A novel mitochondrial targeting fluorescent probe for ratiometric imaging SO2 derivatives in living cells

Article abstract of DOI:10.1016/j.jphotochem.2019.112339

As an important endogenous signaling molecule, SO₂ plays a key role in many physiological processes. However, excessive intake of SO₂ and its derivatives lead to serious health complications of various diseases. In order to study the role of SO₂ and its derivatives in the biological environment, it is of great significance to develop new and effective monitoring methods. Here, a new red emission fluorescent probe (TNPI) based on pyrazoline and hemicyanine dyes for the high selective detection of SO₂ derivatives was developed. The probe TNPI showed some advantages such as long emission wavelength (640 nm), obvious color and fluorescence changes after reaction with SO₂ derivatives, high selectivity and sensitivity toward SO₂ derivatives (detection limit = 80 nM), and large fluorescence emission shift (160 nm) and signal ratio change (from 0.45–445, about 989 times). Intriguingly, the probe was successfully exploited for the fluorescence imaging of SO₂ derivatives in the mitochondria of living cells with low cytotoxicity.

Full text of DOI:10.1016/j.jphotochem.2019.112339

JournalofPhotochemistry&PhotobiologyA:Chemistry390(2020)112339
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
A novel mitochondrial targeting fluorescent probe for ratiometric imaging
SO2 derivatives in living cells
Xiao-Bo Wanga, Hui-Jing Lia,**, Zhenxing Chia, Xianshun Zengb,**, Li-Juan Wangc,
Yun-Fei Chenga, Yan-Chao Wua,d,
*
a School of Marine Science and Technology, Harbin Institute of Technology, Weihai 264209, China
b Tianjin Key Laboratory for Photoelectric Materials and Devices, Key Laboratory of Display Materials & Photoelectric Devices, Ministry of Education, School of Materials
Science & Engineering, Tianjin University of Technology, Tianjin 300384, China
c School of Materials Science and Engineering, Harbin Institute of Technology, Weihai 264209, China
d Weihai Institute of Marine Biomedical Industrial Technology, Wendeng District, Weihai 264400, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
As an important endogenous signaling molecule, SO2 plays a key role in many physiological processes. However,
excessive intake of SO2 and its derivatives lead to serious health complications of various diseases. In order to
study the role of SO2 and its derivatives in the biological environment, it is of great significance to develop new
and effective monitoring methods. Here, a new red emission fluorescent probe (TNPI) based on pyrazoline and
hemicyanine dyes for the high selective detection of SO2 derivatives was developed. The probe TNPI showed
some advantages such as long emission wavelength (640 nm), obvious color and fluorescence changes after
reaction with SO2 derivatives, high selectivity and sensitivity toward SO2 derivatives (detection limit = 80 nM),
and large fluorescence emission shift (160 nm) and signal ratio change (from 0.45–445, about 989 times).
Intriguingly, the probe was successfully exploited for the fluorescence imaging of SO2 derivatives in the mi-
tochondria of living cells with low cytotoxicity.
Pyrazoline-cyanine dye
SO2 derivatives
Color
Emission shift
Imaging
Mitochondria
1. Introduction
seen as one of the most desirable methods because of its simplicity, high
sensitivity and low detection limits [17–22]. Until now, majority of the
Sulfur dioxide (SO2), a colorless, pungent and decaying odor gas,
reported fluorescent probes for SO2 derivatives are based on nucleo-
philic addition of SO2 derivatives to C]C, C]N, C]O and N]N
bonds. But there are still many difficulties to overcome. First, some
fluorescent probes can only detect SO2 derivatives in a single wave-
length, which cannot avoid the influence of instrument and environ-
ment fluctuations [23–26]. Second, some fluorescent probes have short
emission wavelengths (< 600 nm) that limit their biological applica-
tions [27–35]. Next, some ratio probes have short emission shifts and
small signal emission ratio after reacting with SO2 derivatives, which
could not avoid the overlap of two emission wavelengths [36,37]. Some
fluorescent probes are required to detect sulfur dioxide in a high pro-
portion of organic solvents [38,39]. Finally, the role of SO2 derivatives
in bodies is unclear due to a lack of suitable detection methods in cells
and subcellular organelles. Therefore, it is of great significance to de-
velop an organelle targeting long wavelength emission fluorescence
probe with high signal ratio for detection of SO2 derivatives in the
environment and biological systems.
exists in the form of sulfites (SO32−) and bisulfite (HSO3−) in aqueous
media. Epidemiological studies have demonstrated that the excessive
2−
intake of SO2 derivatives (SO3
and HSO3-) may lead to may be as-
sociated with many diseases such as respiratory system diseases, ner-
vous system diseases and lung cancer [1–3]. It is worth noting that
sulfites were classified as carcinogens by the world health organization
and the international agency for research on cancer (IARC) on 27 Oc-
tober 2017. Despite their numerous harmful properties, SO2 derivatives
are widely involved in our daily life [4–6]. With the increasing con-
sumption of petroleum, SO2 are becoming as more and more serious
environmental pollutants [7,8]. SO2 derivatives can also be produced
endogenously in mitochondria during the biosynthesis of thiol-con-
taining amino acids [9–11]. Therefore, the development of highly se-
lective and sensitive methods for the detection of SO2 derivatives in
living cells becomes a high priority.
In contrast with other methods [12–16], fluorescence detection is
⁎ Corresponding author at: School of Marine Science and Technology, Harbin Institute of Technology, Weihai 264209, China.
⁎⁎ Corresponding authors.
E-mail addresses: lihuijing@iccas.ac.cn (H.-J. Li), xshzeng@tjut.edu.cn (X. Zeng), ycwu@iccas.ac.cn (Y.-C. Wu).
https://doi.org/10.1016/j.jphotochem.2019.112339
Received 28 October 2019; Received in revised form 17 December 2019; Accepted 22 December 2019
Availableonline24December2019
1010-6030/©2019ElsevierB.V.Allrightsreserved.

X.-B. Wang, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry390(2020)112339
Scheme 1. Design of the Ratiometric Fluorescent Probe TNPI and Sensing Mechanism.
Herein, a novel mitochondrial-targeted fluorescent probe (TNPI)
0.5 % DMSO) for 30 min as a control group. The other three groups
were incubated with different concentrations of sulfite (10 μM, 30 μM,
150 μM) in PBS buffer (containing 0.5 % DMSO) for 30 min, washed
with PBS buffer solution for three times, and then incubated with 10 μM
probe TNPI in PBS buffer (containing 0.5 % DMSO) for another 30 min.
The cells were washed with PBS buffer solution 3 times before fluor-
escence imaging. The fluorescence images were obtained using
Olympus FV1000 laser scanning microscope. The green light was ex-
cited at 405 nm and the emission spectrum was 500–550 nm. The red
light is excited by an excitation light of 552 nm with an emission
spectrum of 560–600 nm.
with red emission (640 nm) for monitoring SO2 derivatives was devel-
oped in this work. In this probe, the hemicyanine moiety was selected
because it can be used as an extended conjugated group and an iden-
tification group of the SO2 derivatives. The probe TNPI has some ad-
vantages shown as follows: (1) a long emission wavelength (640 nm);
(2) a ratio of two channels to detect SO2 derivatives; (3) large emission
shift (160 nm); (4) remarkable limit of detection (80 nM); (5) up to 989-
fold signal ratio enhancement (from 0.45–445). Moreover, to demon-
strate its potential practical applications value, the probe TNPI was also
applied for monitoring SO2 derivatives in the mitochondria of living
cells through the method of dual-channel ratio imaging.
3. Results and discussion
2. Experimental section
3.1. Design and synthesis of TNPI
2.1. Main instruments and reagents
The pyrazoline moiety of the probe TNPI has been selected as a
fluorophore due to its high quantum yield, low toxicity, and effective
cell permeability [40–42]. Hemicyanine dye is a cationic dye that not
only increases the water solubility of the probe TNPI, but also acts as an
identification group of the SO2 derivative. Moreover, hemicyanine ca-
tionic dyes can be easily absorbed by cell mitochondria, which makes
the probe have a certain cell targeting property [34,43,44]. Thus, the
combination of pyrazoline and hemicyanine moiety might be used to
construct a mitochondria-targeted fluorescent probe with long wave-
length emission for sensing SO2 derivatives.
As shown in Scheme 1, addition of SO2 derivatives interrupts the
conjugation between pyrazoline and hemicyanine moieties based on the
1, 4-nucleophilic addition reaction in the vinyl group of TNPI. The
synthesis of TNPI is illustrated in Scheme 2. The probe and inter-
mediate structures were identified by 1H NMR, 13C NMR and/or HRMS
spectra (see Supporting Information).
All reagents are from commercial suppliers and no further pur-
ification was required. Column chromatography silica gel used in the
experiment was 200–300 mesh. Melting points were measured by using
a Gongyi X-5 microscopy digital melting point apparatus. IR spectra
were recorded by using an Electrothemal Nicolet 380 spectrometer. The
NMR spectra of 1H and 13C were measured on the AVANCE II spec-
trometer with tetramethylsilane (TMS) as the internal reference mate-
rial. Bruker APEX IV spectrometer was used for high resolution mass
spectrometry analysis (HRMS). UV–vis spectrum were obtained from T6
new century spectrometer. Model PHS-3C pH meter was used for pH
measurement. Fluorescence spectra measurements were performed on
the F-2700 fluorescence spectrophotometer. Olympus FV1000 laser-
scanning confocal microscope was used for fluorescence imaging.
2.2. Procedure for spectral measurements
The synthesis of probe TNPI is shown in Scheme 2. Compound 1 can
be obtained through previous literature reports [36].
A solution of TNPI (1 mM) was prepared in DMF. Other testing
analytes (KF, NaCl, KBr, KI, Na2CO3, NaHCO3, NaNO3, NaNO2, AcONa,
Na2S, NaHSO3, Na2SO3, Na2SO4, Na2S2O4, Na2S2O5, KSCN, CaCl2,
MgCl2, ZnCl2, Cys, Hcy, GSH, H2O2 and NaClO4) were prepared as
10 mM aqueous solution with deionized water.
The stock solution of probe TNPI (100 μL) was added to a 10 ml of
volumetric flask. Different amounts of analytes were added and diluted
with PBS buffer (10 mM, pH 7.4) containing 5 % DMF to corresponding
concentrations. Spectral acquisition time was 30 min.
1-(Naphthalen-2-yl)-3-phenylprop-2-en-1-one (2). To a solution of
benzaldehyde (0.75 g, 7.05 mmol) in EtOH (10 mL) was added drop-
wise 5 mL of aqueous NaOH solution (4 mol/L) at 0 °C, and then was
added 2-acetylnaphthalene (1.00 g, 5.88 mmol). The resulting mixture
was stirred for 3 h at room temperature, filtrated. The residue was
washed and recrystallized from EtOH to afford 2 as a yellow solid
(1.38 g, 90.9 %). M.P. 163–165 °C; IR (film) υmax: 3057.51, 1658.33,
1626.42, 1598.42, 1448.69, 1328.24, 1184.74, 1123.99, 760.88 cm−1
;
1H NMR (400 MHz, DMSO-d6): δ 8.96 (s, 1 H), 8.17 (d, J =3.6 Hz, 1 H),
8.15 (d, J =1.6 Hz, 1 H), 8.12–8.13 (m, 1 H), 8.05 (d, J =8.4 Hz,1 H),
8.01 (d, J =7.6 Hz, 1 H), 7.92–7.94 (m, 2 H), 7.83 (d, J =15.6 Hz,4 H),
7.64–7.68 (m, 2 H), 7.47–7.49 (m, 3 H); 13C NMR (100 MHz, DMSO-d6):
δ 189.4, 144.5, 135.6, 135.4, 135.3, 132.9, 131.2, 131.1, 130.2, 129.5,
129.2, 129.0, 128.3, 127.5, 124.7, 122.6; HRMS (C19H14O) m/z: cal-
culated for [M+H]+: 259.1117; Found [M+H]+: 259.1127.
3-(Naphthalen-2-yl)-1,5-diphenyl-4,5-dihydro-1H-pyrazole(3). A mix-
ture of compound 2 (0.50 g, 1.94 mmol) and phenyl hydrazine (0.25 g,
2.32 mmol) in 10 mL of glacial acetic acid was stirred at 90 °C for 5 h,
cooled to room temperature, and then collected the solids. The residue
was recrystallized with EtOH to give 3 as pale yellow solid (0.51 g,
2.3. Cell culture and fluorescence imaging
HeLa cells were incubated in medium (MEM, minimum essential
medium, supplemented with 10 % fetal bovine serum at 37 °C, in 5 %
CO2). The cell suspension of 100 μL per well was then inoculated into a
96-well plate containing a cap slide and cultured overnight. Cells were
treated with TNPI. Treated cells were further incubated for 24 h. 100 μL
of CCK8 was added to each well and incubated at 37 °C for 30 min. The
optical absorbance at 450 nm was measured.
The cultured HeLa cells were divided into four groups. The first
group was incubated with 10 μM probe TNPI in PBS buffer (containing
2

X.-B. Wang, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry390(2020)112339
Scheme 2. Synthetic procedures of probe TNPI.
75.61 %). M.P. 187–189 °C; IR (film) υmax: 2919.96, 1595.70, 1498.51,
1358.75, 1129.76, 827.95, 744.48, 692.29 cm−1 1H NMR (400 MHz,
127.0, 126.7, 126.6, 125.4, 124.6, 123.2, 122.2, 113.9, 113.1, 105.9,
63.0, 51.1, 43.4, 35.4, 27.3; HRMS (C38H34N3I) m/z: calculated for [M-
I]+: 532.2747; Found [M-I]+: 532.2734.
;
CDCl3): δ 8.18 (dd, J = 1.6, 8.4 Hz, 1 H), 7.79–7.86 (m, 4 H), 7.48 (d, J
=2.8 Hz, 1 H), 7.47 (d, J =3.6 Hz,1 H), 7.36 (s, 2 H), 7.35 (d, J
=1.2 Hz, 2 H), 7.28–7.29 (m, 1 H), 7.19–7.23 (m, 2 H), 7.12–7.14 (m,
2 H), 6.81 (t, J =6 Hz, 1 H), 5.33 (dd, J = 7.2, 12.4 Hz, 1 H), 3.95 (dd,
J = 12.4, 17.2 Hz, 1 H), 3.27 (dd, J = 7.2, 17.2 Hz, 1 H); 13C NMR
(100 MHz, CDCl3): δ 147.2, 145.2, 143.0, 133.80, 133.7, 130.8, 129.6,
129.4, 128.6, 128.5, 128.3, 128.0, 126.9, 126.8, 126.3, 125.5, 123.9,
119.6, 113.9, 65.0, 43.9; HRMS (C25H20N2) m/z: calculated for [M
+H]+: 349.1699; Found [M+H]+: 349.1713.
3.2. Spectral changes of TNPI toward sulfite
The pH value is generally considered to be an important factor af-
fecting the interaction between the substances. In order to obtain the
influence of pH on the detection process, the fluorescence intensity of
the probe TNPI solution was tested under different pH values (Fig.S1).
The fluorescent intensity ratio of free probe TNPI (10 μM) was almost
unchanged between pH 2 and pH 12. However, the solution of probe
4-(3-(Naphthalen-2-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ben-
zaldehyde (4). POCl3 (0.21 ml, 2.30 mmol) was added to 2 mL of DMF
dropwise at room temperature. The mixture was stirred for 15 min at
room temperature, added with compound 3 (0.40 g, 1.15 mmol, in 2 mL
of DMF) at 0 °C, stirred at room temperature for 5 h, and poured into
10 mL of ice-water. The pH of the reaction mixture was adjusted to
neutral with 30 % ammonia water. The reaction solution was extracted
with ethyl acetate (3 × 20 mL). Organic layer was dried with anhy-
drous Na2SO4 and evaporated to dryness. The crude products were
purified by silica gel column chromatography to give 4 as a yellow solid
(0.35 g, yield: 81.7 %). M.P. 171–173 °C; IR (film) υmax: 2924.10,
1681.10, 1592.74, 1519.15, 1406.13, 1222.99, 1162.57, 1129.21,
TNPI displayed a strong fluorescent intensity ratio enhancement in the
2−
presence of SO3
over a wide pH range (5–9), which suggested that
the probe can detect sulfite under physiological conditions. In order to
get detection time of TNPI with sulfite, fluorescence kinetics experi-
ments were carried out. As depicted Fig. 1, different concentrations of
2−
2−
SO3
were added to TNPI (10 μM). Although 200 μM SO3
could
complete the reaction with reaching an intensity plateau in 10 min, a
low concentration of SO3 took a longer time to achieve fluorescence
2-
intensity saturation. In addition, the fluorescence intensity ratio
changes with time after adding lower concentrations of SO32- were also
2−
investigated (Figure S2). Thus, 30 min after the addition of SO3 was
821.09, 700.20 cm−1 1H NMR (400 MHz, CDCl3): δ 9.78 (s, 1 H), 8.19
;
selected as the test time point in the experiments.
(dd, J = 1.6, 8.4 Hz, 1 H), 7.82–7.91 (m, 4 H), 7.72 (d, J =8.8 Hz, 2 H),
7.49–7.55 (m, 2 H), 7.36–7.40 (m, 2 H), 7.28–7.33 (m, 3 H), 7.18 (d, J
=8.6 Hz, 2 H), 5.45 (dd, J = 5.6, 12 Hz, 1 H), 4.01 (dd, J = 12,
17.2 Hz, 1 H), 3.36 (dd, J = 5.6, 17.2 Hz, 1 H); 13C NMR (100 MHz,
CDCl3): δ 190.7, 150.2, 148.6, 141.4, 133.9, 133.3, 131.8, 129.6, 129.6,
128.5, 128.4, 128.2, 128.0, 127.9, 127.0, 126.8, 126.2, 125.7, 123.5,
In order to investigate the selectivity, UV response experiments
were carried out. The free probe TNPI (10 μM) showed a strong ab-
2−
sorption peak at 558 nm (Fig. 2). However, after the addition of SO3
,
the maximum absorption was shifted to 380 nm. No similar spectral
changes were observed in other analytes. Simultaneously, the color of
112.9, 63.6, 43.6; HRMS (C26H20N2O) m/z: calculated for [M+H]+
:
377.1648; Found [M+H]+: 377.1661.
1,3,3-Trimethyl-2-(4-(3-(naphthalen-2-yl)-5-phenyl-4,5-dihy-dro-1H-
pyrazol-1-yl)styryl)-3H-indol-1-Ium iodide (TNPI). Compound 1 (0.08 g,
1.94 mmol) and compound 4 (0.10 g, 2.32 mmol) were dissolved in
5 mL of ethanol in a 25 mL of round bottom flask. The reaction mixture
was stirred at 80 °C for 12 h. The solvent was then evaporated. The
residue was purified by column chromatography using CH2Cl2/MeOH
(v/v = 1:20) to give TNPI as a purple solid (0.11 g, yield: 63.9 %). M.P.
201–204 °C; IR (film) υmax: 3361.55, 2924.10, 1569.60, 1516.22,
1402.81, 1291.68, 1170.67, 1113.84, 855.57, 708.72 cm−1
；
1H NMR
(400 MHz, CDCl3): δ 8.29 (d, J =16 Hz, 1 H), 8.18–8.21 (m, 2 H), 8.09
(d, J =9.2 Hz, 2 H), 8.00 (d, J =8.0 Hz, 1 H), 7.96–7.98 (m, 2 H), 7.79
(d, J =8.0 Hz, 1 H), 7.75 (d, J =8.0 Hz, 1 H), 7.56–7.59 (m, 3 H),
7.49–7.51 (m, 1 H), 7.36–7.40 (m, 2 H). 7.29–7.33 (m, 3 H), 7.21–7.29
(m, 3 H), 5.92 (dd, J = 4.4, 12 Hz, 1 H), 4.17 (dd, J = 12, 20 Hz, 1 H),
4.00 (s, 3 H), 3.43 (dd, J = 4.4, 18 Hz, 1 H), 1.74 (d, J =4.4 Hz, 6 H);
13C NMR (100 MHz, CDCl3) δ 179.4, 154.4, 152.9, 148.5, 141.9, 141.4,
140.3, 134.4, 133.8, 132.8, 129.2, 129.0, 128.7, 128.2, 128.0, 127.6,
Fig. 1. Time-dependent fluorescence intensity ratio changes (I480 nm/I640 nm) of
2−
TNPI (10 μM) upon addition of varied concentrations of SO3
in PBS buffer
(10 mM, pH 7.4) containing 5 % DMF at room temperature.
3

X.-B. Wang, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry390(2020)112339
2−
Fig. 4. Fluorescence response of probe TNPI (10 μM) toward SO3
(200 μM)
Fig. 2. Absorption spectra changes of the probe TNPI (10 μM) and the presence
of 200 μM various analytes in PBS buffer (10 mM, pH 7.4) containing 5 % DMF
as a co-solvent at room temperature. The color changes were shown in the inset.
and other small biological species (200 μM) at fluorescence intensity ratios (I480
nm/I640 nm) in PBS buffer (10 mM, pH 7.4) containing 5 % DMF as a co-solvent
at room temperature. The mixture was kept for 30 min before the fluorescence
intensity was measured.
TNPI solution changed from purple to colorless, whereas no obvious
changes were observed in other analytes (Fig. 2 inset). The selective
change indicated that TNPI could act as a "naked eye" colorimetric
indicator for sulfite detection.
The sensing behavior of probe TNPI toward various analytes was
also tested with fluorescence spectroscopy in PBS buffer (10 mM, pH
7.4) containing 5 % DMF as a co-solvent, as shown in Fig. 3. The so-
lution of free probe had an obvious red fluorescence at around 640 nm
very little influence on sensing of SO32−. In addition, lower con-
2−
centration of SO3
(30 μM) was tested in the presence of 200 μM
competing analytes (Figure S3). We can see that even if the con-
centration of interfering analytes was much higher than SO32−, SO3
2−
can also significantly increase the fluorescence ratio of the probe TNPI
solution (10 μM). These results proved that TNPI had good anti-inter-
ference ability for sulfite recognition.
with excitation at 558 nm (fluorescence quantum yield: 0.13). Upon the
2−
addition of SO3
and HSO3-, the solution of TNPI showed a marked
blue-green fluorescence with strong emission at 480 nm with excitation
at 380 nm (fluorescence quantum yield of the product: 0.28), indicating
the interruption of the π-conjugation between pyrazoline and hemi-
cyanine moiety. This large emission shift (160 nm) resulted in the
emission peaks being well distinguished before and after reacting with
sulfite, and thus ratiometric sensing can be carried out, which provided
a built in correction for biological environmental impacts. Only HS- led
to a slight fluorescence change. The addition of other tested species
3.3. Quantitative responses of TNPI toward sulfite
To study the sensitivity of the probe TNPI to sulfite, fluorescence
spectra were recorded by adding different amounts of SO3
2−
into the
solution of TNPI (Fig. 5). The probe TNPI had a weak fluorescence in
the absence of SO32-. Upon addition of increasing concentrations of
SO32-, the solution of TNPI showed a gradual enhancement in the
fluorescence emission bond at 480 nm. In contrast, the fluorescence
intensity of solution gradually decreases at 640 nm. The fluorescence
intensity ratio (I480 nm/I640 nm) increased from 0.45–445 after adding 20
equiv. of Na2SO3. Furthermore, the fluorescence intensity ratios
induced negligible changes in the fluorescence intensity. The results
2−
demonstrated that TNPI showed a high selectivity toward SO3
HSO3- over other analytes. SO3
/
2−
and HSO3- showed the same fluor-
escent detection effect. Therefore, SO32−was selected as the re-
2-
showed a good linear relationship with the concentration of SO3
presentative of SO2 derivatives in the latter experiments. In order to
(0–30 μM) (Fig. 6). The detection limit of TNPI for SO32- was calculated
to be 80 nM based on 3σ/slope method (see ESI). It can be concluded
that TNPI was a sensitive indicator for sulfite in aqueous media from
these results.
detect the sulfite accurately, the sensing process of TNPI should not be
2−
interfered. Then, interference experiments were tested. SO3
was
added into to the solution of TNPI pretreated with a competing analyte.
The results are shown in Fig. 4, all the relevant analytes measured have
Fig. 3. Fluorescence spectra changes of the probe TNPI (10 μM) and the presence of 200 μM various analytes in PBS buffer (10 mM, pH 7.4) containing 5 % DMF as a
co-solventat room temperature under excitation at 380 nm (a) and 558 nm (b).
4

X.-B. Wang, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry390(2020)112339
2−
Fig. 5. The fluorescence spectra changes of probe TNPI (10 μM) with increasing amount of SO32−. Each spectrum was obtained 30 min after SO3
addition.
3.4. The proposed sensing mechanism
In order to study the sensing mechanism, the Gaussian 09 program
was firstly used for theoretical calculation [45]. As shown in Fig. 7,
optimized molecular geometry of TNPI clearly showed π-conjugation
between pyrazoline and hemicyanine moieties through a C]C double
bond. This π-conjugation structure was corrupted in TNPI-SO3, nu-
cleophilic addition reaction product of C]C double bond of TNPI with
SO32−. We then further compared the highest occupied molecular or-
bital (HOMO) and lowest unoccupied molecular orbital (LUMO) of
TNPI and TNPI-SO3 [71]. In TNPI, HOMO is mainly localized on the
pyrazoline moiety with small distribution on hemicyanine, while LUMO
is mainly localized on hemicyanine moiety. ICT from pyrazoline to
hemicyanine moieties is clearly observed in this “D-π-A” structure. In
TNPI-SO3, HOMO is totally dominated by hemicyanine moiety and
LUMO is dominated by pyrazoline moiety, respectively. The π-con-
junction between pyrazoline and hemicyanine moieties is destroyed.
Thus, the ICT process between the pyrazoline and hemicyanine of
TNPI-SO3 is inhibited. Therefore, this could lead to a blue-shift in the
absorption and emission spectra [46].
Fig. 6. Fluorescence intensity ratio changes (I480 nm/I640 nm) of probe TNPI
2−
(10 μM) against SO3
concentrations from 0 μM–350 μM. Inset: The linear
2−
relationship between fluorescent intensity ratio and SO3
(0−30 μM).
concentration
In order to understand the reaction mechanism of TNPI toward
SO32−, we verified the process of reaction by some tracking method.
Fig. 7. Theoretical computation of TNPI and TNPI-SO3. Optimized molecular geometries of TNPI and TNPI-SO3, their HOMO and LUMO orbital distributions, and
the corresponding electric energies.
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JournalofPhotochemistry&PhotobiologyA:Chemistry390(2020)112339
Firstly, 1H NMR titration was carefully performed (Fig. S4). An obvious
difference was observed before and after the reaction of TNPI and
SO32−. Signals corresponding to the protons (7.79 ppm for Hb and
7.75 ppm for Ha) assigned to the double bond hydrogens in the probe
TNPI disappeared and new signals emerged at 6.59 ppm (Hb,) and
6.51 ppm (Ha,) after the reaction with SO32−. Moreover, the proton
signal for methyl group (Hc) in the structure of TNPI changed from
4.00 ppm to 2.94 ppm. Secondly, the product of reaction was confirmed
using HRMS, where a dominant peak at m/z value of 635.2218 corre-
sponds to [TNPI + HSO3- + Na]+ (Fig. S5). These experimental results
implied that the sensing mechanism involves the nucleophilic attack of
Tracker Green) was almost identical from line profile. In order to pre-
liminarily verify that the mitochondrial targeting of the probe TNPI was
due to the cationic hemicyanine moiety, mitochondrial uncoupling
agent 2,4-dinitrophenol (DNP) was used to eliminate the negative
charge of the mitochondrial inner membrane of the cells. The result
showed that the Pearson coefficient was significantly reduced (Figure
S7). Therefore, the mitochondrial targeting of the probe TNPI may be
due to the charge attraction between cationic hemicyanine moiety in
the structure of probe TNPI with a positive charge and cellular mi-
tochondrial membrane with the negative potential.
2-
SO3 on the vinyl group of the probe TNPI, which are in good ac-
4. Conclusion
cordance with the reports in literature [44,47]. The structure of con-
jugation was interrupted, causing a blue-shift phenomenon in the
emission wavelength (Scheme 1).
In summary, a novel red emission fluorescent probe (TNPI) has been
developed for two-channel ratio detection of subcellular SO2 deriva-
tives. The probe exhibited not only outstanding sensitivity (80 nM)
toward SO2 derivatives but also high selectivity upon other species in a
physiological pH aqueous solution. A large emission shift (160 nm) and
a sharp emission ratio increase (from 0.45–445) were observed during
the monitoring of SO2 derivatives. The probe was also successfully used
to monitor the changes of SO2 derivatives in the mitochondria of cells
through the formation of dual-channel ratio imaging. This probe would
provide a promising chemical analysis tool for studying the application
of SO2 derivatives in medical diagnosis and disease research.
3.5. Cellular imaging
Based on the above excellent results, the bioimaging application of
probe TNPI was further explored. To evaluate cytotoxicity of TNPI, we
conducted CCK8 assay in HeLa cells. Compared with the control, the
cells incubated with 10 μM TNPI for 24 h still maintained a survival rate
of 92.3 % (Fig. S6), which clearly indicated that TNPI could be suitably
applied to the cultured cells.
To demonstrate the abilities of the probe to enter cells and image
SO2 derivatives in living cells, we performed a separate emission
window using a confocal microscope [21,48]. As shown in Fig. 8, after
incubating HeLa cells with TNPI, strong fluorescence in the red channel
could be readily observed and slight fluorescence was captured in the
Author statement
Xiao-Bo Wang carried out the experiments and wrote the manu-
script. Hui-Jing Li and Yan-Chao Wu designed experiments and revised
the manuscript; Zhenxing Chi and Yun-Fei Cheng analyzed sequencing
data and developed analysis tools; Xianshun Zeng directed the cell
imaging experiments; Li-Juan Wang performed theoretical calculations.
green channel. However, when Hela cells were pre-incubated with
2−
different concentrations of SO3
(10 μM, 30 μM, 150 μM) for 30 min
before incubating with 10 μM TNPI under the same conditions, the red
fluorescence receded while the green fluorescence gradually increased.
Therefore, the probe has the potential application in detecting the
distribution and concentration of SO2 derivatives in cells through the
method of dual-channel ratio imaging.
Declaration of Competing Interest
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.
After the SO32− imaging in cells, the distribution of the probe at the
subcellular level was determined by the co-localization experiments in
Hela cells [49,50]. The cells were pretreated with 0.5 μM TNPI and
subsequently 200 nM Mito-Tracker Green (or 200 nM Lyso-Tracker
Green). As expected, the fluorescence of TNPI in red channel also
overlapped exactly with that of Mito-Tracker Green in green channel
(Pearson’s correlation of 0.95) but not with that of lysosome (Fig. 9).
The intensity profile of the red (TNPI) and green channels (Mito-
Acknowledgements
This work was supported by the Key Research and Development
Program of Shandong Province (2019GSF108089), the Natural Science
Foundation of Shandong Province (ZR2019MB009), the National
Fig. 8. The confocal fluorescence imaging of
2−
exogenous SO3
in Hela cells. (A) Hela cells
were only incubated with the probe TNPI
(10 μM) for 30 min (a–d). Hela cells were pre-
treated with Na2SO3 10 μM (e–h), 30 μM (j–m),
150 μM (o–r) for 30 min, then incubated with
TNPI (10 μM) for 30 min. Red-channel
λ
em = 560−600 nm (λex =552 nm), green
channel λem = 500−550 nm (λex =405 nm);
(B) Analysis of the quantitative fluorescence
intensity in (A) using ImageJ software；(C)
The ratios of IGreen/IRed in (A). Error bars de-
note standard deviation ( S.D.), n = 3. Scale
bar: 20 μm.
6

X.-B. Wang, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry390(2020)112339
Fig. 9. Co-localization imaging in Hela cells. Cells were pretreated probe TNPI (5 μM, λex =552 nm, λem = 560−600 nm) for 30 min and then treated Mito-Tracker
Green (200 nM, λex =488 nm, λem = 500−550 nm) and Lyso-tracker Green DND-26 (200 nM, λex=488 nm, λem = 500–550 nm) for 30 min. a and f represent green
channel of commercial dye; b and g represent red channel of probe TNPI; c and h represent merged imaging between commercial dye and probe TNPI; e and j
represent Pearson’s correlation; Line profile: intensity profile of the yellow line in image overlap. Scale bar: 10 μm.
Natural Science Foundation of China (21672046, 21372054), and the
Found from the Huancui District of Weihai City.
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Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jphotochem.2019.
112339.
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8