

Z.-Y. Li et al.
Dyes and Pigments 188 (2021) 109180
Scheme 1. Sensing mechanism of probe NBIS toward HSOꢀ3 /SO32-
Scheme 2. Synthesis of probe NBIS.
improvement, such as response time, sensitivity, energy transfer effi-
ciency [38], the distance between two emission bands [39], excitation
wavelength [40]. Therefore, it is imperative to develop new FRET
platforms. Notably, over the past decade, the design and development of
organelle-targeted fluorescent probes have been attractive for precise
monitoring of subcellular microenvironments. Though many probes
with diverse targeting groups have been used to detect bioactive species,
such as reactive oxygen species, reactive nitrogen species, reactive sulfur
species, in specific organelles, endoplasmic reticulum (ER)-targeted
fluorescence probes are very rare and the ER-targeted intrinsic molec-
ular mechanism remains unclear [41]. To the best of our knowledge,
ER-targeted fluorescent probe for monitoring of SO2 derivatives has not
been reported yet.
2. Experimental section
2.1. Apparatus and material
1H NMR and 13C NMR spectra were determined by Bruker Avance
300 spectrometer. Mass spectra were recorded by a Q-TOF6510 spec-
trograph (Agilent). IR spectra were obtained using IR spectrophotometer
VERTEX 70 FT-IR (Bruker Optics). UV–Vis absorption spectra were ob-
tained by a Cary 5000 spectrophotometer (Agilent) and fluorescence
spectra were measured on a LS-55 fluorescence spectrophotometer
(PerkinElmer). The fluorescence lifetime was performed on an Edin-
burgh Instruments FLS920 Fluorescence Spectrometer. All pH of solu-
tions were obtained by a PHS-3C pH-meter (YouKe). Thin-layer
chromatography (TLC) was conducted on silica gel 60F254 plates (Merck
KGaA) and column chromatography was carried out over silica gel
(200–300 mesh). Unless otherwise stated, all reagents and solvents were
As a continuation of our efforts to develop new fluorescent probes,
herein, we report a novel ER-targeted ratiometric fluorescent probe
based on FRET for the detecting of SO2 derivatives (Scheme 1).
2

Z.-Y. Li et al.
Dyes and Pigments 188 (2021) 109180
Fig. 1. (a) Probe NBIS (5
μ
M) responded toward HSOꢀ3 /SO32ꢀ (0–2.0 equiv.). (b) The linear relationship between the ratio values of fluorescence intensity (I534/I610).
Data are mean ± SD (n = 3). λex = 440 nm, slit: 7.5/7.5 nm, speed: 1200 nm/min.
purchased from commercial provider. Twice-distilled water was used
throughout all experiments.
2.4. Calculation of energy transfer efficiency (ETE)
ETE was calculated by following equation:
2.2. Synthesis of probe NBIS
ηETE = 1-FDA/FD
As shown in Scheme 2, the mixture of compound 3 (523 mg, 1.0
mmol), compound 5 (387 mg, 1.0 mmol), 4-dimethylaminopyridine (15
mg, 0.12 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (191 mg, 1.0 mmol) in dichloromethane (140 mL) was
stirred at room temperature for 18 h. The solvent was removed under
reduced pressure and then the residue was purified by column chro-
matography on silica gel to obtain probe NBIS (408 mg, dark violet
solid) in 46% yield, which was characterized by NMR, HRMS and IR
(Figs. S1–4). m. p. = 224–226 ◦C. 1H NMR (300 MHz, CDCl3) δ 8.66 (d, J
= 8.4 Hz, 1H), 8.59–8.57 (m, 1H), 8.45–8.41 (m, 1H), 8.26–8.17 (m,
4H), 8.08–8.01 (m, 2H), 7.73–7.68 (m, 2H), 7.63–7.54 (m, 5H),
7.42–7.39 (m, 2H), 6.97 (d, J = 3.9 Hz, 2H), 6.86–6.83 (m, 1H), 4.42 (d,
J = 4.5 Hz, 3H), 3.82–3.73 (m, 8H), 3.56–3.48 (m, 4H), 2.13 (m, 4H),
2.07 (s, 6H). 13CNMR (75 MHz, DMSO‑d6) δ 181.72, 169.30, 164.45,
163.42, 154.35, 153.08, 152.94, 140.03, 138.17, 137.18, 135.60,
133.98, 133.47, 133.17, 131.59, 131.22, 131.10, 130.46, 129.92,
128.69, 128.06, 127.28, 126.97, 124.39, 123.71, 123.31, 122.37,
114.16, 113.33, 109.42, 108.99, 107.10, 56.49, 55.37, 53.40, 53.37,
46.62, 34.50, 26.26, 26.04, 19.01. IR (KBr, cmꢀ 1): 3443, 2925, 2858,
1637, 1577, 1523, 1458, 1398, 1361, 1299, 1238, 1183, 1124, 1087.
HRMS (ESI): m/z found 764.3530, calculated for [C50H46O3N5]+:
764.3595.
Where, FDA and FD denote the maximum of fluorescence intensity with
and without an acceptor, respectively.
3. Results and discussion
3.1. Design of fluorescent probe NBIS
The energy transfer efficiency of FRET probes is highly dependent on
the substantial overlap of the donor emission spectrum with the acceptor
absorption spectrum. In addition, two well-separated emission bands
with comparable intensities can ensure detection accuracy. However,
because the larger the spectral overlap, the shorter the emission shifts of
FRET systems, to reconcile the two parameters is difficult. Therefore, it
is still a current challenge to construct new FRET pairs.
Naphthalimide derivatives are classical fluorophores and widely
used to construct fluorescent probes including single emission or ratio-
metric signal. Due to the maximum emission of naphthalimide fluo-
rophore is around 560 nm, it can be used as both donor and acceptor in
the different FRET pairs, depending on the spectra of a second compo-
nent [20,28,29,32]. Hemicyanine dyes are widely used in fluorescent
probes based on ICT mechanism, because of its good solubility in water,
high molar extinction coefficients and lower cell toxicity. As a part of
ICT system, benzoindole moiety, generally in the cation form, due to the
excellent electron-withdrawing ability, is often used [27,42–44]. Ac-
cording to the spectral property of hemicyanine dyes, they should be
suitable as the acceptor for the construction of FRET platform. However,
benzoindole-based hemicyanine dyes for FRET platform are still rare
[45]. Therefore, in present work, a new FRET platform is constructed
(named as NBIS), in which naphthalimide derivatives is selected as the
donor and benzoindole-based hemicyanine is selected as the acceptor
(Scheme 1). When probe NBIS in a buffer solution (DMSO/PBS = 3:7,
pH = 7.4) was excited at 440 nm, the maximum emission centered 610
nm from acceptor moiety was observed because FRET process occurred.
However, after the probe reacted with HSOꢀ3 /SO32ꢀ , by a nucleophilic
2.3. Preparation of testing solution and cells culturing
A stock solution of probe NBIS (1 mM) was prepared in DMSO. The
stock solutions of other analytes were prepared by using distilled water.
50 μL stock solution of probe NBIS was transferred into volumetric flask
and diluted to 10 mL by the buffer solution (DMSO:PBS = 3:7, v/v, pH =
7.4) at room temperature. Then, various equivalents of HSOꢀ3 /SO32ꢀ or
other analytes was added.
Henrietta Lacks cells (HeLa cells), human liver cancer cells (HepG2
cells), human normal liver cells (L-O2 cells) were cultured with DMEM
(Dulbecco’s modified Eagle medium) which contains supplement of
10% FBS (Fetal Bovine Serum). Human Dermal fibroblasts (HDFs) were
cultured in DMEM Basic medium supplemented with 10% (v/v) bovine
calf serum. All cell lines were cultured in a humidified incubator at 37 ◦C
under 5% CO2 atmosphere. Images were acquired using a confocal mi-
croscope (Zeiss LSM900).
–
addition at C C double bond in acceptor moiety, ICT process in the
–
acceptor moiety was destroyed and FRET could no longer operate,
leading to the donor emission at 534 nm was recorded. The visible light
excitation (440 nm) and the spacing between two emission peaks (76
nm) could meet the requirements for the ratiometric detection.
3

Z.-Y. Li et al.
Dyes and Pigments 188 (2021) 109180
3.2. Synthesis of probe NBIS
As shown in Scheme 2, compound 1 (1,1,2,3-tetramethyl-1H-benzo
[e]indol-3-ium iodide) was easily synthesized from commercial 1,1,2-
trimethyl-1H-benzo [e]indole by methylation with methyl iodide.
Compound 1 condensed with 4-(piperazin-1-yl)benzaldehyde (2) to
afford (E)-1,1,3-trimethyl-2-(4-(piperazin-1-yl)styryl)-1H-benzo [e]
indol-3-ium iodide (compound 3). The reaction of compound 4 and
pyrrolidine gave 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo [de]iso-
quinolin-2(3H)-yl)benzoic acid (compound 5). Finally, compound 3
reacted with compound 5 to furnish probe NBIS (compound 6), (E)-2-(4-
(4-(4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo [de]isoquinolin-2(3H)-yl)
benzoyl)piperazin-1-yl)styryl)-1,1,3-trimethyl-1H-benzo [e]indol-3-ium
iodide.
3.3. Fluorescence and UV–Vis absorption spectral of probe NBIS
After a series of tests for a suitable solvent, a buffer solution (DMSO/
PBS = 3:7, pH = 7.4) was chosen for all spectral property measurements.
Probe NBIS alone in the buffer solution displayed a strong fluorescence
band centered at 610 nm when it was excited with 440 nm light due to
FRET process. The fluorescence quantum yield is Φ = 0.011 (quinine
sulfate as standard). The addition of HSOꢀ3 /SO32ꢀ led to a new fluores-
cence band centered at 534 nm (Φ = 0.006, quinine sulfate as standard).
It can be observed that fluorescence intensity at 610 nm abated gradu-
ally and the fluorescence intensity at 534 nm increased as the dosage of
HSOꢀ3 /SO23ꢀ increased (Fig. 1 (a)). The phenomena fitted a typical FRET
character. The fluorescence lifetime was measured and the results were
shown in Fig. S5 and Table S1. The average fluorescence lifetime of
donor moiety in the probe at 534 nm was shorter than that of the donor
only, which confirmed the FRET process [46]. A preferable linearity
between the fluorescence intensity ratios (I534/I610) and the concentra-
tions of HSOꢀ3 /SO23ꢀ from 0 to 0.9 equiv. Was obtained (Fig. 1 (b)). The
limit of detection was calculated to be 16.2 nM, which is superior to
previous reports (Table S2). The good sensitivity offers potential for the
sensing of intracellular SO2 derivatives. Similarly, the absorption spec-
trum showed that the absorbance at the longer wavelength decreased
gradually and the absorbance in shorter wavelength changed little when
added HSOꢀ3 /SO23ꢀ in the range of 0–1.6 equiv. (Fig. S6). The addition
reaction product of probe NBIS with HSOꢀ3 /SO32ꢀ was verified by HRMS
data (Fig. S7), in which the peak appeared at m/z 846.3257 was
attributed to the addition product (calculated m/z [M+H]+: 846.3320).
Notably, the fluorescence emission spectrum of the donor could effi-
ciently overlap with the absorption spectrum of the acceptor (Fig. S8).
The energy transfer efficiency of the probe was calculated to be 89%
(Fig. S9). Moreover, two emission peaks separated obviously with a 76
nm emission shift.
Fig. 2. Fluorescence and bright field images of exogenous HSOꢀ3 /SO32ꢀ in HeLa
cells. (a) HeLa cells were pre-incubated with probe NBIS (5
μ
M) for 1 h, fol-
lowed by NaHSO3 (0, 50, 100, 250, 500 and 1000
μM) for another 1 h. Scale
bar: 20 μm. (b) The relative fluorescence intensity ratio of green/red. (λex =
405 nm; green channel: 405–580 nm; red channel: 580–700 nm; data are
presented as the mean ± SEM, *p < 0.05, **p < 0.01, n = 3).
compared with that in the absence of other species (Fig. S10). Thus,
probe NBIS showed high selectivity and good anti-interference ability
for HSOꢀ3 /SO23ꢀ , suggesting the applicability of the probe to detection of
HSOꢀ3 /SO23ꢀ in complex environments.
3.5. Effect of pH and time on NBIS toward HSOꢀ3 /SO32ꢀ
The properties of fluorescence probes depend strongly on solvent
system and its pH because the reaction rate and equilibrium of nucleo-
philic addition is changed in the different solvent and pH. Therefore, in
the condition of a selected solvent system, the effect of pH and time on
NBIS toward HSOꢀ3 /SO32ꢀ was determined. The fluorescence intensity
ratios (I534/I610) of the probe was stable over a wide pH range (pH =
4–8). In addition, in the presence of HSOꢀ3 /SO32ꢀ , the fluorescence in-
tensity ratios (I534/I610) of the reaction solution increased with increases
in pH from 5 to 7, however, no obvious change was observed in the
range of pH 7–8 (Fig. S11), which should be suitable for bioimaging in
living cells.
3.4. Selectivity of probe NBIS toward HSOꢀ3 /SO32ꢀ by fluorescence
spectrometry
The selectivity of a fluorescent probe toward analyte is one of the
most important parameters. Therefore, the selectivity of probe NBIS
toward HSOꢀ3 /SO23ꢀ and other species (Clꢀ , Brꢀ , Iꢀ , AcOꢀ , NO2ꢀ , NOꢀ3 ,
SCNꢀ , S2O32ꢀ , HCO3ꢀ , SO24ꢀ , HSꢀ , Cys, GSH, Hcy) was evaluated in the
buffer solution. The fluorescence intensity ratio of the probe has few
changes in the presence of Clꢀ , Brꢀ , Iꢀ , AcOꢀ , NOꢀ2 , NO3ꢀ , SCNꢀ , S2O23ꢀ
,
The response time of the probe toward HSOꢀ3 /SO32ꢀ was recorded
(Fig. S12). At room temperature, the reaction of the probe with HSOꢀ3 /
SO23ꢀ proceeded in the buffer solution leading to the fluorescence in-
tensity ratios (I534/I610) gradually increasing and no longer change after
12 min. The response of the probe toward HSOꢀ3 /SO32ꢀ was in the better
category (Table S2), which could meet well the requirement of real-time
detection.
HCOꢀ3 , SO24ꢀ , HSꢀ , Cys, GSH, Hcy, respectively. Moreover, the ratio
(I534/I610) value dramatically increases 7.4-fold in the presence of
HSOꢀ3 /SO23ꢀ . The results indicate a high sensitivity of the probe toward
HSOꢀ3 /SO23ꢀ (Fig. S10). To appraise whether the probe can be preferable
for the detection of HSOꢀ3 /SO32ꢀ in the presence of other species (Clꢀ ,
Brꢀ , Iꢀ , AcOꢀ , NO2ꢀ , NO3ꢀ , SCNꢀ , S2O23ꢀ , HCOꢀ3 , SO24ꢀ , HSꢀ , Cys, GSH,
Hcy), the fluorescence spectra of the probe toward HSOꢀ3 /SO32ꢀ (2
equiv.) in the presence of various species (2 equiv.), respectively, were
measured and the fluorescence intensity ratios were almost not changed
4

Z.-Y. Li et al.
Dyes and Pigments 188 (2021) 109180
Fig. 3. (a) The first column: HepG2 cells were treated with probe NBIS (5
μ
M)
M)
for 1 h; The second column: HepG2 cells were pre-treated with GSH (500
μ
and Na2S2O3 (250
μ
M) for 1 h before incubated with probe NBIS (5
μ
M) for
another 1 h; The third column: HepG2 cells were pre-cultured with GSH (500
M) for 1 h, and then cultured with probe NBIS (5 M) for 1 h; The fourth
column: HepG2 cells were treated for 1 h with GSH (500 M) and Na2S2O3
(250 M) after pre-treatment with TNBS (10 mM) for 20 min, followed by probe
NBIS (5
μ
μ
μ
Fig. 4. (a) L-O2 cells were incubated with probe NBIS (5
GSH (500 M) and Na2S2O3 (250 M) or NaHSO3 (100 and 500
1 h. (b) The relative ratio (Green/Red) of fluorescence intensity of column 1, 2,
μ
M) for 1 h, then with
μ
μ
μ
μ
M) for another
μ
M) for 1 h. Scale bar: 20 μm. (b) The fluorescence ratio of Green/Red
of column 1, 2, 3 and 4 in (a). (λex = 405 nm; green channel: 405–580 nm; red
channel: 580–700 nm; data are presented as the mean ± SEM, ***p < 0.001, n
= 3).
3 and 4 in (a). Scale bar: 20 μm. (λex = 405 nm; green channel: 405–580 nm; red
channel: 580–700 nm; data are presented as the mean ± SEM, **p < 0.01, ***p
< 0.001, n = 3).
3.6. Cell imaging of NBIS
bioimaging application.
The real time imaging of exogenous HSOꢀ3 /SO32ꢀ in HeLa cells was
investigated by the use of probe NBIS. HeLa cells were incubated with
After fluorescence properties of probe NBIS in the buffer solution for
HSOꢀ3 /SO23ꢀ was evaluated, the real-time detection of intracellular
HSOꢀ3 /SO23ꢀ was further achieved. According to SRB assay [47], the
cytotoxicity of probe NBIS was primarily evaluated. The results revealed
that the probe had no obvious cytotoxicity in HeLa cells (Fig. S13). The
photostability of a probe in living cells is another crucial factor.
Therefore, the photostability of probe NBIS in HeLa cells was put to the
test, and the results revealed that the probe was photostable within 3
min (Fig. S14). The results showed that probe NBIS was suitable for
probe NBIS (5
μ
M) for 1 h and treated with exogenous HSOꢀ3 /SO32ꢀ (0,
50, 100, 250, 500 and 1000 μM) respectively for 1 h. The imaging
showed that the red fluorescence in 580–700 nm channel decreased
gradually and the green fluorescence in 405–580 nm channel enhanced
obviously as the increment of HSOꢀ3 /SO32ꢀ (Fig. 2). The fluorescence
intensity ratio (green/red) enlarged clearly along with the concentration
increase of HSOꢀ3 /SO32ꢀ , and even 6-fold enhancements (Fig. 2).
Therefore, probe NBIS could realize real time and ratiometric detection
5

Z.-Y. Li et al.
Dyes and Pigments 188 (2021) 109180
Fig. 5. HeLa cells, HDFs, HepG2 cells and L-O2 cells
were incubated with probe NBIS (5 μM) for 1 h, af-
terward with ER-Tracker Red (1:200) for 30 min. a1-
4: Fluorescence images of HeLa cells (a1), HDFs (a2),
HepG2 cells (a3) and L-O2 cells (a4) with probe NBIS
from the green channel (λex = 405 nm; green channel:
405–580 nm). b1-4: Fluorescence images of HeLa
cells (b1), HDFs (b2), HepG2 cells (b3) and L-O2 cells
(b4) with ER-Tracker Red channel (λex = 639 nm; red
channel: 640–700 nm). c1-4: Merged images with
(a1-4) and (b1-4). d1-4: Co-localization images of
HeLa cells (d1) (MCC = 0.97), HDFs (d2) (MCC =
0.93), HepG2 cells (d3) (MCC = 0.96) and L-O2 cells
(d4) (MCC = 0.97). Scale bar: 20 μm.
of HSOꢀ3 /SO23ꢀ in living cells.
monitoring of HSOꢀ3 /SO32ꢀ was discovered unexpectedly (Fig. 5). The
endoplasmic reticulum (ER) is an important site for protein and lipid
synthesis, calcium homeostasis and detoxification of poisonous sub-
stances [50]. It is reported that SO2 preconditioning has a protective
effect on rat myocardial ischemia/reperfusion injury and this process
involves endoplasmic reticulum stress (ERS) [51]. SO2 derivatives in
liver tissue increased xanthine oxidase activity, induced ER stress and
caused caspase activation [52]. Therefore, ER-targeted fluorescent
probe NBIS could be useful for exploring physiological and pathological
functions of SO2 derivatives.
The ratiometric imaging of endogenous HSOꢀ3 /SO32ꢀ in living HepG2
cells was fulfilled by probe NBIS. It is well known that endogenous SO2
derivatives in mammalian liver cells could generate through thiosulfate
sulfurtransferase (TST) catalyzed reaction of thiosulfate (Na2S2O3) and
glutathione (GSH). Thus, HepG2 cells were firstly pre-cultured with
probe NBIS (5
Na2S2O3 (250
μ
M) for 1 h, and incubated with GSH (500
μM) and
μ
M) for 1 h, then confocal imaging was conducted. The
results indicated that the cells showed an obvious bright green fluores-
cence and receded red fluorescence (Fig. 3 (a)). As a comparison, no
obvious changes in the fluorescence of HepG2 cells loaded with probe
NBIS and hatched with only GSH were observed. To further verify
endogenous HSOꢀ3 /SO23ꢀ in living HepG2 cells leading the changes in the
fluorescence, 2,4,6-trinitrobenzenesulphonate (TNBS), a common in-
hibitor of the TST enzyme, was used. After HepG2 cells were hatched
with TNBS (10 mM) for 20 min, followed by the treatment with GSH
4. Conclusion
A new FRET-based fluorescent probe was constructed by the union of
a naphthalimide fluorophore and a benzoindole-based hemicyanine
fluorophore. The probe could detect HSOꢀ3 /SO32ꢀ in ratiometric fluo-
rescence manner with good selectivity and sensitivity. The probe could
be excited by visible light and showed a satisfied energy transfer effi-
ciency and clear spacing of two emission peaks. Moreover, the probe
could target effectively the endoplasmic reticulum and was successfully
used to image the endogenous and exogenous HSOꢀ3 /SO32ꢀ in living cells.
(500
μ
M) and Na2S2O3 (250
μM) for 1 h, the cells were incubated with
probe NBIS (5
μ
M) for 1 h. The imaging showed no clear fluorescence
variation. Compared with other test groups, the relative ratio (green/
red) of the fluorescence intensity of HepG2 cells treated with Na2S2O3
and GSH exhibited clear variations and even 5-fold enhancements (Fig. 3
(b)). Therefore, probe NBIS could be successfully applied to image
exogenous and endogenous HSOꢀ3 /SO32ꢀ in living cells.
Credit author statement
Our previous work illustrated that the concentration of the endoge-
nous HSOꢀ3 /SO23ꢀ was higher in liver cancer cells than that in normal
liver cells [48,49]. In the present, for L-O2 cells loaded with probe NBIS
and incubated with GSH and Na2S2O3 for 1 h no obvious changes were
recorded (Fig. 4). Therefore, the level difference of endogenous
HSOꢀ3 /SO23ꢀ from liver cancer cells and normal liver cells was further
verified by probe NBIS.
Zhang-Yi Li: Writing – original draft, Writing – review & editing,
Synthesis and spectral property test of the probe. Xiao-Ling Cui: Cell
imaging assay. Ye-Hao Yan: Spectral property test of the probe. Qiao-
Ling Che: Synthesis of the probe. Jun-Ying Miao: Supervision. Bao-Xiang
Zhao: Design of the probe, Supervision. Zhao-Min Lin: Cell imaging
assay, Supervision.
Organelle-targeted fluorescent probes for bioactive species imaging
are of great significance to research the biological roles of these mole-
cules [41]. In this work, ER-targeted fluorescent probe NBIS for
6

Z.-Y. Li et al.
Dyes and Pigments 188 (2021) 109180
[18] Samanta S, Halder S, Dey P, Manna U, Ramesh A, Das G. A ratiometric fluorogenic
probe for the real-time detection of SO23- in aqueous medium: application in a
cellulose paper based device and potential to sense SO23- in mitochondria. Analyst
2018;143:250–7. https://doi.org/10.1039/C7AN01368J.
Declaration of competing interest
The authors declare no conflicts of interest.
[19] Xu W, Teoh CL, Peng J, Su D, Yuan L, Chang YT. A mitochondria-targeted
ratiometric fluorescent probe to monitor endogenously generated sulfur dioxide
derivatives in living cells. Biomaterials 2015;56:1–9. https://doi.org/10.1016/j.
biomaterials.2015.03.038.
Acknowledgments
[20] Sun SG, Ding H, Yuan G, Zhou L. An efficient TP-FRET-based lysosome-targetable
fluorescent probe for imaging peroxynitrite with two well-resolved emission
channels in living cells, tissues and zebrafish. Anal Chim Acta 2020;1100:200–7.
https://doi.org/10.1016/j.aca.2019.11.065.
This study was supported by the National Key Research and Devel-
opment Program of China (2017YFA0104604), the National Natural
Science Foundation of China (No.31871407, 31741083, 31870831), the
Natural Science Foundation of Shandong Province (ZR2018MB042).
The authors acknowledge the assistance of Shandong University Struc-
tural Constituent and Physical Property Research Facilities.
[21] Xia S, Zhang Y, Fang M, Mikesell L, Steenwinkel TE, Wan S, Phillips T, Luck RL,
Werner T, Liu H. A FRET-based near-infrared fluorescent probe for ratiometric
detection of cysteine in mitochondria. ChemBioChem 2019;20:1986–94. https://
doi.org/10.1002/cbic.201900071.
[22] Ren M, Wang L, Lv X, Sun Y, Chen H, Zhang K, Wu Q, Bai Y, Guo W. A rhodol-
hemicyanine based ratiometric fluorescent probe for real-time monitoring of
glutathione dynamics in living cells. Analyst 2019;144:7457–62. https://doi.org/
10.1039/C9AN01852B.
Appendix A. Supplementary data
[23] Tamima U, Song CW, Santra M, Reo YJ, Banna H, Islam MR, Ahn KH. A benzo[b]
xanthene-derived fluorescent probe capable of two-photon ratiometric imaging of
lysosomal cysteine with high specificity. Sensor Actuator B Chem 2020;322:
128588. https://doi.org/10.1016/j.snb.2020.128588.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dyepig.2021.109180.
[24] Hu Q, Qin C, Huang L, Wang H, Liu Q, Zeng L. Selective visualization of
hypochlorite and its fluctuation in cancer cells by a mitochondria-targeting
ratiometric fluorescent probe. Dyes Pigments 2018;149:253–60. https://doi.org/
10.1016/j.dyepig.2017.10.002.
References
[1] Huang Y, Tang C, Du J, Jin H. Endogenous sulfur dioxide: a new member of
gasotransmitter family in the cardiovascular system. Oxid. Med. Cell Longev. 2016:
8961951. https://doi.org/10.1155/2016/8961951. 2016.
[25] Jun YW, Sarkar S, Singha S, Reo YJ, Kim HR, Kim JJ, Chang YT, Ahn KH. A two-
photon fluorescent probe for ratiometric imaging of endogenous hypochlorous acid
in live cells and tissues. Chem Commun 2017;53:10800–3. https://doi.org/
10.1039/C7CC05834A.
[2] Song Y, Peng H, Bu D, Ding X, Yang F, Zhu Z, Tian X, Zhang L, Wang X, Tang C,
Huang Y, Du J, Jin H. Negative auto-regulation of sulfur dioxide generation in
vascular endothelial cells: AAT1 S-sulfenylation. Biochem Biophys Res Commun
2020;525:231–7. https://doi.org/10.1016/j.bbrc.2020.02.040.
[26] Gao J, He Y, Chen Y, Song D, Zhang Y, Qi F, Guo Z, He W. Reversible FRET
fluorescent probe for ratiometric tracking of endogenous Fe3+ in ferroptosis. Inorg
Chem 2020;59:10920–7. https://doi.org/10.1021/acs.inorgchem.0c01412.
[27] Roberts S, Seeger M, Jiang Y, Mishra A, Sigmund F, Stelzl A, Lauri A,
[3] Pisoschi AM, Pop A, Gajaila I, Iordache F, Dobre R, Cazimir I, Serban A. Analytical
methods applied to the assay of sulfur-containing preserving agents. Microchem J
2020;155:104681. https://doi.org/10.1016/j.microc.2020.104681.
[4] Yu W, Jin H, Tang C, Du J, Zhang Z. Sulfur-containing gaseous signal molecules,
ion channels and cardiovascular diseases. Br J Pharmacol 2018;175:1114–25.
https://doi.org/10.1111/bph.13829.
´
Symvoulidis P, Rolbieski H, Preller M, Dean-Ben XL, Razansky D, Orschmann T,
Desbordes SC, Vetschera P, Bach T, Ntziachristos V, Westmeyer GG. Calcium sensor
for photoacoustic imaging. J Am Chem Soc 2018;140:2718–21. https://doi.org/
10.1021/jacs.7b03064.
[5] Niu M, Han Y, Li Q, Zhang J. Endogenous sulfur dioxide regulates hippocampal
neuron apoptosis in developing epileptic rats and is associated with the PERK
signaling pathway. Neurosci Lett 2018;665:22–8. https://doi.org/10.1016/j.
neulet.2017.11.036.
[28] Kong X, Li M, Dong B, Yin Y, Song W, Lin W. An ultrasensitivity fluorescent probe
based on the ICT-FRET dual mechanisms for imaging β-galactosidase in vitro and
ex vivo. Anal Chem 2019;91:15591–8. https://doi.org/10.1021/acs.
analchem.9b03639.
[6] Xu J, Yuan H, Zeng L, Bao G. Recent progress in Michael addition-based fluorescent
probes for sulfur dioxide and its derivatives. Chin Chem Lett 2018;29:1456–64.
https://doi.org/10.1016/j.cclet.2018.08.012.
[29] Zhang Y, Chen Y, Bai Y, Xue X, He W, Guo Z. FRET-based fluorescent ratiometric
probes for the rapid detection of endogenous hydrogen sulphide in living cells.
Analyst 2020;145:4233–8. https://doi.org/10.1039/D0AN00531B.
[30] Qian M, Zhang L, Pu Z, Xia J, Chen L, Xia Y, Cui H, Wang J, Peng X. A NIR
fluorescent probe for the detection and visualization of hydrogen sulfide using the
aldehyde group assisted thiolysis of dinitrophenyl ether strategy. J Mater Chem B
2018;6:7916–25. https://doi.org/10.1039/C8TB02218F.
[7] Li K, Li LL, Zhou Q, Yu KK, Kim JS, Yu XQ. Reaction-based fluorescent probes for
SO2 derivatives and their biological applications. Coord Chem Rev 2019;388:
310–33. https://doi.org/10.1016/j.ccr.2019.03.001.
[8] Yang B, Xu J, Zhu HL. Recent progress in the small-molecule fluorescent probes for
the detection of sulfur dioxide derivatives (HSO-3/SO32-). Free Radical Biol Med
2019;145:42–60. https://doi.org/10.1016/j.freeradbiomed.2019.09.007.
[9] Dong B, Song W, Lu Y, Kong X, Mehmood AH, Lin W. An ultrasensitive ratiometric
fluorescent probe based on the ICT-PET-FRET mechanism for the quantitative
measurement of pH values in the endoplasmic reticulum (ER). Chem Commun
2019;55:10776–9. https://doi.org/10.1039/C9CC03114F.
[31] Zhang H, Wang B, Ye Y, Chen W, Song X. A ratiometric fluorescent probe for
simultaneous detection of Cys/Hcy and GSH. Org Biomol Chem 2019;17:9631–5.
https://doi.org/10.1039/C9OB01960J.
[32] Zhao X, Yuan G, Ding H, Zhou L, Lin Q. A TP-FRET-based fluorescent sensor for
ratiometric visualization of selenocysteine derivatives in living cells, tissues and
zebrafish. J Hazard Mater 2020;381:120918. https://doi.org/10.1016/j.
jhazmat.2019.120918.
[10] Zhang Y, Xia S, Mikesell L, Whisman N, Fang M, Steenwinkel TE, Chen K, Luck RL,
Werner T, Liu H. Near-infrared hybrid rhodol dyes with spiropyran switches for
sensitive ratiometric sensing of pH changes in mitochondria and drosophila
melanogaster first-instar larvae. ACS Appl. Bio Mater. 2019;2:4986–97. https://
doi.org/10.1021/acsabm.9b00710.
[33] Zhang W, Huo F, Cheng F, Yin C. Employing an ICT-FRET integration platform for
the real-time tracking of SO2 metabolism in cancer cells and tumor models. J Am
Chem Soc 2020;142:6324–31. https://doi.org/10.1021/jacs.0c00992.
[34] Yan Y, Zhang X, Zhang X, Li N, Man H, Chen L, Xiao Y. Ratiometric sensing
lysosomal pH in inflammatory macrophages by a BODIPY-rhodamine dyad with
restrained FRET. Chin Chem Lett 2020;31:1091–4. https://doi.org/10.1016/j.
cclet.2019.10.025.
[11] Lu X, Zhang G, Li D, Tian X, Ma W, Li S, Zhang Q, Zhou H, Wu J, Tian Y. Thiophene
aromatic amine derivatives with two-photon activities as probes for the detection
of picric acid and pH. Dyes Pigments 2019;170:107641. https://doi.org/10.1016/j.
dyepig.2019.107641.
[35] Jia X, Li W, Guo Z, Guo Z, Li Y, Zhang P, Wei C, Li X. An NBD-based mitochondrial
targeting ratiometric fluorescent probe for hydrogen sulfide detection. Chemistry
2019;4:8671–5. https://doi.org/10.1002/slct.201901991.
[12] Sun YQ, Liu J, Zhang J, Yang T, Guo W. Fluorescent probe for biological gas SO2
derivatives bisulfite and sulfite. Chem Commun 2013;49:2637–9. https://doi.org/
10.1039/C3CC39161B.
[36] Zhang D, Liu A, Ji R, Dong J, Ge Y. A mitochondria-targeted and FRET-based
ratiometric fluorescent probe for detection of SO2 derivatives in water. Anal Chim
Acta 2019;1055:133–9. https://doi.org/10.1016/j.aca.2018.12.042.
[13] Zhu L, Xu J, Sun Z, Fu B, Qin C, Zeng L, Hu X. A twisted intramolecular charge
transfer probe for rapid and specific detection of trace biological SO2 derivatives
and bio-imaging applications. Chem Commun 2015;51:1154–6. https://doi.org/
10.1039/C4CC08258C.
¨
[37] Dong B, Tian M, Kong X, Song W, Lu Y, Lin W. Forster resonance energy transfer-
based fluorescent probe for the selective imaging of hydroxylamine in living cells.
Anal Chem 2019;91:11397–402. https://doi.org/10.1021/acs.analchem.9b02737.
[38] Wu WL, Wang ZY, Dai X, Miao JY, Zhao BX. An effective colorimetric and
ratiometric fluorescent probe based FRET with a large Stokes shift for bisulfite. Sci
Rep 2016;6:25315. https://doi.org/10.1038/srep25315.
[14] Li H, Fan J, Long S, Du J, Wang J, Peng X. A fluorescent and colorimetric probe for
imaging the mitochondrial sulfur dioxide in living cells. Sensor Actuator B Chem
2018;273:899–905. https://doi.org/10.1016/j.snb.2018.07.008.
[15] Zhao M, Liu D, Zhou L, Wu B, Tian X, Zhang Q, Zhou H, Yang J, Wu J, Tian Y. Two
water-soluble two-photon fluorescence probes for ratiometric imaging endogenous
SO2 derivatives in mitochondria. Sensor Actuator B Chem 2018;255:1228–37.
https://doi.org/10.1016/j.snb.2017.08.053.
[39] Wu WL, Ma HL, Huang MF, Miao JY, Zhao BX. Mitochondria-targeted ratiometric
fluorescent probe based on FRET for bisulfite. Sensor Actuator B Chem 2017;241:
239–44. https://doi.org/10.1016/j.snb.2016.10.028.
[16] Tamima U, Santra M, Song CW, Reo YJ, Ahn KH. A benzopyronin-based two-
photon fluorescent probe for ratiometric imaging of lysosomal bisulfite with
complete spectral separation. Anal Chem 2019;91:10779–85. https://doi.org/
10.1021/acs.analchem.9b02384.
[40] Li DP, Han XJ, Yan ZQ, Cui Y, Miao JY, Zhao BX. A far-red ratiometric fluorescent
probe for SO2 derivatives based on the ESIPT enhanced FRET platform with
improved performance. Dyes Pigments 2018;151:95–101. https://doi.org/
10.1016/j.dyepig.2017.12.056.
[17] Lu Y, Li H, Yao Q, Sun W, Fan J, Du J, Wang J, Peng X. Lysozyme-targeted
ratiometric fluorescent probe for SO2 in living cells. Dyes Pigments 2020;180:
108440. https://doi.org/10.1016/j.dyepig.2020.108440.
7

Z.-Y. Li et al.
Dyes and Pigments 188 (2021) 109180
[41] Gao P, Pan W, Li N, Tang B. Fluorescent probes for organelle-targeted bioactive
species imaging. Chem Sci 2019;10:6035–71. https://doi.org/10.1039/
C9SC01652J.
[47] Skehan P, Storeng R, Scudiero D, Monks A, McMahon J. New colorimetric
cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 1990;82:
1107–12. https://doi.org/10.1093/jnci/82.13.1107.
[42] Zhu L, Fu M, Yin B, Wang L, Chen Y, Zhu Q. A red-emitting fluorescent probe for
mitochondria-target microviscosity in living cells and blood viscosity detection in
hyperglycemia mice. Dyes Pigments 2020;172:107859. https://doi.org/10.1016/j.
dyepig.2019.107859.
[48] Yan YH, Cui XL, Li ZY, Ding MM, Che QL, Miao JY, Zhao BX, Lin ZM. A synergetic
FRET/ICT platform-based fluorescence probe for ratiometric imaging of bisulfite in
lipid droplets. Anal Chim Acta 2020;1137:47–55. https://doi.org/10.1016/j.
aca.2020.09.002.
[43] Zhao X, He F, Dai Y, Ma F, Qi Z. A single fluorescent probe for one- and two-photon
imaging hydrogen sulfide and hydrogen polysulfides with different fluorescence
signals. Dyes Pigments 2020;172:107818. https://doi.org/10.1016/j.
dyepig.2019.107818.
[49] Li DP, Wang ZY, Cao XJ, Cui J, Wang X, Cui HZ, Miao JY, Zhao BX.
A mitochondria-targeted fluorescent probe for ratiometric detection of endogenous
sulfur dioxide derivatives in cancer cells. Chem Commun 2016;52:2760–3. https://
doi.org/10.1039/C5CC09092J.
[44] Chen B, Li C, Zhang J, Kan J, Jiang T, Zhou J, Ma H. Sensing and imaging of
mitochondrial viscosity in living cells using a red fluorescent probe with a long
lifetime. Chem Commun 2019;55:7410–3. https://doi.org/10.1039/C9CC03977E.
[45] Yan YH, Wu QR, Che QL, Ding MM, Xu M, Miao JY, Zhao BX, Lin ZM.
A mitochondria-targeted fluorescent probe for the detection of endogenous SO2
derivatives in living cells. Analyst 2020;145:2937–44. https://doi.org/10.1039/
D0AN00086H.
[50] Sitia R, Braakman I. Quality control in the endoplasmic reticulum protein factory.
Nature 2003;426:891–4. https://doi.org/10.1038/nature02262.
[51] Wang XB, Huang XM, Ochs T, Li XY, Jin HF, Tang CS, Du JB. Effect of sulfur
dioxide preconditioning on rat myocardial ischemia/reperfusion injury by
inducing endoplasmic reticulum stress. Basic Res Cardiol 2011;106:865. https://
doi.org/10.1007/s00395-011-0176-x.
[52] Ercan S, Kencebay C, Basaranlar G, Derin N, Aslan M. Induction of xanthine
oxidase activity, endoplasmic reticulum stress and caspase activation by sodium
metabisulfite in rat liver and their attenuation by Ghrelin. Food Chem Toxicol
2015;76:27–32. https://doi.org/10.1016/j.fct.2014.11.021.
¨
[46] Clegg RM. Forster resonance energy transfer—FRET what is it, why do it, and how
it’s done. In: Laboratory techniques in biochemistry and molecular biology.
Elsevier; 2009. p. 1–57. https://doi.org/10.1016/S0075-7535(08)00001-6.
8