Two-Photon Fluorescence Probe for Selective Monitoring of Superoxide in Live Cells and Tissues

Article abstract of DOI:10.1021/acs.analchem.9b03937

The abnormal location or generation of superoxide radical anion (O₂ ^?-) are implicated in many diseases, including cancers; thus, development of an efficient method to detect O₂ ^?- is of great importance. Inspired by the fluorophore-governed selective manner to O₂ ^?- and peroxynitrite (ONOO^-) of previously reported phosphinate-based fluorescence probes, in this contribution, a phosphinothioate-containing probe, TPP, was designed. The probe exhibited easy accessibility through a one-step sequence and good photostability and biocompatibility. Interestingly, TPP showed high specificity and sensitivity to O₂ ^?- over other reactive oxygen species/nitrogen species including ONOO^-. Furthermore, with the assistance of two-photon microscopy, TPP was successfully applied for imaging endogenous O₂ ^?- in live cells and tissues.

Full text of DOI:10.1021/acs.analchem.9b03937

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Article
A two-photon fluorescence probe for selective
monitoring of superoxide in live cells and tissues
Liyan Chen, Myoung Ki Cho, Di Wu, Hwan Myung Kim, and Juyoung Yoon
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b03937 • Publication Date (Web): 21 Oct 2019
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Analytical Chemistry
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A two-photon fluorescence probe for selective monitoring of
superoxide in live cells and tissues
Liyan Chen, †§ Myoung Ki Cho, ‡§ Di Wu,† Hwan Myung Kim*‡ and Juyoung Yoon*†
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† Department of Chemistry and Nano Science, Ewha Womans University, Seoul, 120-750, Korea
‡ Department of Chemistry and Energy Systems Research, Ajou University, Suwon, 16499, Korea
*Correspondence :
(J. Yoon) Email: jyoon@ewha.ac.kr, Fax: 82-2-3277-2385
(H. M. Kim) Email: kimhm@ajou.ac.kr
•-
ABSTRACT: The abnormal location or generation of superoxide radical anion (O2 ) are implicated in many diseases, including
•-
cancers, thus development of an efficient method to detect O2 is of great importance. Inspired by the fluorophore governed
•-
selective manner to O2 and peroxynitrite (ONOO-) of previously reported phosphinate based fluorescence probes, in this
contribution, a phosphinothioate containing probe, TPP was designed. The probe exhibited easy accessibility through a one-step
sequence and good photostability and biocompatibility. Interestingly, TPP showed high specificity and sensitivity to O2•- over other
ROS/RNS including ONOO-. Furthermore, with the assistance of two-photon microscopy, TPP was successfully applied for
imaging endogenous O2•- in live cells and tissues.
•-
Reactive oxygen species (ROS) and nitrogen species (RNS)
including hydrogen peroxide (H2O2), peroxynitrite (ONOO-),
hypochlorite (ClO-), singlet oxygen (1O2), and superoxide
fluorescence O2 probes, owing to their own merits such as
high specificity and low detection limit, have attracted
particular attention. Thanks to the lower toxicity of
organophosphorus compounds as well as the remarkable
•-
radical anion (O2 ) play pivotal roles in many physiological
•-
and pathological processes.1-3 Among these ROS/RNS,
performance for sensing O2 of phosphinate group, a series of
O2 probes based on this reactive group have been developed
•-
•-
superoxide radical anion (O2 ), which is generated through the
one-electron reduction of oxygen has been recognized as the
precursor of most of the reactive oxygen and nitrogen
using diverse fluorophores such as fluorescein, 2-
(benzothiazol-2-yl)-phenol (HBT), hemi-cynine, dibenz[a,c]-
phenazine (DBP), naphthalimide, etc.50-58 However, recently
Churchill’s group reported a “Turn-on” fluorescent sensing
system in which phosphinate group exhibited excellent
•-
species.4-6 The abnormal expression of O2 apparently reflects
the level of other reactive species and leads to many diseases,
such as inflammation, neurodegeneration, autoimmune
disorders, and even cancer.7-10 Therefore, the development of
•- 59
specificity towards peroxynitrite instead of O2 .
•-
efficient methods for sensitive and selective monitoring of O2
Subsequently, the specific property to peroxynitrite was also
found by Fan’s group.60 These discoveries aroused our interest
to further explore the property and application of the
phosphinate group. Our extreme curiousness led to
is of great importance. However, due to its extremely short
half-life and multifunction, the recognition and quantification
of O2•- in vitro and in vivo still remains a big challenge.11
investigation whether changing the phosphinate to
a
Over the past couple of decades, several technologies have
been applied to monitor generation and level of O2 , such as
•-
phosphinothioate group will still exhibit high reactivity
towards O2•- or will it specifically respond to ONOO-.
electron spin resonance (ESR) spectroscopy, mass
spectrometry (MS), electrochemical determination, and
chromatography.12-16 However, these traditional methods had
their own disadvantages such as time consumption,
complicated sample pretreatment, expensive instrument cost,
etc. Moreover, the reported methods exhibited difficulties in
application to real-time detection in the living systems. In
contrast, fluorescent bioimaging technology has been found to
be more efficient since it possesses good sensitivity and
selectivity, less invasiveness, more convenience, and powerful
potential in bioanalysis.17-24
On the contrary, although various elegant fluorescence probes
have been reported, there exist still some limitations. For
example, some probes need a sophisticated procedure for
•-
synthesis, some probes are merely applied to detect O2 at the
cellular level, and tissue imaging still needs to be exploited.
More importantly, many probes find limited applications in
one-photon microscopy due to disadvantages of
photobleaching, autofluorescence of biosamples, and shallow
tissue-penetration depth resulting from the short excitation
wavelength. In contrast, two-photon (TP) fluorescence probes
are hypothesized to overcome the above-mentioned
disadvantages to a certain extent as they are excited by two
near-infrared (NIR) laser pulses.61-66
Basically, the well-known small molecular fluorescence
•-
probes for the detection of O2 were implemented through
redox,25-32 protection-deprotection process,33-43 or other
nucleophilic reactions44-49. The protection-deprotection-based
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Taking all the aforementioned issues into consideration, herein,
Page 2 of 8
(ONOO−)69 were prepared using the previously reported
procedure and the concentration was confirmed by UV
spectroscopy. Nitric oxide (NO•) was generated using
DEA•NONOate.
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the phosphinothioate containing probe TPP was designed and
synthesized in one step using the commercially available
compounds dansyl chloride and diphenylphosphine oxide as
the starting materials (Scheme 1, S1).67
Two-photon Fluorescence Microscopy. Two-photon
fluorescence microscopy images were acquired using
multiphoton microscopes (Leica TCS SP8 MP) and samples
were excited through titanium sapphire laser (Mai Tai HP).
Output power of two-photon excitation was 2.49 W at 740 nm
and the mean power of the focal plane was calculated to be
approximately 1.94 × 106 W cm-2.
N
N
CH3CN
+
H
P O
S
P
R.T
O
O
S
O
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Cl
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Cell Culture. Cells were cultured in glass-bottomed dishes
(NEST). Before imaging, the medium was replaced from
growth medium to serum-free medium. After TPP (5 μM)
treatment, cells were incubated in 5 % CO2 for 30 min at
37 °C. DMEM (WelGene) to which 10 % FBS, 100 μg mL-1
streptomycin, and 100 units mL-1 penicillin were added was
used for culture medium.
Scheme 1. Synthesis of probe TPP.
EXPERIMENTAL SECTION
Materials and Instruments. Commercially available starting
reagents were used without further purification. 1H NMR
spectra were recorded on a Bruker DPX spectrometer at 300
MHz. Chemical shifts were reported on the delta (δ) scale in
parts per million (ppm) relative to the singlet (0 ppm) for
tetramethylsilane (TMS). 13C NMR spectra were recorded at
125 MHz with complete proton decoupling. Chemical shifts
are reported in ppm relative to the central line at 77 ppm for
CDCl3.
Cell viability. Cells were seeded in a 96-well plate with
culture media. After overnight culture, cells were incubated
with samples for 24 hr. To identify cytotoxicity, 0.5 mg/mL of
MTT (Sigma) media was added to the cells for 4 hr, and the
produced formazan was dissolved in 0.1 mL of
dimethylsulfoxide (DMSO) and read at OD 650 nm with a
Spectramax Microwell plate reader. The viability of no probe
treated cells was calculated as 100 % live state.
Synthesis of Probe TPP. Dansyl chloride (0.5 mmol, 128 mg)
and diphenylphosphine oxide (2.0 mmol, 404 mg) were
dissolved in 10 mL CH3CN in a round-bottom flask and
reaction was initiated at 80 oC for 8 h. After completion of the
reaction, the reaction was cooled down to the room
temperature and diphenylphosphinic acid was filtered off. The
solid was washed with CH3CN (25 mL) for 3 times.
Subsequently, the filtrate was evaporated under vacuum. The
crude product was purified by silica gel column
chromatography using petroleum Hexane-DCM (3:1) as the
Photostability. Three areas that were chosen randomly as the
same size were monitored to detect the changes in the two-
photon excited fluorescence (TPEF) intensities. Photostability
of TPP was acquired by monitoring the time course of TPEF
intensities during 1 h at 2.00-sec intervals using xyt mode.
Detecting window was adjusted as 400−600 nm for collecting
the TPEF intensities by exciting at 740 nm.
Preparation and Imaging of Rat Hippocampus. Typically,
14 old male SD rats were used to obtain rat hippocampal
tissues. The tissues were sliced to 400 μm thickness through
vibrating blade microtome in DPBS (Gibco). After TPP (20
μM) was loaded, sliced tissues were incubated in 5% CO2, for
1
eluent. H NMR (300 MHz, CDCl3): δ 8.37-8.24 (m , 2H),
8.21-8.07 (m, 4H), 7.35 – 7.28 (m, 4H), 7.16-7.01 (m, 6H),
3.95 (s, 6H) (Figure S8); 13C NMR (125 MHz, CDCl3): δ
151.37, 136.64, 135.62, 135.59, 133.29, 132.39, 132.37,
131.83, 131.75, 129.91, 128.63, 128.53, 126.80, 126.37,
124.85, 124.06, 121.06, 114.64, 45.57 (Figure S9); HRMS
(ESI) m/z = 404.1239, calcd for [M+H]+ C24H23NOPS =
404.1238 (Figure S10).
o
1 h at 37 C. Before imaging, tissues stained with TPP were
washed with DPBS. TPM tissue imaging was carried out at
70−190 μm depth.
RESULTS AND DISCUSSION
Spectroscopic Characterization. Fluorescence emission
•-
Spectral Response of TPP to O2 . With the probe TPP in
spectra
were
obtained
using
an
UV
RF-5301/PC
absorption
hand, the spectral properties of probe TPP in the presence of
spectrofluorophotometer
(Shimadzu).
•-
O2 under simulated physiological conditions was evaluated
spectroscopy measurements were carried out on Scinco S-
3100 using a 1 cm optical path length cell at room temperature.
A stock solution of probe TPP (1 mM) was prepared in N,N-
dimethylformamide. Superoxide solution (O2•−) was prepared
by adding KO2 to dry dimethylsulfoxide. A solution of tert-
butyl hydroperoxide (TBHP), hypochlorous acid (HOCl), and
hydrogen peroxide (H2O2) was prepared in ultrapure water by
diluting 70% tert-butyl hydroperoxide, 5% NaClO, and 30%
H2O2 solutions, respectively. Glutathione (GSH), cysteine
initially. As can be seen in Figure 1A, the free probe TPP
showed no fluorescence with excitation at 345 nm in PBS
buffer/DMF (v:v = 7:3) solutions. In contrast, upon addition of
•-
various amounts of O2 , the emission focused at 470 nm
gradually enhanced. When the concentration of O2•- was above
60 μM, the fluorescence intensity at 470 nm reached the
maximum (Figure S1). Excellent linearity was observed
between the fluorescence intensity at 470 nm and the
•−
concentrations of O2 ranging from 0 to 20 equivalents
-
(Cys), homocysteine (Hcy), hydrogen sulfide (H2S), and NO2
(Figure S2). The detection limit was determined as 150 nM
according to the 3δ/k criterion, where δ is the standard
deviation of the blank (n = 11) and k is the slope for the range
of linearity. Also, the maximum absorption peak for the free
solutions were prepared in ultrapure water. Hydroxyl radical
(•OH) was generated by adding hydrogen peroxide (H2O2, 100
µM) to ammonium iron(II) sulfate (100 µM) solution. Singlet
oxygen (1O2) was prepared by adding H2O2 stock solution to
HOCl solution (10 equivalents). HOBr68 and peroxynitrite
•-
probe TPP was observed at 330 nm. The addition of O2 also
led to a slight enhancement (Figure 1B).
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Wavelength / nm
t
0.20
0.15
0.10
0.05
Figure 2. Fluorescent intensity at 470 nm of probe TPP in the
mixture of PBS/DMF (v:v = 7:3) solutions to different kinds of
reactive species (200 μM), λex = 345 nm; slits : 5/5 nm.
(B)
Mechanism Study. Based on the well-established sensing
mechanism reported previously,50-58 the reaction process of
probe TPP towards O2•- has been proposed in Scheme 2. It is
20 EQ O2-
0 EQ O2-
•-
hypothesized that the nucleophilic O2 first reacted with the
electron-deficient phosphinyl phosphorus. Subsequently, the
relatively weak π bond of the phosphinyl group was broken
leading to the formation of the negative-charged oxygen. Then
P=O band was reformed accompanied by the generation of
side products, diphenylphosphinic acid (DPPA) and highly
fluorescent 5-(dimethylamino)naphthalene-1-thiol NAT. In
order to further verify this proposed mechanism, the mixture
300
400
500
Wavelength / nm
•-
of probe TPP and O2 in PBS/DMF solutions was monitored
Figure 1. (A) Fluorescence spectra of probe TPP in the mixture
of PBS/DMF (v:v = 7:3) was obtained upon additions of O2 (0-
200 μM); λex = 345 nm; slit : 5/5 nm; (B) UV-Vis spectra of probe
TPP in the mixture of PBS/DMF (v:v = 7:3) was obtained upon
additions of O2•- (0-200 μM).
•-
under high-resolution mass spectrometry (HRMS) (Figure S4).
The appearance of an evident peak located at m/z 203.1337 in
the mass spectrum is well consistent with the formed highly
fluorescent product NAT (calculated as 203.0769).
Importantly, the observed formation of peaks at m/z 219.1160
in the HRMS spectrum is deduced for the side product DPPA
(calcd. m/z 219.0575 for C12H12PO2).
Specificity Evaluation. Presence of excellent specificity is
another necessary condition for a qualified fluorescence probe, so
selective sensing property was carefully verified by screening
probe TPP with other relevant bioanalytes including ROS (1O2,
O
O
P
O
N
2Ph2P(O)OH
-
N
H2O2, HOCl, HOBr, TBO•, •OH), RNS (ONOO•, NO2 , NO•),
DPPA
biothiols (GSH, Cys, Hcy, H2S) and metal ions (Cu2+, Fe2+, Fe3+,
Mg2+, Zn2+). As shown in Figure 2, the evident fluorescence
enhancement was only observed in the presence of O2•-, while
other reactive species did not promote apparent phenomena. The
results indicate that probe TPP exhibited superior specificity
towards O2•- compared to other species.
N
-
O2
S
P
S
P
O
O
O
O
SH
NAT
•-
Scheme 2. The sensing mechanism of probe TPP and O2 .
Kinetic Study. To determine the reaction rate of probe TPP
•-
•-
towards O2 , time-dependent optical response was investigated.
O2 Sensing in living cells under TPM. Encouraged by the
good performance of probe TPP, the potential of the probe in
monitoring O2 inside the cells was further investigated.
Initially, the effects of different pH values on the fluorescence
response to O2 were explored. As the result shown in Figure
S5, the free probe displayed no fluorescence in the range of 3-
10. However, additons of O2 lead to the dramatic
enhancement of intensity at 470 nm in the range of 5 to 10.
As shown in Figure S3a, the free probe TPP displayed steady
fluorescence intensity with increasing time, which
demonstrated stable photostability. In contrast, the addition of
O2 caused a dramatic enhancement at the fluorescence
intensity of 470 nm, which reached the plateau after about 25
•-
•-
•-
•-
min (Figure S3a). The pseudo-first-order rate constant (kobs
)
•-
for the monitoring response of probe TPP towards O2 was
calculated as 1.0× 10−2 min−1 based on the kinetic data (Figure
S3b).
•-
The broad pH tolerance of probe TPP for the detection of O2
demonstrates TPP might have potential applications in the
biological systems.
The cytotoxicity test is another important component of
determining the use of the probe in biological systems. Thus,
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Page 4 of 8
range was used as detection window.
the survival rate of HeLa cells under different concentrations
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7
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of probe TPP was investigated. As the result in Figure S6, the
probe displayed low toxicity towards cells in the range of 0 to
10 μΜ. Subsequently, we conducted cell study to confirm the
applicability of TPP in the live system using two-photon
microscopy (TPM). The two-photon excited fluorescence
•-
O2 Sensing in living tissues under TPM. Finally,
•-
endogenous O2 measurement with probe TPP was attempted
in fresh rat hippocampal tissues. The average fluorescence
intensity of tissues incubated with probe TPP for 1 h in the
hippocampal CA3 regions was 10.94 while the intensity of the
tissues pretreated with PMA for 30 min was 24.44. When
stimulated with PMA, the average fluorescence intensity was
2-fold higher than in control tissues (Figure 4). This result
•-
spectra of TPP with O2 in vitro displayed similar behaviors
to those obtained by one-photon excitation (Figure 3h). After
staining of TPP in the cells, a maximum emission wavelength
at 452 nm was observed (Figure S7). Consequently, we could
observe the change in fluorescence in the cells at the emission
window of 400-600 nm. In addition, the two-photon excited
fluorescence (TPEF) intensity was maintained constant during
irradiation for 1 hour, demonstrating high photostability
(Figure S7).
showed
a similar tendency as in cells. These results
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demonstrate that probe TPP could sensitively monitor the
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endogenous O2•- level in live specimens.
(a)
(b)
•-
Subsequently, changes in endogenous O2 concentration were
monitored using probe TPP. The TPM imaging study was
conducted in Raw 264.7 macrophages. Phorbol myristate
•-
acetate (PMA) was used for generating endogenous O2 by
stimulation.70 The average fluorescence intensity of Raw 264.7
cells stained with probe TPP was 31 and cells stained with
probe TPP after PMA stimulation was 63 respectively (Figure
3a and 3b). As expected, cells labeled with probe TPP after
PMA stimulation showed 2-fold greater fluorescence intensity
than without PMA pre-treatment. Fluorescence intensity of
cells, which was treated with superoxide scavenger (Tiron),
was similar with the control (Figure 3c and g). Based on these
results, we confirmed that probe TPP can be applied to detect
endogenous O2•- levels (Figure 3).
45 μm
(c)
(d)
35
30
25
20
15
10
5
Control
8 g/mL PMA
(a) Control
(b) PMA
(c) PMA + Tiron
Figure 4. (a), (b) TPM images of 20 μM probe TPP stained to rat
hippocampal slice tissues; (a) Tissues stained with 20 μM probe
TPP for 1 h at 37 C; (b) Tissues stained with 20 μM probe TPP
o
for 1 h at 37 oC after PMA stimulation for 30 min; (c) Bright-field
images (10X magnification) of hippocampal slice. Regions of
CA1 and CA3 were marked.; (d) Bar graphs about mean TPEF
intensities in (a), (b); For acquiring TPM tissue imaging in the
regions of CA3, 400-600 nm range was used as detection window
and the excitation was 740 nm.
(d)
(e)
(f)
CONCLUSION
In conclusion, a new type of turn-on fluorescence probe TPP
was
(dimethylamino)naphthalene-1-thiol for the detection of
superoxide radical anion (O2 ). The phosphinothioate moiety
has been employed as a reactive group for the first time. The
probe is easily accessible through a one-step sequence. Upon
addition of O2 , a 20-fold enhancement in fluorescent intensity
was induced with high specificity and sensitivity. Moreover,
the valuable biological potential was demonstrated by the fact
that TPP could successfully detect endogenous O2 in the
25 μm
designed
and
synthesized
based
on
5-
(g) 80
(h) 40
20 EQ
35
30
25
20
15
10
5
70
60
50
40
30
20
•-
0
EQ
•-
10
0
300
400
500
600
700
Control
PMA + Trion
1 g/mL PMA
Wavelength / nm
•-
living cells and living tissues under two-photon microscopy.
These results illustrate that TPP could be used as a promising
Figure 3. TPM images of 5 μM TPP stained to Raw 264.7 cells.;
(a) Cells stained with 5 μM probe TPP for 30 min at 37 °C; (b)
Cells stained with 5 μM probe TPP for 30 min at 37 °C after
PMA stimulation for 30 min; (c) Cells stained with 5 μM probe
TPP for 30 min at 37 °C after pretreating with 1 μg/mL PMA and
10 μM Tiron for 30 min.; (d-f) are bright field images of (a-c); (g)
Bar graphs about mean TPEF intensities in (a-c); (h) Two photon
excited emission spectra of probe TPP in the mixture of
PBS/DMF (v:v = 7:3) were obtained upon additions of O2•- (0-200
μM). Spectra were obtained using CCD detector and the
excitation was 740 nm. For collecting TPM images, 400-600 nm
•-
fluorescence probe for the detection of O2 and this work
provides a new design strategy of fluorescence O2•- probes.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
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Extraction of mechanism study, the synthetic pathway, NMR and
Mass spectrum of the probe
(12) Lu, N.; Zhang, Y.; Li, H.; Gao, Z., Oxidative and nitrative
modifications of α-enolase in cardiac proteins from
diabetic rats. Free. Radical. Bio. Med. 2010, 48, 873-881.
(13) Zhuang, X.; Liu, S.; Zhang, R.; Song, F.; Liu, Z.; Liu, S.,
Identification of unfolding and dissociation pathways of
superoxide dismutase in the gas phase by ion-mobility
separation and tandem mass spectrometry. Anal. Chem.
2014, 86, 11599−11605.
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AUTHOR INFORMATION
Corresponding Author
*Juyoung Yoon: jyoon@ewha.ac.kr; Fax: 82-2-3277-2385.
* Hwan Myung Kim: kimhm@ajou.ac.kr
7
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(14) Dondurmacıoğlu, F.; Avana, A. N.; Apak, R.,
Simultaneous detection of superoxide anion radicals and
determination of the superoxide scavenging activity of
Author Contributions
§ These authors contributed equally.
antioxidants
using
a
N,N-dimethyl-p-phenylene
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ACKNOWLEDGMENT
(15) de Campos, R. P. S.; Siegel1, J. M.; Fresta, C. G.; Caruso,
G.; da Silva, J. A. F.; Lunte, S. M., Indirect detection of
superoxide in RAW 264.7 macrophage cells using
microchip electrophoresis coupled to laser-induced
fluorescence. Anal. Bioanal. Chem. 2015, 407, 7003-7012.
(16) Liu, H.; Zhang, L.; Chen, J.; Zhai, Y.; Zeng, Y.; Li, L., A
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This study was supported financially by a National Research
Foundation of Korea (NRF) grant funded by the Korea
government (MSIP) (No. 2012R1A3A2048814 for J. Yoon and
No. 2019R1A2B5B03100278 for H. M. Kim). The Korea Basic
Science Institute (Western Seoul) is acknowledged for the LC/MS
data. FAB mass spectral data were obtained from the Korea Basic
Science Institute (Daegu) on a Jeol JMS 700 high resolution mass
spectrometer.
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20-Fold Turn-On Enhancement
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