Selective detection of peroxynitrite in living cells by a near-infrared diphenyl phosphinate-based dicyanoisophorone probe

Article abstract of DOI:10.1016/j.saa.2020.118890

A new NIR fluorescent probe for detection of ONOO^? has been developed, which possesses a large Stokes shift, good selectivity and low cytotoxicity. This NR-ONOO probe exhibits a strong turn-on near-infrared fluorescence response toward ONOO^? ion under excitation at 560 nm and has been successfully applied in detecting ONOO^? in living HeLa cells.

Full text of DOI:10.1016/j.saa.2020.118890

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118890
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Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Short Communication
Selective detection of peroxynitrite in living cells by a near-infrared
diphenyl phosphinate-based dicyanoisophorone probe
Yibin Zhanga, Dongge Mab,
⁎
a
College of Chemistry and Chemical Engineering, Yangtze Normal University, Fuling, Chongqing 408100. PR China
College of Chemistry and Materials Engineering, Beijing Technology and Business University, Beijing 100048, PR China
b
a r t i c l e i n f o
a b s t r a c t
A new NIR fluorescent probe for detection of ONOO− has been developed, which possesses a large Stokes shift,
good selectivity and low cytotoxicity. This NR-ONOO probe exhibits a strong turn-on near-infrared fluorescence
response toward ONOO− ion under excitation at 560 nm and has been successfully applied in detecting ONOO−
in living HeLa cells.
Article history:
Received 2 July 2020
Received in revised form 25 August 2020
Accepted 26 August 2020
Available online 28 August 2020
© 2020 Elsevier B.V. All rights reserved.
Keywords:
Peroxynitrite detection
NIR diphenylphosphinate-based
dicyanoisophorone fluorescent probe
1. Introduction
peroxynitrite have been reported [16–27], and it is severely desired to
develop new fluorescent probes for the detection of peroxynitrite with
Peroxynitrite (ONOO−) acts as a reactive oxygen species (ROS) in
various pathological and physiological processes, which was endoge-
nously formed by the combination reaction of superoxide (O2•−) and
nitric oxide (NO•) [1–3]. In living biosystem, low level of peroxynitrite
plays a critical role in signal transduction, but high level of peroxynitrite
could induce damage of some certain biomolecules, such as lipids, pro-
teins, and nucleic acids, etc [4,5]. The abnormal fluctuations of the
peroxynitrite concentration eventually cause various diseases including
inflammation, cancers, cardiovascular diseases, atherosclerosis,
ischemia-reperfusion injury, and diabetes [6]. Thus, the development
of peroxynitrite detection methods in living biosystem has become ur-
gently needed.
Compared with other techniques, fluorescent probe-based imaging
technology has higher sensitivity, less invasiveness and greater conve-
nience, thus it attracted a great deal of attention [7,8]. However, the ex-
citation and emission wavelengths of most previously reported probes
for detection of peroxynitrite just cover ranges within 650 nm, which
render them not suitable for detecting peroxynitrite in vivo due to the
autofluorescence interferences and insufficient penetration depth in
the visible region [9–12]. In contrast, the use of near-infrared fluores-
cence (650–900 nm) avoids the influence of background bio-
autofluorescence and allows deep penetration into tissues [13–15].
However, to date, only a few probes of NIR fluorescence for detecting
NIR emission.
Thus, we herein developed a new NIR florescent probe NR-ONOO for
detection of peroxynitrite by using dicyanoisophorone as fluorophore,
which was widely utilized in NIR fluorescence because of its excellent
photophysical property including large Stokes shift, good photostability
and lower biotoxicity. Besides, diphenylphosphinic group was chosen as
a sensitive and selective recognition group for the detection of
peroxynitrite. The fluorescence of probe NR-ONOO was inhibited by
the blocking of the intramolecular charge transfer (ICT). After reaction
with peroxynitrite, phosphinate group departed and released the free
phenolic hydroxyl group as a strong electron-donating group, which re-
covers the ICT effect and the generated dicyanoisophorone exhibits the
NIR fluorescence emission (Scheme 1). The probe NR-ONOO could se-
lectively and sensitively detect peroxynitrite with a large Stokes shift
(133 nm) and a low detection limit (78.70 nM). Besides, this probe
also has good membrane penetrability as well as low cell toxicity, mak-
ing it suitable for the detection of peroxynitrite in living HeLa cells by
using confocal microscope.
2. Material preparation and methods
2.1. Materials and instruments
Unless specifically mentioned, all used chemical reagents were ob-
tained from Sigma-Aldrich chemical company without further purifica-
tion. Deionized water was used for preparing phosphoric acid buffer
solution. UV–Vis-NIR absorption spectra and fluorescence spectra
⁎
Corresponding author.
E-mail addresses: zhangyibin@yznu.edu.cn (Y. Zhang), madongge@btbu.edu.cn
(D. Ma).
https://doi.org/10.1016/j.saa.2020.118890
1386-1425/© 2020 Elsevier B.V. All rights reserved.

2
Y. Zhang, D. Ma / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118890
with deionized water to concentration of 10 mM. Other analytes with
a concentration of 100 mM were prepared in PBS solution including
O2•−, H2O2, NO, ClO−, •OH, tBuO•, H2S, SO23−, Cys, Hcy and GSH.
2.4. Cell maintenance and fluorescence imaging
Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) with fetal bo-
vine serum (Gibco 10%), penicillin and streptomycin (100 μg/mL) were
used for HeLa cells culture at 37 °C in 5% CO2. Cell imaging of HeLa: The
HeLa cells were incubated with probe NR-ONOO for 30 min at 37 °C,
then incubated with peroxynitrite donor SIN-1 (a peroxynitrite genera-
tor) or treated with the ONOO− scavenger, ebselen. Cell imaging in
HepG2: pretreated with PMA and LPS followed by incubating with the
probe. Next, the cells were washed with D-PBS two times and then
were observed by Nikon A1MP confocal laser scanning microscope for
cell imaging. Cell viability assays of probe were determined using MTT
methanol method.
Scheme 1. Detecting peroxynitrite by probe NR-ONOO.
were obtained with LAMBDA 1050+ UV/Vis/NIR and Horiba Scientific
Spectrofluorometer. Bruker 400 MHz spectrometer is used for 1H NMR
and 13C NMR experiments. Nikon A1MP confocal laser scanning micro-
scope was used for living cell imaging.
2.2. Synthesis of probe
Compound 1 was synthesized according to previous literature [28].
Compound 1 (580 mg, 2 mmol), Compound 2 (472 mg, 2 mmol) and
0.1 mL triethylamine was added in 10 mL dichloromethane and stirring
at room temperature for 8 h. After the reaction was finished monitored
by TLC, 10 mL water was added into the crude mixture. The organic
layer was extracted with dichloromethane (5mL*3), dried with rotary
evaporator and then purified by silica gel flash chromatography (eluted
with petroleum ether/dichloromethane 5:1, v/v) to afford the desired
product as a yellow solid (900 mg, 92% yield). (Scheme 2A).
3. Results and discussion
3.1. Design and synthesis
Fluorophore is an important member of fluorescent probes. In recent
years, isophorone-fused fluorophore (compound 1) has attracted wide
attention due to its excellent optical property such as near-infrared fluo-
rescence emission [29], large Stokes shift and the convenience in mod-
ifying phenolic hydroxyl group. Besides, diphenylphosphinate group
has been reported as ONOO−-specific reaction model [30].Thus, it is
highly probable to regulate the switching of fluorescence of probe NR-
ONOO by the protection and deprotection of phenolic hydroxyl group
with diphenyl phosphinate group.
The detailed synthetic procedures of the probe and the sensing
mechanism toward detecting ONOO− were outlined in Scheme 2.
Firstly, compound 1 was readily synthesized according to previous liter-
ature. Then, the probe was prepared by the reaction of compound 1
2.3. Spectra measurements
All fluorescence measurement was performed in PBS (10 mM,
pH 7.4, 10% DMSO, 25 °C) with excitation at 560 nm and slit width of
5.0 nm. The stock solution of ONOO− was dissolved in deionized
water to concentration of 2 mM. And the stock solution of probe NR-
ONOO was prepared according to previous literature and dissolved
Scheme 2. (A) The synthetic route of probe NR-ONOO; (B) the possible mechanism of sensing peroxynitrite by probe NR-ONOO.

Y. Zhang, D. Ma / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118890
3
with compound 2 in the presence of triethylamine at room temperature
via a one-step reaction, with dichloromethane as solvent. The molecule
structures of the probe were characterized and confirmed by 1H NMR,
13C NMR, and MALDI-TOF-MS, as shown in SI. 1H NMR (400 MHz,
Chloroform‑d) δ 7.93–7.79 (m, 5H), 7.54 (d, J = 8.6 Hz, 2H), 7.46 (d,
J = 7.0 Hz, 4H), 7.40–7.36 (m, 2H), 7.24–7.19 (m, 2H), 6.96 (s, 1H),
6.86 (s, 1H), 6.78 (s, 1H), 2.57 (s, 2H), 2.41 (s, 2H), 1.04 (s, 6H). 13C
NMR (100 MHz, Chloroform‑d) δ 169.34, 153.81, 152.10, 136.05,
132.82, 131.96, 131.85, 130.03, 129.11, 128.98, 123.69, 121.52, 113.68,
112.89, 78.90, 43.26, 39.44, 32.34, 28.32. MALDI-TOF mass
spectrometry:491.33.
After obtaining the probe, we first evaluated the possibility of the
probe to detect ONOO− by UV–Vis absorption and fluorescence-
emission spectra under physiological conditions. As shown in Fig. 1
(left), the UV-absorption peak at 400 nm decreased accompanied with
an obviously increasing signal at 545 nm in the presence of ONOO−
.
As shown in Fig. 1 (right), probe NR-ONOO displayed negligible fluores-
cence emission owing to the blocked ICT effect, because the electron-
donating effect of the phenolic hydroxyl group was suppressed by
diphenyl phosphinate protecting group. However, the fluorescence
peak around 678 nm was gradually enhanced and reached saturation
under 560 nm excitation in the presence of ONOO−. Noteworthy,
probe NR-ONOO shows a large Stokes shift (133 nm) in response to
ONOO−, which is much larger than other reported probes. This large
Stokes shift is beneficial to minimize the interferences originated from
self-absorption. Besides, it can be observed that the fluorescence spec-
trum of probe NR-ONOO after reaction with ONOO− was almost consis-
tent to that of compound 1. MALDI-TOF mass spectrometry was applied
to investigate the recognition mechanism and the peak at m/z 291.59
[M + H]+ corresponding to compound 1 was observed after mixing
probe NR-ONOO with ONOO−. Additionally, the reaction product of
mixing probe and ONOO− was monitored by TLC plate (Fig. S9), and
was fully characterized by 1H NMR and 13C NMR (Figs. S10–11)
confirming that the product is compound 1. The above data testifies
that the significantly increased fluorescence signal was induced by the
removal of the ONOO− responsive diphenyl phosphinate group (see
Scheme 1) and the reaction mechanism of probe NR-ONOO accorded
with the previous literature [31–42].
Fig. 2. The fluorescent recognition (slit: 5 nm) of probe NR-ONOO (10 μM) to
ONOO−(100 μM) and other various species (100 μM).
of other analytes made negligible changes in the emission spectra.
These results suggested that the probe NR-ONOO has high selectivity
for ONOO−. Besides, the competing experiments in the presence of
other ROS/RNS were also conducted and there are no obvious fluores-
cence fluctuations with other analytes (see Fig. S5), and the negligible
effect of physiological concentrations (GSH and Cys) at 2 mM level
was shown in Fig. S12. In short, all these results indicate that the
diphenyl phosphinate group of this probe was specifically reactive to
ONOO− over other analytes, causing the excellent selectivity for
ONOO−. Because the normal physiological pH value is about 7.4, we fur-
ther discussed the effect of pH on the fluorescence response of NR-
ONOO to ONOO− in the revised manuscript. The results are shown in
Fig. S8. The probe had great change on the performance with pH
changes (lower than 6.5 and higher than 8.5), but slight effect at phys-
iological pH (6.5–8.5). Those experiments results demonstrated that
the probe showed high prospect for biological utilization.
3.3. Time-dependent fluorescence response
3.2. Selectivity and pH value effect of probe NR-ONOO toward ONOO−
After the selectivity study was completed, time-dependent fluores-
cence experiments of probe for ONOO− were carried out. As shown in
Fig. S6, the fluorescence intensity changing at 678 nm was saturated
within ~25 min when 10.0 equiv. of ONOO− was added to 10 μM
probe, ~40 min when 5.0 equiv. of ONOO−, and ~60 min when 1.0
equiv. of ONOO− was added, respectively (Fig. S6). These data show
The selectivity experiments of probe NR-ONOO toward other ROS/
RNS such as O2•−, H2O2, NO, ClO−, •OH, tBuO•, H2S, SO23−, Cys, Hcy and
GSH were conducted with fluorescence spectrometry. As shown in
Fig. 2, upon the addition of ONOO−, an obvious signal change in the
fluorescence spectrum of probe NR-ONOO was observed under
560 nm excitation. In contrast, even with the addition of 10 equivalent
that although the probe needs a certain period to respond to ONOO−
,
3.5x106
3.0x106
2.5x106
2.0x106
1.5x106
1.0x106
5.0x105
0.0
0.6
0.5
0.4
0.3
0.2
0.1
0.0
300
400
500
600
700
600
650
700
750
800
Wavelength(nm)
Wavelength(nm)
Fig. 1. (left) The UV–vis spectra of probe NR-ONOO (10.0 μM) in the absence (red line) and presence of ONOO−(20 equiv.) (blue line). (right) The fluorescence emission spectra of probe
NR-ONOO (10.0 μM) in the absence (black line) and presence of ONOO− (20 equiv.) (red line).

4
Y. Zhang, D. Ma / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118890
but less than an hour recognition time is acceptable for monitoring
ONOO− in vivo.
3.4. Linear relationship between the fluorescence response of NR-ONOO
and total ONOO− ion concentration
Next, the fluorescence titration studies were carried out to investi-
gate the sensitivity of probe NR-ONOO toward ONOO−. As shown in
Fig. 3 (top), the increasing concentration of ONOO− caused an obvious
fluorescence turn-on at 678 nm in a concentration-dependent manner.
Fig. 3 (down) shows that the fluorescence response has a good linear re-
lationship between the emission intensity and ONOO− concentration
(R2 = 0.99293). Then, we use the formula DL = 3 s/k (s for the standard
deviation of the fluorescence spectrometer and k for the slope of the
correction curve) to get the detection limit as low as 78.70 nM, indicat-
ing that probe NR-ONOO has excellent sensitivity for the quantitative
detection of ONOO−.
3.5. Cytotoxicity of probe NR-ONOO
To validate the cytotoxicity of the probe NR-ONOO, MTT method
was used in living HeLa cells. As shown in Fig. S7, HeLa cells remained
high activity after incubating with various concentrations of probe NR-
ONOO over 24 h (10, 20, 30, 40, 50 μM), and the highest viability
about 80% can be found even at 50 μM concentration, which means
that this probe has slight cytotoxicity and satisfactory bio-
Fig. 4. Fluorescence images for exogenous ONOO− in HeLa cells. a: HeLa cells were
incubated with 10 μM probe, control group; b: HeLa cells were incubated with 5 μM
probe, then incubated by SIN-1 (100 μM, 0.5 h); c: HeLa cells were incubated with
10 μM probe, then incubated by SIN-1 (100 μM, 0.5 h); d: HeLa cells were incubated
with 10 μM probe, pretreated with 100 μM ebselen, and then treated with 100 μM SIN-1
for 30 min.
compatibility. The low cytotoxicity is in favor of gaining a better under-
standing for biological utilization.
3.6. Imaging ONOO− in living cells
Encouraged by the selective and sensitive characteristic of probe NR-
ONOO toward ONOO−, we evaluated the bioimaging applications of this
probe for detection of ONOO− in living HeLa cells. First, HeLa cells were
treated with probe, and further incubated with ONOO− peroxynitrite
donor SIN-1 (a peroxynitrite generator), as shown in Fig. 4b and c, a
strong fluorescence can be observed in red channel. To verify if the fluo-
rescence increase is resulting from ONOO−, ebselen, an ONOO− scaven-
ger, was applied in a blocking experiment, the red fluorescence is
significantly reduced by the pretreatment with the ebselen (see
Fig. 4d). Additionally, HepG2 cells was used to monitor the generation
of endogenous ONOO−. HepG2 cells were pretreated with phorbol 12-
myristate 13-acetate (PMA) and lipopolysaccharide (LPS) for 1 h to pro-
duce endogenous ONOO− followed by incubating probe. As shown in
Fig. S13, the HepG2 cells stimulated with PMA and LPS also exhibited in-
tense fluorescence. This result indicated that the probe could achieve vi-
sually tracing the changes of endogenous or exogenous ONOO− levels in
living cells.
Determination of ONOO− in living cells.
4. Conclusion
A new NIR fluorescent probe for detection of ONOO− through an ICT
process has been developed and successful applied in detecting ONOO−
in living HeLa cells. This probe has a large Stokes shift, good selectivity
and low cytotoxicity. The addition-elimination reaction process by nu-
cleophilic attack of ONOO− to diphenyl phosphinate motif of the
Fig. 3. (top) Fluorescence response of probe (10 μM) to various concentrations of ONOO−
(reaction time is 1 h) (down) Linear correlation between the enhancement of emission
intensity at 638 nm. λex = 560 nm, λem = 678 nm.

Y. Zhang, D. Ma / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118890
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