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Accepted Manuscript
A novel mitochondria-targeted two-photon fluorescent probe for dynamic and
reversible detection of the redox cycles between peroxynitrite and glutathione
Chunlong Sun, Wen Du, Peng Wang, Yang Wu, Baoqin Wang, Jun Wang, Wenjun
Xie
PII:
S0006-291X(17)32101-0
DOI:
10.1016/j.bbrc.2017.10.123
Reference:
YBBRC 38740
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 22 October 2017
Accepted Date: 23 October 2017
Please cite this article as: C. Sun, W. Du, P. Wang, Y. Wu, B. Wang, J. Wang, W. Xie, A novel
mitochondria-targeted two-photon fluorescent probe for dynamic and reversible detection of the redox
cycles between peroxynitrite and glutathione, Biochemical and Biophysical Research Communications
(2017), doi: 10.1016/j.bbrc.2017.10.123.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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A novel mitochondria-targeted two-photon fluorescent probe for
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dynamic and reversible detection of the redox cycles between
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peroxynitrite and glutathione
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Chunlong Suna, 1∗, Wen Dua, 1∗∗, Peng Wangb, Yang Wuc, Baoqin Wanga, Jun Wanga,
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Wenjun Xiea
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a. School of biotechnology, Key Laboratory of Instrumental Analysis of Binzhou City, Shandong
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Provincial Key Laboratory of Eco-environmental Science for Yellow River Delta, Binzhou
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University, Binzhou 256603, China
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b.Department of Biomedical Engineering, School of Engineering, China Pharmaceutical University,
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Nanjing, 210009, China
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c. Research Center of Clinical Oncology, Jiangsu Cancer Hospital, Nanjing 210009, China
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Abbreviations: ONOO−, peroxynitrite; GSH, glutathione; GSSG, glutathione
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disulfide; ROS reactive oxygen species; D-π-A, donor-π-acceptor; OP, One-photon;
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TP, Two-photon; MTT, 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium
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bromide; L929, Mouse fibroblast cell line; MCF-7, human breast adenocarcinoma cell
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line; Colo 205, human colon adenocarcinoma cells; PET, photoinduced electron
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transfer; SIN-1, 3-morpholinosydnonimine
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*Corresponding author. Tel.: +86-0543-3190096; E-mail address: sunchunlong2016@163.com
(Chunlong Sun)
** Corresponding author. Tel.: +86-0543-3190096; E-mail address:duwen6688@163.com (Wen
Du)
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These authors contribute equally.
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Abstract
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Redox homeostasis is important for maintenance of normal physiological functions
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within cells. Redox state of cells is primarily a consequence of precise balance between
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levels of reducing equivalents and reactive oxygen species. Redox homeostasis
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between peroxynitrite (ONOO−) and glutathione (GSH) is closely associated with
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physiological and pathological processes, such as prolonged relaxation in vascular
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tissues and smooth muscle preparations, attenuation of hepatic necrosis, and activation
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of matrix metalloproteinase-2. We report a two-photon fluorescent probe (TP-Se)
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based on water-soluble carbazole-based compound, which integrates with organic
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selenium, to monitor changes in ONOO−/GSH levels in cells. This probe can reversibly
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respond to ONOO− and GSH and exhibits high selectivity, sensitivity, and
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mitochondrial targeting. The probe was successfully applied to visualize changes in
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redox cycles during ONOO− outbreak and antioxidant GSH repair in cells. The probe
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will lead to significant development on redox events involved in cellular redox
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regulation.
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Key Words
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Redox; Two-photon fluorescent probe; Mitochondria; TP-Se; Fluorescence imaging
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1. Introduction
Mitochondria, the principal energy-producing compartments in most cells, play roles
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in numerous vital cellular processes [1]. Endogenous peroxynitrite (ONOO−) has been
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recognized as a strong oxidant agent in cells, especially in mitochondria. In vivo, high
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concentrations of ONOO− are produced from the fast reaction with nitric oxide (NO)
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and superoxide anion (O2•−), and this reaction doesn't have to be enzymatic. The
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ONOO− is relatively stable, but the acid form (ONOOH) rapidly become to nitrate.
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Although the half-life of the acid form (ONOOH) is very short, the oxidative species
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comes into being a unique biological oxidation_reduction cycle indicating human
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health and disease [2, 3]. ONOO− has also been shown as a nitrating agent that causes
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nitrative stress in cells, but more investigations concerning endogenous ONOO− have
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been devoted to modulating signal transduction pathways via its ability to form nitrate
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biomolecules, including nitrated tyrosine residues, 8-nitroguanosine, and nitro fatty
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acids, to influence cellular processes[4-7]. Intracellular thiols provide abundant
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reducing sources, which are central to cellular redox homeostasis in antioxidant defense
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systems. Glutathione (GSH) is the most abundant endogenous thiol, whose
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concentration ranges from 1 mM to 15 mM depending on cell type [8-11]. GSH
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controls redox homeostasis through equilibrium between its reduced (GSH) and
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disulfide forms (GSH disulfide (GSSG)). Formation and reactions of intracellular
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peroxynitrite primarily occur mitochondria [12-15], wherein GSH exists predominantly
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in reduced form at a GSH/GSSG molar ratio of >100:1[16, 17]. ONOO− can
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significantly perturb mitochondrial GSH/GSSG ratio and cause irreversible damage to
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respiration. Interpreting regulation and interplay between peroxynitrite and Glutathione
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can also reveal physiological and pathological roles of endogenous peroxynitrite.
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ONOO− is modulated by cellular antioxidant defense systems, and the selenium (Se)
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plays an important role as an active target of the antioxidant enzyme glutathione
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peroxidase (GPx) [18, 19]. GPx can catalyze the reduction of ONOO− by GSH via a
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particular ping-pong mechanism [20]. Small artificial organic molecule probes
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emerged as one of the most powerful biotechniques for detecting physiologically active
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species in living systems with high temporal and spatial resolution [21]. Not only are
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these probes confirmed useful in detection of disease states, but they also allow for
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screening-type analysis of potential signal transduction pathways in cells. Therefore,
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we strive to develop a probe that can respond reversibly to changes in redox
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homeostasis regulation via a redox-based mechanism for visualizing states of these
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redox cycles.
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One-photon (OP) fluorescence imaging offers a powerful approach to monitor
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intracellular ONOO− levels owing to its high sensitivity [22]. However, existing
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ONOO− fluorescent probes used for cell imaging feature some drawbacks. For instance,
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small molecular fluorescent probes frequently suffer from interference from other
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reactive oxygen species (ROS) [23], delayed response time, and irreversible reactions
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[24]. Two-photon (TP) imaging possesses distinct advantages, including increased
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penetration depth and reduced specimen photodamage, due to its excitation that utilizes
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two low-energy photons [25]. Although several TP probes have been used for imaging
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thiols [26], H2O2 [27], and metal ions [28], no report exists on TP imaging of ONOO− in
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live cells. For ideal monitoring of ONOO− fluxes, fluorescent probes must offer more
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substantial penetration depth, less photodamage, and high sensitivity, instantaneous
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response, and reversibility considering the extremely short lifetime and low
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concentrations of intracellular ONOO−.
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Therefore, a novel probe with features of TP fluorescence imaging should be
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for dynamically monitoring fluctuations
of redox
selectively,
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instantaneously, and reversibly. This imaging method not only can considerably
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improve accuracy of ONOO− detection but can also be adopted flexibly according to
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depths of various samples. To date, no reports have been published regarding the use of
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TP fluorescent probes in imaging redox, especially in mitochondria. After confirming
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the key point of our research, we designed and synthesized a single fluorescent TP-Se
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probe for reversible detection of ONOO− and GSH (Scheme 1) [29-31].
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O
Se
Se
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N
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ONOO
N
N
GSH
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N
TP-Se
TP-SeO
Scheme 1. Structures of TP-Se and TP-SeO.
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A novel TP fluorophore was utilized based on electron-donating carbazole and
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electron-withdrawing cationic pyridinium moiety. This excellent intermolecular charge
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transfer system will guarantee TP activity of the probe [32-35]. As for TP-Se, methyl
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pyridinium moiety as mitochondria-targeted functional group was conjugated with
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carbazole to identify location of mitochondria [36, 37] and also provided the probe with
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good water solubility. Considering these advantages, we mimicked the catalytic cycle
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and developed a TP probe containing an organoselenium moiety that can be used for
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reversible peroxynitrite detection.
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2. Experimental
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2.1. Apparatus and Materials
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Two-photon excited fluorescence spectra were measured using a Tsunami 3941: Ti:
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sapphire femtosecond laser as exciting light source (865 nm) with a pulse width of
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<145 fs and a repetition rate of 75 MHz, and USB2000 (OCEAN OPTICS) was used as
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the recorder. A UV-Vis Spectrophotometer (SHIMADZU, Japan) was used to perform
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the absorbance measurements. A Nikon confocal laser-scanning microscope (NIKON,
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Japan) with an objective lens was used to perform the florescence measurements.
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Compounds 2 and 3 were synthesized in our laboratory.
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2.2. Design and Synthesis of two-photon fluorescent probe TP-Se
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Scheme 2. Synthetic route of two-photon fluorescent probe TP-Se.
Scheme 2 shows the synthetic route of designed two-photon fluorescent probe
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TP-Se. Compound 2 (0.6 mmol) and Compound 3 (1.80 mmol) were dissolved in
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Methanol (150 ml). The solution was stirred under argon at 55°C for 3 h. The crude
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product was collected as powder. After recrystalization from methanol, title products
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(TP-Se) were obtained as a brick-red solid. (Yield, 56.1%). 1H-NMR (300MHz,
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DMSO-d6), δ (ppm): 8.94 (s, 1H), 8.78 (d, 2H), 8.66 (s, 1H), 8.18 (d, 1H), 8.09 (m, 3H),
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7.97 (d, 2H), 7.50 (d, 1H), 7.44 (m, 4H), 7.33 (d, 2H), 7.05 (s, 1H), 6.95 (s, 2H), 4.53 (s,
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2H), 4.39 (s, 3H), 1.29(t, 3H). TOF-MS m/z: calcd. For C35H30N3Se+: 571.6. (1H NMR
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and TOP mass spectra see Supplementary Fig. 1)
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2.3. Cell Culture
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Human hepatocellular liver carcinoma cell line and mouse macrophage cell line
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(RAW264.7) were maintained following protocols provided by the American-type
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Tissue Culture Collection. Cells were seeded at a density of 1 × 106 cells mL-1 for
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confocal imaging in Roswell Park Memorial Institute 1640 medium supplemented with
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10% fetal bovine serum, NaHCO3 (2 g/L), and 1% antibiotics (penicillin/streptomycin,
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100 U/ml). Cultures were maintained at 37 °C under a humidified atmosphere
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containing 5% CO2.
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3. Results and discussion
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3.1. Investigation of detecting mechanism
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As shown in Scheme 1, structure of TP-Se was confirmed by time-of-flight (TOF)
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mass analysis. To confirm reaction mechanisms, TOF mass spectra were used to
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investigate oxidation of TP-Se by ONOO−. A peak corresponding to [TP-Se-I−]+
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appeared at m/z = 572.2 (Supplementary Fig. 1e). When 20 equiv. of ONOO− was
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added to the solution, a peak corresponding to [TP-SeO-I−]+ appeared at m/z=588.1,
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demonstrating complete oxidation of Se (Scheme 1) (Supplementary Fig. 1f). Mass
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results confirmed that the Se-containing probe (TP-Se) was converted to corresponding
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TP-SeO under addition of ONOO−.
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Following the ping-pong mechanism [31], we confirmed a new TP-reversible
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fluorescent probe (TP-Se) for detection of ONOO− in living cells through rapid
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photoinduced electron transfer (PET). Fluorescence of TP-Se was quenched as a result
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of PET between the modulator and transducer, but Se oxidation prevented PET,
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“switching on” fluorescence emission (Scheme 1).
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3.2. Absorption analysis
Absorption spectra were obtained using UV-visible spectrophotometer at 25 °C.
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TP-Se probe (1 ml, 0.01 mM) was added to 1 ml color comparison tubes and
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investigated through absorption spectroscopy in PBS buffer (50 mM, pH 7.4) with 1%
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DMSO. Mixtures were equilibrated for 5 min before measurement. All tests were
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performed in the presence of 0.10 M NaCl to maintain constant ionic strength. As
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shown in Figure 1a, absorption of TP-Se was 445 nm, showing a blue excitative peak
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(TP-Se) similar to the red one (TP-Se+ 20 equiv. ONOO−).
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3.3. Fluorescence analysis
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Fluorescence spectra were determined using a spectrofluorometer. TP-Se probe (1
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ml, 0.01 mM) was added to 1 ml color comparison tubes and investigated through
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fluorescence spectroscopy in PBS buffer (50 mM, pH 7.4) with 1% DMSO. Mixtures
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were equilibrated for 5 min before measurement. Fluorescence intensity was measured
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at λex=565 nm. All tests were performed in the presence of 0.10 M NaCl to maintain
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constant ionic strength. As shown in Figure 1 (b, c, and d), TP-Se displayed almost no
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fluorescence in the solution possibly due to weakly radiative deactivation of the excited
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state through the Se group. Accordingly, emission intensity at 565 nm increased with
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continuous addition of ONOO− to TP-Se solution and upon excitation at 430 nm and
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reached its maximum when 25 equiv. of ONOO− was added. UV spectra (Figure 1a) of
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ONOO− displayed a similar sensing behavior toward addition of more ONOO−. A good
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linear relationship of emission intensity against ONOO− was observed from 0 equiv. to
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25 equiv. (inset of Figure 1c), and detection limit (3 σ/k, where σ refers to standard
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deviation of blank measurement, and k is the slope between fluorescence intensity
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versus ONOO− concentration.) was as low as 31 nM. Results showed that ONOO−
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probe possesses good sensitivity for detecting ONOO−. We also monitored
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fluorescence changes with time when ONOO− (25 equiv.) was added to the solution of
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TP-Se probe. As shown in Figure 1a, maximum fluorescence intensity was obtained in
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2 min. All fluorescence tests were performed after 30 min.
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3.4. TP fluorescence measurement
TP cross-sections were calculated using TP-induced fluorescence measurement. In
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the range of 750 nm to 940 nm, maximum TP absorption of cross-sections (δmax) of
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TP-Se appearing at 865 nm reached 1775 GM (Figure 1e). Emission spectra of TP-Se
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(10 µ M) with addition of ONOO− (0–25 equiv.) were tested under TP excitation (λex=
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865 nm) (Figure 1f). Results indicated that TP-Se can function well as TP fluorescent
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probe for ONOO− in living systems. TP-excited fluorescence titration experiment was
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also conducted for TP-Se. Under TP excitation (λex= 865 nm), TP-excited
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fluorescence spectra were enhanced upon addition of ONOO−.
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Photophysical properties of TP-Se were tested under simulated physiological
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conditions (Tris buffer, pH 7.4). TP-Se emits at around 565 nm and is excited at 430
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(OP, Figure 1d) and 865 nm (TP, Figure 1f).
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3.5. Selectivity toward peroxynitrite at various pH values
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We also evaluated the effect of pH on fluorescence. In living cells, different
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organelles feature varying pH values (Figure 2a). (1) Cytosol works at pH values of
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6.80 to 7.40, whereas (2) acidic organelles (e.g., lysosomes and endosomes) function in
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the pH range of 4.5 to 6.0 [31]. In the absence of ONOO−, fluorescence intensity of
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TP-Se remained unchanged at a pH range of 5.0 to 10.0, indicating that TP-Se was
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hardly influenced by pH. Meanwhile, upon addition of ONOO−, unchanged and strong
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fluorescence signals were detected in the pH range of 5.0 to 10.0, which also covers the
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pH range of physiological environments. Therefore, combined with results of its
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outstanding response behavior, TP-Se exhibits robust analytical potential for biological
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applications.
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3.6. Selectivity of TP-Se and in vitro cytotoxicity studies
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To confirm selectivity of TP-Se for ONOO−, we investigated its responses toward
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competing ROS (ClO−, O2•−, •OH, 1O2, •NO, H2O2, and ONOO−) and metal ions
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(including Fe3+, Fe2+, Cu2+, Cu+, Zn2+, and Mg2+). Test solutions of TP-Se (10 µM) in
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pH 7.4 phosphate-buffered saline (PBS) (containing 1% DMSO) were prepared.
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Solutions of various test species were prepared by diluting the stock solution with
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PBS buffer solution. Different anions were prepared with metal ions (10-500 µM)
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forming ZnCl2, MgSO4, CuCl2, CuNO3, FeCl3, FeCl2, and ONOO− (10-500 µM).
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Various ROS (10-100 µM), including ClO−, •OH, O2•−, 1O2, •NO, H2O2, and ONOO−,
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were prepared according to a previously reported procedure. Results showed that
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TP-Se exhibited high selectivity for ONOO− and was unperturbed upon addition of
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other ROS and metal ions (Figures 2b, c). TP-Se displayed an instantaneous response
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and good photostability (Figure 2b, c, and d). Thus, between TP-Se and intracellular
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ONOO−, reaction features, including selectivity, reversibility, and instantaneity, endow
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TP-Se with capability to probe ONOO− dynamically in cells. And in vitro cytotoxicity
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studies of TP-Se were performed on L929, MCF-7, and Colo 205 cells by MTT assays.
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These results suggest that TP-Se is a safe and non-toxic probe (Supplementary Fig. 2).
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3.7. Fluorescence recovery rate with various reducing materials.
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Given that various types of antioxidants are contained in cells, an additional test was
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performed on the probe to determine whether other reducing species will act as
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interferents. As shown in Figure 3a, TP-Se probe displayed excellent selectivity
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response to thiols, which are the main antioxidants in vivo at an intracellular
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concentration of ~5 mM [38, 39]. Average fluorescence recovery rate reached up to
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~96.0% for reduction by GSH and L-cysteine.
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3.8. Reversibility of probe in Solution
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Fluorescence responses of TP-Se (10 µM) to redox cycles were normalized. TP-Se
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was oxidized by 1 equiv. of added ONOO−. After 7 min, the solution was treated with 2
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equiv. of GSH. When fluorescence returned to initial levels, another 1 equiv. of
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ONOO− was added to the mixture. Redox cycle was repeated four times. ONOO−
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originates from a peroxynitrite donor, 3-morpholinosydnonimine (SIN-1). All data
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were acquired from 50 mM PBS (acetonitrile 5% v/v) at pH 7.4 (λex= 445 nm, λem = 565
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nm). Figure 3b shows that reversible oxidation–reduction cycle can be repeated at least
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four times with only a modest fluorescence decrement (18% of TP-SeO was bleached
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during the process). Altogether, results showed that TP-Se is suitable for mitochondrial
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peroxynitrite-reversible monitoring in living cells.
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3.9. TP bio-imaging in mitochondria
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To further investigate subcellular localization of TP-Se, HeLa cells and a
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commercially available mitochondrial dye (MitoTracker Red FM) were employed for
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co-localization study. Cells were treated with TP-Se (10 µM) for 30 min and
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subsequently with ONOO− (20 equiv.) for 15 min and then treated with MitoTracker
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Red FM (0.6 µM) for 30 min. Repeatability of correlation plot, which describes the
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distribution between green (Figure 4A-a) and red channels (Figure 4A-b), revealed high
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co-localization in mitochondria. Results indicated that TP-Se was specifically
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site-internalized in mitochondria, and it can be used to detect mitochondrial ONOO−
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under TP excitation in living cells.
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3.10. Confocal fluorescence images of reversible redox cycles in living cells.
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We then tested whether the probe can be used to monitor ONOO−-reversible redox
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cycles in living cells. Living RAW264.7 cells loaded with 10.0 µM TP-Se for 5 min
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showed only faint fluorescence (Figure 4B-a). However, incubating the same
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TP-Se-loaded cells with 10.0 µM 3-morpholinosydnonimine (SIN-1), a peroxynitrite
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donor [40], resulted in substantial increase in intracellular fluorescence after 10 min as
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the probe detected oxidative stress (Figure 4B-b). Cells were then treated with the ROS
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scavenger GSH S-transferase (GST, 250 units/mL). After 10 min, intracellular
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fluorescence decreased to faint levels as GST reduced cell environment (Figure 4B-c).
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Addition of a second aliquot of SIN-1 oxidant resulted in another burst of oxidative
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stress and an increase in intracellular fluorescence (Figure 4B-d). These experiments
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demonstrated that low levels of intracellular fluorescence, as shown in Figure 4B, were
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not due to photobleaching or dye leakage. Findings suggest that TP-Se can sense redox
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cycles through reversible fluorescence responses in living cells.
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4. Discussion
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To conclude, we have developed a new Two-photon reversible fluorescent probe
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(TP-Se), that exhibits good sensitivity and selectivity in monitoring ONOO− oxidation
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and GSH reduction events under physiological conditions in living cells without
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interference from other biologically relevant species. The probe effectively hinders
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influence of autofluorescence in biological systems and poses minimal toxicity to cells.
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TP-Se can also be used for real-time imaging of living cells. Our results show that
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TP-Se probe can be used to visualize mitochondrial peroxynitrite levels with negligible
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background fluorescence and cellular toxicity.
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Funding
This work was supported by Shandong Provincial Natural Science Foundation
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[ZR2016BL16, ZR2016CL01], China; A project of Shandong Province Higher
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Educational Science and Technology Program [J17KA120]; Doctor Foundation of
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Binzhou University [2016Y17, 2016Y02].
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Supporting information
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H NMR and Mass spectra see Supplementary Fig. 1. Measurement of TP
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cross-sections, Confocal Imaging, and MTT assay see Supplementary Information.
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Figure 1. (a) Absorption spectra of TP-Se (1 ml, 0.01 mM) in the absence and presence of ONOO−
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(20 equiv.) in PBS buffer (50 mM, pH 7.4) with 1% DMSO. (b) Time-dependent fluorescence
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intensity changes of TP-Se (1 ml, 0.01 mM) with addition of ONOO− (25 equiv.) in PBS buffer. (c)
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The relationship between fluorescence intensity and ONOO− concentrations and under UV light
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(λex= 430 nm). (d) Fluorescence spectra of TP-Se (1 ml, 0.01 mM) in the presence of increasing
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amount of ONOO− (0–25 equiv.) in PBS buffer (50 mM, pH 7.4) with 1% DMSO, λex= 430 nm). (e)
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TP absorption of TP-Se cross-sections (10 µM) with addition of ONOO− (25 equiv.). (f) Emission
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spectra of TP-Se (10 µM) with addition of ONOO− (0-25 equiv.) under TP excitation (λex= 865 nm).
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Figure 2. (a) Fluorescence intensity change in TP-Se (1 µM) with addition of ONOO− (15 µM)
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under different pH values (5.0, 6.0, 7.0, 7.4, 8.0, 9.0, and 10.0) by using universal buffer solution
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(0.1 mM citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M Tris, 0.1 M KCl) with 1% DMSO.
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λex/λem = 445/565 nm. (b and c) Fluorescence responses of TP-Se (10 µM) to various metal ions.
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Data were acquired in 0.03 M Tris, pH 7.4, with λex = 430 nm. (d) Fluorescence responses of
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TP-Se (10 µM) to various ROS and nitrogen species. Data were acquired in 0.03 M Tris, pH 7.4,
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with λex = 430 nm.
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Figure 3. (a) Fluorescence recovery rate with various reducing materials. TP-Se (10.0 µM) was
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oxidized by addition of 1 equiv. of ONOO−. Then, the solution was treated with various reducing
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materials for 20 min: 1. metallothionein (80.0 µM); 2. vitamin C (80 µM); 3. L-cysteine (25.0 µM);
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4. GSH (25.0 µM); 5. vitamin E (80 µM); 6. uric acid (80 µM); 7. tyrosine (80 µM); 8. histidine (80
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µM). Fluorescence recovery percentage is defined as (F–F0)/F ×100%, here F corresponds to
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fluorescence intensity of TP-SeO (10.0 µM), and F0 is fluorescence intensity of the probe after
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addition of reducing material. (b) Normalized fluorescence responses of TP-Se (10 µM) to redox
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cycles. TP-Se was oxidized by 1 equiv. of added ONOO− (blue lines). After 7 min, the solution was
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treated with 2 equiv. of GSH (red lines). Red lines represent normalized intensities obtained after
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treatment with 2 equiv. of GSH.
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Figure 4. Confocal fluorescence images of HeLa cells. Confocal fluorescence images of HeLa
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cells after treatment with TP-Se (10 µM) for 30 min and subsequently with ONOO− (20 equiv.) for
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15 min and then with MitoTracker Red FM (0.6 µM) for 30 min. (A-a) Green emission (530–570
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nm), λex= 865 nm; (A-b) red emission (580–665 nm), λex= 644 nm. Confocal fluorescence images
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of reversible redox cycles in living RAW264.7 cells. (B-a) RAW264.7 cells loaded with 10.0 µM
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TP-Se for 5 min. (B-b) Dye-loaded cells treated with 10.0 µM SIN-1 for 10 min. (B-c) Dye-loaded,
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SIN-1-treated cells incubated with GST for 10 min. (B-d) Cells exposed to a second dose of SIN-1
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for an additional 10 min. (B-e) Dye-loaded, SIN-1-treated cells incubated with a second dose of
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GST (250 units/mL) for 10 min. (B-f) Bright-field image of (B-a). (B-g) Relative the fluorescent
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intensity of TP-Se-labeled cells in panels B(a−e). Cells shown are representative images from
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replicate experiments (n = 5).
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TP-Se
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TP Fluorescence
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← ONOO¯ added
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Figure 2.
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TP-Se
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OClˉ
Fe²⁺
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Highlights
1. We report a two-photon fluorescent probe (TP-Se) for optical imaging.
2. TP-Se can monitor reversibly changes in ONOO−/GSH levels in mitochondria.
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3. TP-Se will lead to significant development on redox events in cellular redox
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regulation.
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