Biosensors and Bioelectronics 64 (2015) 285–291
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A ratiometric fluorescent probe based on a coumarin–hemicyanine scaffold for sensitive and selective detection of endogenous peroxynitrite Xin Zhou a,b, Younghee Kwon c,d, Gyoungmi Kim a, Ji-Hwan Ryu c,d,n, Juyoung Yoon a,nn a
Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Republic of Korea Department of Chemistry, Faculty of Science, Yanbian University, People's Republic of China c Research Center for Human Natural Defense System, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea d BK 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 19 June 2014 Received in revised form 17 August 2014 Accepted 29 August 2014 Available online 8 September 2014
In the study described herein, the red emitting probe CHCN, which possesses a linked coumarin– hemicyanine scaffold, was developed for detection of peroxynitrite (ONOO ) under physiological conditions. The studies show that CHCN displays a dual ratiometric and colorimetric response to ONOO that is caused by an oxidation process. A possible mechanism of this oxidation process was proposed and confirmed by ESI-MS spectra for the first time. CHCN shown highly selective and sensitive towards ONOO with a low limit of detection LOD (49.7 nM). Moreover, CHCN has appreciable cell permeability and, as a result, it is applicable to ratiometric detection of exogenous and endogenous ONOO in living cells during phagocytic immune response. We anticipate that, owing to their ideal properties, probes of this type will find great use in explorations of the role played by ONOO in biology. & 2014 Elsevier B.V. All rights reserved.
Keywords: Peroxynitrite sensor ONOO sensor Reactive oxygen species probe Fluorescent chemosensor Cell imaging
1. Introduction Reactive oxygen species (ROS) and nitrogen species (RNS) play important roles in physiology and pathology (Chen et al., 2011; Dickinson and Chang, 2011; Pacher et al., 2007; Winterbourn, 2008; Xu et al., 2013; Yang et al., 2010). Among ROSs and RNSs, peroxynitrite (ONOO ) has received special attention owing to its unusually potent oxidizing ability and strongly nucleophilic character (Radi, 2013). As pointed out in recent reports, ONOO is widely considered to be an important factor in the onset and progression of many cellular processes (Dickinson and Chang, 2011). For example, as a strong nucleophile, this species reacts rapidly with carbon dioxide in vivo to form the nitrosoperoxycarbonate (ONOOCO2 ) intermediate. ONOOCO2 then rapidly decomposes homolytically to generate carbonate radical (CO3 ), which is believed to cause specific cellular damage (apoptotic or necrotic cell death) (Radi, 2013). As a potent oxidant, ONOO reacts directly with electron-rich moieties. This process causes n Corresponding author at: Research Center for Human Natural Defense System, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea. nn Corresponding author at: Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120120-750, Republic of Korea. Fax: þ 82 232773419. E-mail addresses: [email protected] (J.-H. Ryu), [email protected] (J. Yoon).
http://dx.doi.org/10.1016/j.bios.2014.08.089 0956-5663/& 2014 Elsevier B.V. All rights reserved.
damage to a wide array of biomolecules (e.g., DNA and proteins) (Kryston et al., 2011; Soon et al., 2011), which leads to many human diseases, such as septic stroke, cancer, diabetes, inflammatory diseases, and neurodegenerative disorders (Szabo et al., 2007). In contrast, growing evidence suggests that the wellcontrolled generation of certain ONOO serves as a signaling transduction factor for a wide range of normal cellular functions (Dickinson and Chang, 2011). Therefore, it is important to develop a clear understanding of the role played by ONOO in cellular functions. For this purpose, it is essential to develop highly sensitive and selective methods for accurate and direct detection of ONOO . However, as a consequence of its short half-life (o 20 ms) under typical physiological conditions (Yang et al., 2006), direct and unambiguous detection of ONOO in living cells can not be easily made by using traditional analytical methods (Chen et al., 2013b; Oushiki et al., 2010; Yu et al., 2012). In the past decade, a large number of fluorescent probes have been designed and constructed for specific detection of ONOO (Chen et al., 2011; Dickinson et al., 2010; Kalyanaraman et al., 2012). These probes are based on various strategies that rely on direct oxidation reactions of boronates (Chen et al., 2013b; Sikora et al., 2009; Sun et al., 2014), active ketones (Peng and Yang, 2010; Sun et al., 2009; Yang et al., 2006), metal complexes (Rausaria et al., 2011), selenium (Xu
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et al., 2011), tellurium (Koide et al., 2012; Yu et al., 2013), and agents (Ueno et al., 2006; Zhang et al., 2012). Despite the encouraging progress that has been made, a great challenge still exists in developing fluorescent probes with high sensitivities and selectivities that are able to distinguish between ONOO and other ROSs (e.g. ClO and H2O2) (Kalyanaraman et al., 2012; Zhang et al., 2012). Moreover, devising probes of this type, which selectively detect endogenous ONOO in vivo without disturbance being offered by the presence of innate reducing biomolecules such as biothiols, is a particular demanding task (Chen et al., 2013b; Yu et al., 2013). Recently, ratiometric type fluorescent probes, especially those that emit light in the near-infrared (NIR) region, have attracted increasing attention because of their intrinsic self-calibrating character and ability to be used for cell and body imaging (Bozdemir et al., 2011; Erbas-Cakmak et al., 2013; Guo et al., 2014; Kolemen et al., 2011; Wu et al., 2014; Yuan et al., 2010). However, compared to the numerous turn on/off fluorescence ONOO probes that have been developed to date, only a few ratiometric probes of this type have been described. In 2010, Nagano et al. reported that cyanine dyes (e.g. Cy5 and Cy7) are readily oxidized by various ROSs to form products that do not fluorescence. More importantly, the studies demonstrated that cyanine dyes display reactivities towards ROS that are governed by the lengths of their conjugated central polymethine chains. For example, Cy7 is more susceptible to ROS promoted oxidation than Cy5 (Oushiki et al., 2010). In spite of this phenomenon, the poor selectivities and the fluorescence quenching response of cyanine dye towards ROSs hinders their broad usage in biology. In order to overcome this limitation, we embarked on a program aimed at the development of a new kind of ratiometric florescence platform that relies on the utilization of a hybrid coumarin–hemicyanine dye. As has been reported recently, these types of hybrid dyes have been explored in the context of ratiometric fluorescent sensors for some common nucleophilic reagents, such as cyanide, sulfite and sulfide (Chen et al., 2013a; Lv et al., 2011; Sun et al., 2013). However, to our knowledge, hybrid coumarin–hemicyanine dyes have not been explored as sensing platforms for ROSs. Taking these factors into account, we have developed the ROS probe CHCN, a conjugated hybrid dye composed by a coumarin group and a hemi-cyanine moiety linked through a carbon-carbon double bond. The results of this effort show that CHCN can be employed to detect ONOO in a dual ratiometric and colorimetric manner with high sensitivity and selectivity. In addition, we have demonstrated an application of CHCN for ratiometric detection of exogenous and endogenous ONOO in living cells.
2.2. Generation of ROS/RNS H2O2 was from dilution of 28% solution in water. Tert-butyl hydroperoxide was from dilution of 70% solution in water. ROO∙ was generated from 2,2′-Azobis (2-amidinopropane) dihydrochloride. NO∙ was generated from SNP (Sodium Nitroferricyanide (III) Dihydrate). ∙O2 was generated by mixing xanthine (25 μM) and xanthine oxidase (3.2 mU/mL). ∙OH was from the reaction of Ammonium iron (II) sulfate (100 μM) and H2O2 (100 μM). NaClO was from dilution of 12% solution in water. ONOO was prepared according to previous literature and the concentrated was determined by absorbance at 302 nm (Xu et al., 2013). The above ROS or RNS were incubated with FBS in 50 mM KH2PO4 (pH 7.4) for 30 min respectively.
2.3. Cell-culture and confocal microscopy experiments The Human normal lung cell (WI38 VA13) cells and a macrophage cell line murine (RAW 264.7) were cultured on the surface of a glass slide in SPP medium (1% proteose peptone, 0.2% glucose, 0.1% yeast extract, 0.003% EDTA ferric sodium salt) at 37 °C in 5% CO2. The cells were subcultured by scraping and seeding on sixwell plates according to the instructions from the manufacturer. Cells were grown to confluence prior to experiment. For confocal microscopy experiments, WI38 VA13 cells were stained with CHCN (5 μM) for 30 min and washed with DPBS, and then treated with various concentration of exogenous ONOO . Then the cells were investigated by using a dual emission imaging mode. The green fluorescence channel images were collected at 490–540 nm with an excitation wavelength at 473 nm, and the red fluorescence channel images were collected at 575–675 nm with an excitation wavelength at 559 nm, respectively. Murine RAW 264.7 macrophage cells were then stimulated with lipopolysaccharide (LPS, 1 μg/mL) for 16 h, and interferon-γ (IFN-γ, 50 ng/mL) for 4 h, followed by phorbol 12-myristate 13-acetate (PMA, 10 nM) for further 30 min for production of endogens ONOO , or pretreated the cells with either a scavenger of superoxide, 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO, 100 μM), or an NO synthase inhibitor, aminoguanidine (AG, 1 mM), to reduce cellular endogenous ONOO . After that, the cells were stained with CHCN (5 μM) for 30 min, and then washed with DPBS for three times before imaging. The fluorescence images were obtained from a dual emission imaging mode as abovementioned.
2.4. Synthesis of CHCN 2. Materials and methods 2.1. General information of materials and methods Unless otherwise noted, all materials were obtained from commercial sources and were used without further purification. Solvents were dried according to standard procedures. 1H NMR and 13C NMR in CDCl3 were measured on a Bruker AM-300 spectrometer with tetramethylsilane (TMS) as internal standard. Mass spectra were obtained using a JMS-HX 110A/110A tandem mass spectrometer (JEOL). UV–vis spectra were obtained using a Scinco 3000 spectrophotometer (1 cm quartz cell) at 25 °C. Fluorescence spectra were recorded on RF-5301/PC (Shimada) fluorescence spectrophotometer (1 cm quartz cell) at 25 °C. Deionized water was used to prepare all aqueous solutions.
Diethylaminocoumarin-3-aldehyde (50 mg, 2 mmol) and 1,3,3trimethyl-2-methyl-eneindoline (60 mg, 2 mmol) were dissolved in absolute ethanol (20 mL) and refluxed for 10 h. After being cooled to room temperature, remove the ethanol under reduce pressure. The corresponding solid was further purified by silica gel column chromatography using DCM/Methanol (20/1, v/v) as eluent to afford CHCN as a deep violet solid (0.312 g, yield: 68%). 1 H NMR (300 MHz, CDCl3-d3) δ 10.05 (s, 1H), 8.60 (d, J¼ 16.2 Hz, 1H), 8.13 (d, J ¼9.3 Hz, 1H), 8.10 (d, J¼ 16.2 Hz, 1H), 7.52–7.63 (m, 4H), 6.72 (dd, J¼ 9.3 Hz, 1H), 6.47 (s, 1H), 4.31 (s, 3H), 3.54 (q, J¼ 7.1 Hz, 4H), 1.85 (s, 6H), 1.34 (t, J ¼7.1 Hz, 6H). 13C NMR (75 MHz, MeOD-d3): δ 158.1, 154.7, 150.3, 143.1, 140.3, 132.3, 128.7, 122.7, 113.8, 112.4, 110.0, 96.4, 78.1, 51.7, 45.1, 32.6, 25.4, 11.4. MS: [M þ ] at 401.23.
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3. Results and discussion 3.1. Design concept of CHCN The conceptual basis for the design of CHCN included the consideration that it be appropriately water soluble so that the sensing processes can be performed under physiological conditions. In addition, the extended π-conjugated system in CHCN was designed to ensure a considerably red shifted emission. It was also envisaged that the bridging double bond of CHCN would be oxidized by ONOO , leading to destruction of the extended conjugation, which would results in disruption of an intramolecular charge-transfer (ICT) process. As a result, the initial red emission of CHCN should disappear in the presence of ONOO and be replaced by green emission corresponding to coumarin moieties in the oxidation products. Therefore, a colorimetric, ratiometric fluorescent dual response to ONOO is expected using this probe. It is important to note that dyes of this type typically have appreciable cell permeability, as shown by mitochondria specific staining during subcellular imaging, and that they are inert to biothiols, such as Cys and GSH (Sun et al., 2013). 3.2. Ratiometric and colorimetric response towards ONOO As seen by viewing the pathway displayed in Scheme S1, CHCN was conveniently synthesized by direct condensation of an amino3-benzaldehyde derived coumarin-aldehyde and an indolium derivative by using a reported method (Sun et al., 2013). The water solubility and spectroscopic characteristics of CHCN were explored initially. As expected, CHCN was observed to display satisfactory water solubility. The fluorescence response of CHCN to ONOO was examined next. As shown in Fig. 1a, in PBS buffer solution (pH 7.4), CHCN emits red fluorescent with an emission maximum at 635 nm, which is a consequence of the extended πconjugated system present in this linked coumarin–hemicyanine dye. Upon addition of ONOO , the emission peak of CHCN at 635 nm decreases in concert with an increase of a new, more emissive band at 515 nm that corresponds to a blue color (Fig. 1a inset). Moreover, the ratio of the fluorescence intensities of CHCN at 635 and 515 nm linearly correlate with the concentration of ONOO with a correlation coefficient of R¼0.9939 and a detection limit of 49.7 nM (Fig. 1c) (Zhou et al., 2013). Furthermore, the results of absorption titration studies (Fig. 1b) show that upon addition of increasing amounts of ONOO , the main absorption band of CHCN at 568 nm decreases with a concurrent increase in a new band at 422 nm, corresponding to a coumarin chromophore. A clear isosbestic point occurs at 467 nm. As can be seen by viewing the inset in Fig. 1b, addition of ONOO to a PBS buffer solution of CHCN causes a color change from blue violet to faint yellow. The combined results demonstrate that CHCN serves as probe for ratiometric and colorimetric detection of ONOO . 3.3. Selectivity of CHCN towards ONOO To evaluate the selectivity of CHCN towards ONOO , fluorescence and absorption spectra of CHCN before and after addition of common ROSs, including ∙NO, ∙O2, H2O2, OCl , ∙OH, t-BuOO∙, as well as biothiols (Cys, Hcy and GSH), were recorded. As shown in Fig. 2a, under identical conditions, only ONOO causes an observable ratiometric fluorescent response of CHCN. Importantly, the other ROSs and biothiols do not promote observable changes in the spectra of CHCN even when present in 100 equivalent excesses. Moreover, the fluorescence intensity ratio (F515nm/F635nm) of CHCN (Fig. 2c) is much more greatly enhanced (474-fold higher) upon addition of ONOO in contrast to the other agents tested. The absorption spectra displays similar responses (Fig. 2b) as
Fig. 1. (a) Fluorescence spectra of CHCN (1 μM) upon addition of various amounts of ONOO (ranging from 0 to 20 μM) in PBS buffer solution (pH 7.4), each data set was recorded after 10 min with an excitation wavelength at 475 nm. Inset: fluorescent images of CHCN (1 μM) in the absence (A) and presence (B) of ONOO . (b) Absorption spectra of CHCN (1 μM) upon gradual addition of ONOO (0–20 μM) in PBS buffer solution (pH 7.4). Inset: colors of CHCN (10 μM) in the absence (A) and presence (B) of ONOO . (c) Linear correlation between the emission intensity ratio (F515 nm/F635 nm) and the concentration of ONOO . (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
exemplified by the observation that ONOO induced a distinct decrease in the absorption band at 568 nm, while hypochlorite (OCl ) causes only a slight reduction in the intensity of this band
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causes a similar ratiometric and colorimetric dual response of CHCN as does ONOO , but the rates at which the responses occur are much smaller, and excess amounts of OCl (up to more than 200 equiv.) is required to achieve the same spectra responses. These results indicate that, at low concentrations, CHCN displays a high selectivity toward ONOO over other ROSs. 3.4. Proposed mechanism for sensing of ONOO by CHCN As has been well demonstrated in previous studies (Oushiki et al., 2010), electron rich conjugated C ¼C double bonds of cyanine dyes undergo ready oxidative cleavage in the presence of ROSs. This process results in formation of a variety of double bond cleavage products, including 1,3,3-trimethyloxindole as the major product along with derivatives such as aldehyde and carboxylic acid species (Lou et al., 2013; Park et al., 2013). Based on the observation summarized above and the results arising from previous investigations, it is plausible that CHCN displays high sensitivity and selectivity towards ONOO because this ROS promotes oxidative cleavage of the central double bond. To gain experimental support for this proposal, the process taking place between CHCN and ONOO was monitored by using mass spectrometry (MS). Inspection of Fig. 3, shows that upon addition of ONOO the parent peak in the spectrum of CHCN at m/z 401.2 disappears and three new peaks at 417.1, 435.0, and 446.2 arise. The new peaks are ascribed to the three products CHCN–O, CHCN–O2H, and CHCN–NO2, generated initially by oxidation of CHCN (Scheme 1). MS monitoring shows that these products partially undergo further oxidation to generate the final products consisting of predominantly 1,3,3-trimethyloxiindole (m/ z 176.2), coumarin-3-aldehyde (m/z 246.3), the latter of which was isolated and identified by using 1H NMR spectroscopy (Fig. S5). The chemical reaction responsible for sensing was also explored by using 1H NMR titration experiments (Fig. S6). The results show that resonances for the double bond protons of CHCN at 8.03 and 7.98 ppm disappear after addition of ONOO . This observation is consistent with the proposal that CHCN double bond oxidation is responsible for sensing process. Based on these results, we propose that the mechanism for sensing in this system, manifested in a change from red-to-green fluorescence, takes place by a pathway in which the double bond linking the coumarin and hemicyanine moieties in CHCN is oxidatively cleaved by ONOO . 3.5. Ratiometric detection of exogenous and endogenous ONOO in living cells
Fig. 2. Fluorescence spectra (a) and absorption spectra (b) of CHCN (1 μM) in PBS buffer solution (pH 7.4) upon addition of various ROSs (30 equiv.), GSH, Cys, and Hcy (100 equiv.). (c) Corresponding fluorescent intensity ratios (F515 nm/F635 nm) of CHCN (1 μM) in PBS buffer solution (pH 7.4) upon addition of various ROSs (30 equiv.), GSH, Cys, and Hcy (100 equiv. respectively).
under identical conditions. It should be noted that, as a consequence of its strong oxidation ability, OCl is the ROS that most strongly interferes with other ONOO sensing systems (Kalyanaraman et al., 2012). To further illustrate the selectivity of CHCN for ONOO over OCl , detailed fluorescent and UV–vis titrations studies were performed. As shown in Fig. S1, OCl
Because of its desirable properties, CHCN was employed for ratiometric imaging of exogenous and endogenous ONOO in vivo. For this purpose, WI38 VA13 cells were stained with CHCN (5 μM) for 30 min and then washed with DPBS. Treatment of the stained cells with various concentrations of exogenous ONOO was followed by dual emission imaging. Green fluorescence channel images were obtained over the wavelength range of 490–540 nm with an excitation wavelength at 473 nm, and the red channel fluorescence images were recorded in the range of 575–675 nm with an excitation wavelength at 559 nm. Inspection of Fig. 4 shows that CHCN has satisfactory cell permeability and that it gives rise to red fluorescence cell images, which indicated that CHCN is mainly stained in cellular cytoplasm. Meanwhile, there is no green fluorescent emission observed in the green channel. After treatment with exogenous ONOO (100 μM and 300 μM, respectively) for 30 min, the red fluorescence emitted from the cells decreases concurrent with formation of green fluorescence. As showed in Fig. 4i–l, after pretreated with 300 μM ONOO , a strong green fluorescent emission was emerged. The merged pictures
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Fig. 3. ESI-MS ( þ) spectra of CHCN after reaction with equivalent (upper) and excess (lower) amount of ONOO .
(yellow color) indicate that CHCN displays good ratiometric responses (green/red) towards concentrations of ONOO in the cells. Cellular endogenous ONOO have been well proved to modulate signal transduction pathways via its ability to nitrate biomolecules including nitrated tyrosine residues, 8-nitroguanosine, and nitro fatty acids and thereby influencing cellular processes (Yu et al., 2013). To further demonstrate the ability of CHCN to image endogenous ONOO , cultured murine RAW 264.7 macrophages were utilized as a bioassay model because of their well-known ability to generate ROS and RNS in immunological and inflammatory processes (Salonen et al., 2006). Cellular endogenous ONOO was generated by stimulating RAW 264.7 cells with lipopolysaccharide LPS (1 μg/ml) for 16 h, interferon-γ (50 ng/ml) for 4 h, and PMA (10 nM) for 30 min. The cells were then stained with 5 μM CHCN for 30 min and then washed three times with DPBS. Fluorescence images of the cells were generated using the dual emission imaging protocol described above. The results (Fig. 5)
show that CHCN emits bright red fluorescence but no green fluorescence in the controls (Fig. 5a–d). However, after treatment with LPS (1 mg/mL) for 16 h and IFN-g (50 ng/ml) for 4 h followed by additional stimulation with PMA (10 nM) for 30 min (Fig. 5e–h), red fluorescence was found to be suppressed and green fluorescence enhanced, which indicated that endogenous ONOO was produced in RAW 264.7 cells. Moreover, after pretreatment of the cells with either a scavenger of superoxide, 2,2,6,6-tetramethyl-1piperidinyloxy (TEMPO, 100 μM) or an NO synthase inhibitor, aminoguanidine (AG, 1 mM) (Muijsers et al., 2000), the ratios of red/green fluorescence are unperturbed (Fig. 5i–l and m–p). These results indicate that CHCN is an ideal sensor for circulation imaging endogenous ONOO in living cells. 4. Conclusions In summary, in the effort described above we designed and synthesized the ratiometric and colorimetric probe CHCN, which
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N N
O
O
N O
CHCN
O N
O
O
O
O
N
O
N
O O
O
N
-NO 2 or -NO
O
CHCN-ONOO
N
N
N O 2H N
O
CHCN-O
N
O
O
O
O
N O
CHCN-NO2
CHCN-O2 H
O H
[O]
O N 1,3,3-trimethyloxindole
N
O
O
Coum-CHO (major)
Scheme 1. Propose sensing mechanism of CHCN for ONOO .
Fig. 4. Confocal ratiometric fluorescence images of WI38 VA13 cells for exogenous ONOO . The cells were stained with 5 μM CHCN for 30 min and then washed with DPBS before imaging. (a) Control; (e) 0.1 mM ONOO ; (i) 0.3 mM ONOO . The green channel (a, e, i) represents the fluorescence collected at 490–540 nm with an excitation wavelength at 473 nm, the red channel (b, f, j) represents the fluorescence collected at 575–675 nm with an excitation wavelength at 559 nm, Images (c, g, k) represent DIC channels (differential interference contrast), and images (d, h, i) represent merged images of red and green channels, respectively. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
contains a hybrid coumarin–hemicyanine scaffold, for detection of ONOO . The probe CHCN undergoes a highly selective and sensitive red-to-green fluorescence change in the presence of ONOO under physiological conditions. Moreover, evidence was gained to support a proposed sensing mechanism that involves
Fig. 5. Confocal ratiometric fluorescence images of RAW 264.7 cells for endogenous ONOO during phagocytic immune response. The cells were stained with 5 μM CHCN for 30 min and then washed with DPBS before imaging. (a) Control; (e) LPS (1 μg/ml) for 16 h, interferon-γ (50 ng/ml) for 4 h, PMA (10 nM) for 30 min; (i) LPS (1 μg/ml) for 16 h, interferon-γ (50 ng/ml) for 4 h, PMA (10 nM) for 30 min, and then AG (1 mM) for 16 h; (m) LPS (1 μg/ml) for 16 h, interferon-γ (50 ng/ml) for 4 h, PMA (10 nM) for 30 min, and then TEMPO (100 μM) for 16 h. The green channel (a, e, I, m) represents the fluorescence collected at 490–540 nm with an excitation wavelength at 473 nm, the red channel (b, f, j, n) represents the fluorescence collected at 575–675 nm with an excitation wavelength at 559 nm, Images (c, g, k, o) represent DIC channels (differential interference contrast), and images (d, h, I, p) represent merged images of red and green channels, respectively.
ONOO promoted oxidative cleavage of linking carbon-carbon double bond in CHCN. Finally, CHCN was successfully applied to ratiometric detection of exogenous and endogenous ONOO in
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living cells during phagocytic immune response. We expect that probes having the desirable properties of CHCN will find applications in living-imaging studies aimed at elucidating the roles played by ONOO in biology.
Acknowledgments This research was supported by a grant from the National Creative Research Initiative programs of the National Research Foundation of Korea (NRF) funded by the Korean Government (Ministry of Science, ICT and Future Planning) (No. 2012R1A3A2048814). J.-H.R. thanks for the Basic Science Research Program through the NRF funded by the Ministry of Education (Grant 2013R1A1A2008511), and the Bio & Medical Technology Development Program of the NRF funded by Ministry of Science, ICT and Future Planning (NRF2013M3A9D5072551).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.08.089.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A ratiometric fluorescent probe based on a coumarin–hemicyanine scaffold for sensitive and selective detection of endogenous peroxynitrite Xin Zhou a,b, Younghee Kwon c,d, Gyoungmi Kim a, Ji-Hwan Ryu c,d,n, Juyoung Yoon a,nn a
Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Republic of Korea Department of Chemistry, Faculty of Science, Yanbian University, People's Republic of China c Research Center for Human Natural Defense System, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea d BK 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 19 June 2014 Received in revised form 17 August 2014 Accepted 29 August 2014 Available online 8 September 2014
In the study described herein, the red emitting probe CHCN, which possesses a linked coumarin– hemicyanine scaffold, was developed for detection of peroxynitrite (ONOO ) under physiological conditions. The studies show that CHCN displays a dual ratiometric and colorimetric response to ONOO that is caused by an oxidation process. A possible mechanism of this oxidation process was proposed and confirmed by ESI-MS spectra for the first time. CHCN shown highly selective and sensitive towards ONOO with a low limit of detection LOD (49.7 nM). Moreover, CHCN has appreciable cell permeability and, as a result, it is applicable to ratiometric detection of exogenous and endogenous ONOO in living cells during phagocytic immune response. We anticipate that, owing to their ideal properties, probes of this type will find great use in explorations of the role played by ONOO in biology. & 2014 Elsevier B.V. All rights reserved.
Keywords: Peroxynitrite sensor ONOO sensor Reactive oxygen species probe Fluorescent chemosensor Cell imaging
1. Introduction Reactive oxygen species (ROS) and nitrogen species (RNS) play important roles in physiology and pathology (Chen et al., 2011; Dickinson and Chang, 2011; Pacher et al., 2007; Winterbourn, 2008; Xu et al., 2013; Yang et al., 2010). Among ROSs and RNSs, peroxynitrite (ONOO ) has received special attention owing to its unusually potent oxidizing ability and strongly nucleophilic character (Radi, 2013). As pointed out in recent reports, ONOO is widely considered to be an important factor in the onset and progression of many cellular processes (Dickinson and Chang, 2011). For example, as a strong nucleophile, this species reacts rapidly with carbon dioxide in vivo to form the nitrosoperoxycarbonate (ONOOCO2 ) intermediate. ONOOCO2 then rapidly decomposes homolytically to generate carbonate radical (CO3 ), which is believed to cause specific cellular damage (apoptotic or necrotic cell death) (Radi, 2013). As a potent oxidant, ONOO reacts directly with electron-rich moieties. This process causes n Corresponding author at: Research Center for Human Natural Defense System, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea. nn Corresponding author at: Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120120-750, Republic of Korea. Fax: þ 82 232773419. E-mail addresses: [email protected] (J.-H. Ryu), [email protected] (J. Yoon).
http://dx.doi.org/10.1016/j.bios.2014.08.089 0956-5663/& 2014 Elsevier B.V. All rights reserved.
damage to a wide array of biomolecules (e.g., DNA and proteins) (Kryston et al., 2011; Soon et al., 2011), which leads to many human diseases, such as septic stroke, cancer, diabetes, inflammatory diseases, and neurodegenerative disorders (Szabo et al., 2007). In contrast, growing evidence suggests that the wellcontrolled generation of certain ONOO serves as a signaling transduction factor for a wide range of normal cellular functions (Dickinson and Chang, 2011). Therefore, it is important to develop a clear understanding of the role played by ONOO in cellular functions. For this purpose, it is essential to develop highly sensitive and selective methods for accurate and direct detection of ONOO . However, as a consequence of its short half-life (o 20 ms) under typical physiological conditions (Yang et al., 2006), direct and unambiguous detection of ONOO in living cells can not be easily made by using traditional analytical methods (Chen et al., 2013b; Oushiki et al., 2010; Yu et al., 2012). In the past decade, a large number of fluorescent probes have been designed and constructed for specific detection of ONOO (Chen et al., 2011; Dickinson et al., 2010; Kalyanaraman et al., 2012). These probes are based on various strategies that rely on direct oxidation reactions of boronates (Chen et al., 2013b; Sikora et al., 2009; Sun et al., 2014), active ketones (Peng and Yang, 2010; Sun et al., 2009; Yang et al., 2006), metal complexes (Rausaria et al., 2011), selenium (Xu
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et al., 2011), tellurium (Koide et al., 2012; Yu et al., 2013), and agents (Ueno et al., 2006; Zhang et al., 2012). Despite the encouraging progress that has been made, a great challenge still exists in developing fluorescent probes with high sensitivities and selectivities that are able to distinguish between ONOO and other ROSs (e.g. ClO and H2O2) (Kalyanaraman et al., 2012; Zhang et al., 2012). Moreover, devising probes of this type, which selectively detect endogenous ONOO in vivo without disturbance being offered by the presence of innate reducing biomolecules such as biothiols, is a particular demanding task (Chen et al., 2013b; Yu et al., 2013). Recently, ratiometric type fluorescent probes, especially those that emit light in the near-infrared (NIR) region, have attracted increasing attention because of their intrinsic self-calibrating character and ability to be used for cell and body imaging (Bozdemir et al., 2011; Erbas-Cakmak et al., 2013; Guo et al., 2014; Kolemen et al., 2011; Wu et al., 2014; Yuan et al., 2010). However, compared to the numerous turn on/off fluorescence ONOO probes that have been developed to date, only a few ratiometric probes of this type have been described. In 2010, Nagano et al. reported that cyanine dyes (e.g. Cy5 and Cy7) are readily oxidized by various ROSs to form products that do not fluorescence. More importantly, the studies demonstrated that cyanine dyes display reactivities towards ROS that are governed by the lengths of their conjugated central polymethine chains. For example, Cy7 is more susceptible to ROS promoted oxidation than Cy5 (Oushiki et al., 2010). In spite of this phenomenon, the poor selectivities and the fluorescence quenching response of cyanine dye towards ROSs hinders their broad usage in biology. In order to overcome this limitation, we embarked on a program aimed at the development of a new kind of ratiometric florescence platform that relies on the utilization of a hybrid coumarin–hemicyanine dye. As has been reported recently, these types of hybrid dyes have been explored in the context of ratiometric fluorescent sensors for some common nucleophilic reagents, such as cyanide, sulfite and sulfide (Chen et al., 2013a; Lv et al., 2011; Sun et al., 2013). However, to our knowledge, hybrid coumarin–hemicyanine dyes have not been explored as sensing platforms for ROSs. Taking these factors into account, we have developed the ROS probe CHCN, a conjugated hybrid dye composed by a coumarin group and a hemi-cyanine moiety linked through a carbon-carbon double bond. The results of this effort show that CHCN can be employed to detect ONOO in a dual ratiometric and colorimetric manner with high sensitivity and selectivity. In addition, we have demonstrated an application of CHCN for ratiometric detection of exogenous and endogenous ONOO in living cells.
2.2. Generation of ROS/RNS H2O2 was from dilution of 28% solution in water. Tert-butyl hydroperoxide was from dilution of 70% solution in water. ROO∙ was generated from 2,2′-Azobis (2-amidinopropane) dihydrochloride. NO∙ was generated from SNP (Sodium Nitroferricyanide (III) Dihydrate). ∙O2 was generated by mixing xanthine (25 μM) and xanthine oxidase (3.2 mU/mL). ∙OH was from the reaction of Ammonium iron (II) sulfate (100 μM) and H2O2 (100 μM). NaClO was from dilution of 12% solution in water. ONOO was prepared according to previous literature and the concentrated was determined by absorbance at 302 nm (Xu et al., 2013). The above ROS or RNS were incubated with FBS in 50 mM KH2PO4 (pH 7.4) for 30 min respectively.
2.3. Cell-culture and confocal microscopy experiments The Human normal lung cell (WI38 VA13) cells and a macrophage cell line murine (RAW 264.7) were cultured on the surface of a glass slide in SPP medium (1% proteose peptone, 0.2% glucose, 0.1% yeast extract, 0.003% EDTA ferric sodium salt) at 37 °C in 5% CO2. The cells were subcultured by scraping and seeding on sixwell plates according to the instructions from the manufacturer. Cells were grown to confluence prior to experiment. For confocal microscopy experiments, WI38 VA13 cells were stained with CHCN (5 μM) for 30 min and washed with DPBS, and then treated with various concentration of exogenous ONOO . Then the cells were investigated by using a dual emission imaging mode. The green fluorescence channel images were collected at 490–540 nm with an excitation wavelength at 473 nm, and the red fluorescence channel images were collected at 575–675 nm with an excitation wavelength at 559 nm, respectively. Murine RAW 264.7 macrophage cells were then stimulated with lipopolysaccharide (LPS, 1 μg/mL) for 16 h, and interferon-γ (IFN-γ, 50 ng/mL) for 4 h, followed by phorbol 12-myristate 13-acetate (PMA, 10 nM) for further 30 min for production of endogens ONOO , or pretreated the cells with either a scavenger of superoxide, 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO, 100 μM), or an NO synthase inhibitor, aminoguanidine (AG, 1 mM), to reduce cellular endogenous ONOO . After that, the cells were stained with CHCN (5 μM) for 30 min, and then washed with DPBS for three times before imaging. The fluorescence images were obtained from a dual emission imaging mode as abovementioned.
2.4. Synthesis of CHCN 2. Materials and methods 2.1. General information of materials and methods Unless otherwise noted, all materials were obtained from commercial sources and were used without further purification. Solvents were dried according to standard procedures. 1H NMR and 13C NMR in CDCl3 were measured on a Bruker AM-300 spectrometer with tetramethylsilane (TMS) as internal standard. Mass spectra were obtained using a JMS-HX 110A/110A tandem mass spectrometer (JEOL). UV–vis spectra were obtained using a Scinco 3000 spectrophotometer (1 cm quartz cell) at 25 °C. Fluorescence spectra were recorded on RF-5301/PC (Shimada) fluorescence spectrophotometer (1 cm quartz cell) at 25 °C. Deionized water was used to prepare all aqueous solutions.
Diethylaminocoumarin-3-aldehyde (50 mg, 2 mmol) and 1,3,3trimethyl-2-methyl-eneindoline (60 mg, 2 mmol) were dissolved in absolute ethanol (20 mL) and refluxed for 10 h. After being cooled to room temperature, remove the ethanol under reduce pressure. The corresponding solid was further purified by silica gel column chromatography using DCM/Methanol (20/1, v/v) as eluent to afford CHCN as a deep violet solid (0.312 g, yield: 68%). 1 H NMR (300 MHz, CDCl3-d3) δ 10.05 (s, 1H), 8.60 (d, J¼ 16.2 Hz, 1H), 8.13 (d, J ¼9.3 Hz, 1H), 8.10 (d, J¼ 16.2 Hz, 1H), 7.52–7.63 (m, 4H), 6.72 (dd, J¼ 9.3 Hz, 1H), 6.47 (s, 1H), 4.31 (s, 3H), 3.54 (q, J¼ 7.1 Hz, 4H), 1.85 (s, 6H), 1.34 (t, J ¼7.1 Hz, 6H). 13C NMR (75 MHz, MeOD-d3): δ 158.1, 154.7, 150.3, 143.1, 140.3, 132.3, 128.7, 122.7, 113.8, 112.4, 110.0, 96.4, 78.1, 51.7, 45.1, 32.6, 25.4, 11.4. MS: [M þ ] at 401.23.
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3. Results and discussion 3.1. Design concept of CHCN The conceptual basis for the design of CHCN included the consideration that it be appropriately water soluble so that the sensing processes can be performed under physiological conditions. In addition, the extended π-conjugated system in CHCN was designed to ensure a considerably red shifted emission. It was also envisaged that the bridging double bond of CHCN would be oxidized by ONOO , leading to destruction of the extended conjugation, which would results in disruption of an intramolecular charge-transfer (ICT) process. As a result, the initial red emission of CHCN should disappear in the presence of ONOO and be replaced by green emission corresponding to coumarin moieties in the oxidation products. Therefore, a colorimetric, ratiometric fluorescent dual response to ONOO is expected using this probe. It is important to note that dyes of this type typically have appreciable cell permeability, as shown by mitochondria specific staining during subcellular imaging, and that they are inert to biothiols, such as Cys and GSH (Sun et al., 2013). 3.2. Ratiometric and colorimetric response towards ONOO As seen by viewing the pathway displayed in Scheme S1, CHCN was conveniently synthesized by direct condensation of an amino3-benzaldehyde derived coumarin-aldehyde and an indolium derivative by using a reported method (Sun et al., 2013). The water solubility and spectroscopic characteristics of CHCN were explored initially. As expected, CHCN was observed to display satisfactory water solubility. The fluorescence response of CHCN to ONOO was examined next. As shown in Fig. 1a, in PBS buffer solution (pH 7.4), CHCN emits red fluorescent with an emission maximum at 635 nm, which is a consequence of the extended πconjugated system present in this linked coumarin–hemicyanine dye. Upon addition of ONOO , the emission peak of CHCN at 635 nm decreases in concert with an increase of a new, more emissive band at 515 nm that corresponds to a blue color (Fig. 1a inset). Moreover, the ratio of the fluorescence intensities of CHCN at 635 and 515 nm linearly correlate with the concentration of ONOO with a correlation coefficient of R¼0.9939 and a detection limit of 49.7 nM (Fig. 1c) (Zhou et al., 2013). Furthermore, the results of absorption titration studies (Fig. 1b) show that upon addition of increasing amounts of ONOO , the main absorption band of CHCN at 568 nm decreases with a concurrent increase in a new band at 422 nm, corresponding to a coumarin chromophore. A clear isosbestic point occurs at 467 nm. As can be seen by viewing the inset in Fig. 1b, addition of ONOO to a PBS buffer solution of CHCN causes a color change from blue violet to faint yellow. The combined results demonstrate that CHCN serves as probe for ratiometric and colorimetric detection of ONOO . 3.3. Selectivity of CHCN towards ONOO To evaluate the selectivity of CHCN towards ONOO , fluorescence and absorption spectra of CHCN before and after addition of common ROSs, including ∙NO, ∙O2, H2O2, OCl , ∙OH, t-BuOO∙, as well as biothiols (Cys, Hcy and GSH), were recorded. As shown in Fig. 2a, under identical conditions, only ONOO causes an observable ratiometric fluorescent response of CHCN. Importantly, the other ROSs and biothiols do not promote observable changes in the spectra of CHCN even when present in 100 equivalent excesses. Moreover, the fluorescence intensity ratio (F515nm/F635nm) of CHCN (Fig. 2c) is much more greatly enhanced (474-fold higher) upon addition of ONOO in contrast to the other agents tested. The absorption spectra displays similar responses (Fig. 2b) as
Fig. 1. (a) Fluorescence spectra of CHCN (1 μM) upon addition of various amounts of ONOO (ranging from 0 to 20 μM) in PBS buffer solution (pH 7.4), each data set was recorded after 10 min with an excitation wavelength at 475 nm. Inset: fluorescent images of CHCN (1 μM) in the absence (A) and presence (B) of ONOO . (b) Absorption spectra of CHCN (1 μM) upon gradual addition of ONOO (0–20 μM) in PBS buffer solution (pH 7.4). Inset: colors of CHCN (10 μM) in the absence (A) and presence (B) of ONOO . (c) Linear correlation between the emission intensity ratio (F515 nm/F635 nm) and the concentration of ONOO . (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
exemplified by the observation that ONOO induced a distinct decrease in the absorption band at 568 nm, while hypochlorite (OCl ) causes only a slight reduction in the intensity of this band
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causes a similar ratiometric and colorimetric dual response of CHCN as does ONOO , but the rates at which the responses occur are much smaller, and excess amounts of OCl (up to more than 200 equiv.) is required to achieve the same spectra responses. These results indicate that, at low concentrations, CHCN displays a high selectivity toward ONOO over other ROSs. 3.4. Proposed mechanism for sensing of ONOO by CHCN As has been well demonstrated in previous studies (Oushiki et al., 2010), electron rich conjugated C ¼C double bonds of cyanine dyes undergo ready oxidative cleavage in the presence of ROSs. This process results in formation of a variety of double bond cleavage products, including 1,3,3-trimethyloxindole as the major product along with derivatives such as aldehyde and carboxylic acid species (Lou et al., 2013; Park et al., 2013). Based on the observation summarized above and the results arising from previous investigations, it is plausible that CHCN displays high sensitivity and selectivity towards ONOO because this ROS promotes oxidative cleavage of the central double bond. To gain experimental support for this proposal, the process taking place between CHCN and ONOO was monitored by using mass spectrometry (MS). Inspection of Fig. 3, shows that upon addition of ONOO the parent peak in the spectrum of CHCN at m/z 401.2 disappears and three new peaks at 417.1, 435.0, and 446.2 arise. The new peaks are ascribed to the three products CHCN–O, CHCN–O2H, and CHCN–NO2, generated initially by oxidation of CHCN (Scheme 1). MS monitoring shows that these products partially undergo further oxidation to generate the final products consisting of predominantly 1,3,3-trimethyloxiindole (m/ z 176.2), coumarin-3-aldehyde (m/z 246.3), the latter of which was isolated and identified by using 1H NMR spectroscopy (Fig. S5). The chemical reaction responsible for sensing was also explored by using 1H NMR titration experiments (Fig. S6). The results show that resonances for the double bond protons of CHCN at 8.03 and 7.98 ppm disappear after addition of ONOO . This observation is consistent with the proposal that CHCN double bond oxidation is responsible for sensing process. Based on these results, we propose that the mechanism for sensing in this system, manifested in a change from red-to-green fluorescence, takes place by a pathway in which the double bond linking the coumarin and hemicyanine moieties in CHCN is oxidatively cleaved by ONOO . 3.5. Ratiometric detection of exogenous and endogenous ONOO in living cells
Fig. 2. Fluorescence spectra (a) and absorption spectra (b) of CHCN (1 μM) in PBS buffer solution (pH 7.4) upon addition of various ROSs (30 equiv.), GSH, Cys, and Hcy (100 equiv.). (c) Corresponding fluorescent intensity ratios (F515 nm/F635 nm) of CHCN (1 μM) in PBS buffer solution (pH 7.4) upon addition of various ROSs (30 equiv.), GSH, Cys, and Hcy (100 equiv. respectively).
under identical conditions. It should be noted that, as a consequence of its strong oxidation ability, OCl is the ROS that most strongly interferes with other ONOO sensing systems (Kalyanaraman et al., 2012). To further illustrate the selectivity of CHCN for ONOO over OCl , detailed fluorescent and UV–vis titrations studies were performed. As shown in Fig. S1, OCl
Because of its desirable properties, CHCN was employed for ratiometric imaging of exogenous and endogenous ONOO in vivo. For this purpose, WI38 VA13 cells were stained with CHCN (5 μM) for 30 min and then washed with DPBS. Treatment of the stained cells with various concentrations of exogenous ONOO was followed by dual emission imaging. Green fluorescence channel images were obtained over the wavelength range of 490–540 nm with an excitation wavelength at 473 nm, and the red channel fluorescence images were recorded in the range of 575–675 nm with an excitation wavelength at 559 nm. Inspection of Fig. 4 shows that CHCN has satisfactory cell permeability and that it gives rise to red fluorescence cell images, which indicated that CHCN is mainly stained in cellular cytoplasm. Meanwhile, there is no green fluorescent emission observed in the green channel. After treatment with exogenous ONOO (100 μM and 300 μM, respectively) for 30 min, the red fluorescence emitted from the cells decreases concurrent with formation of green fluorescence. As showed in Fig. 4i–l, after pretreated with 300 μM ONOO , a strong green fluorescent emission was emerged. The merged pictures
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Fig. 3. ESI-MS ( þ) spectra of CHCN after reaction with equivalent (upper) and excess (lower) amount of ONOO .
(yellow color) indicate that CHCN displays good ratiometric responses (green/red) towards concentrations of ONOO in the cells. Cellular endogenous ONOO have been well proved to modulate signal transduction pathways via its ability to nitrate biomolecules including nitrated tyrosine residues, 8-nitroguanosine, and nitro fatty acids and thereby influencing cellular processes (Yu et al., 2013). To further demonstrate the ability of CHCN to image endogenous ONOO , cultured murine RAW 264.7 macrophages were utilized as a bioassay model because of their well-known ability to generate ROS and RNS in immunological and inflammatory processes (Salonen et al., 2006). Cellular endogenous ONOO was generated by stimulating RAW 264.7 cells with lipopolysaccharide LPS (1 μg/ml) for 16 h, interferon-γ (50 ng/ml) for 4 h, and PMA (10 nM) for 30 min. The cells were then stained with 5 μM CHCN for 30 min and then washed three times with DPBS. Fluorescence images of the cells were generated using the dual emission imaging protocol described above. The results (Fig. 5)
show that CHCN emits bright red fluorescence but no green fluorescence in the controls (Fig. 5a–d). However, after treatment with LPS (1 mg/mL) for 16 h and IFN-g (50 ng/ml) for 4 h followed by additional stimulation with PMA (10 nM) for 30 min (Fig. 5e–h), red fluorescence was found to be suppressed and green fluorescence enhanced, which indicated that endogenous ONOO was produced in RAW 264.7 cells. Moreover, after pretreatment of the cells with either a scavenger of superoxide, 2,2,6,6-tetramethyl-1piperidinyloxy (TEMPO, 100 μM) or an NO synthase inhibitor, aminoguanidine (AG, 1 mM) (Muijsers et al., 2000), the ratios of red/green fluorescence are unperturbed (Fig. 5i–l and m–p). These results indicate that CHCN is an ideal sensor for circulation imaging endogenous ONOO in living cells. 4. Conclusions In summary, in the effort described above we designed and synthesized the ratiometric and colorimetric probe CHCN, which
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N N
O
O
N O
CHCN
O N
O
O
O
O
N
O
N
O O
O
N
-NO 2 or -NO
O
CHCN-ONOO
N
N
N O 2H N
O
CHCN-O
N
O
O
O
O
N O
CHCN-NO2
CHCN-O2 H
O H
[O]
O N 1,3,3-trimethyloxindole
N
O
O
Coum-CHO (major)
Scheme 1. Propose sensing mechanism of CHCN for ONOO .
Fig. 4. Confocal ratiometric fluorescence images of WI38 VA13 cells for exogenous ONOO . The cells were stained with 5 μM CHCN for 30 min and then washed with DPBS before imaging. (a) Control; (e) 0.1 mM ONOO ; (i) 0.3 mM ONOO . The green channel (a, e, i) represents the fluorescence collected at 490–540 nm with an excitation wavelength at 473 nm, the red channel (b, f, j) represents the fluorescence collected at 575–675 nm with an excitation wavelength at 559 nm, Images (c, g, k) represent DIC channels (differential interference contrast), and images (d, h, i) represent merged images of red and green channels, respectively. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
contains a hybrid coumarin–hemicyanine scaffold, for detection of ONOO . The probe CHCN undergoes a highly selective and sensitive red-to-green fluorescence change in the presence of ONOO under physiological conditions. Moreover, evidence was gained to support a proposed sensing mechanism that involves
Fig. 5. Confocal ratiometric fluorescence images of RAW 264.7 cells for endogenous ONOO during phagocytic immune response. The cells were stained with 5 μM CHCN for 30 min and then washed with DPBS before imaging. (a) Control; (e) LPS (1 μg/ml) for 16 h, interferon-γ (50 ng/ml) for 4 h, PMA (10 nM) for 30 min; (i) LPS (1 μg/ml) for 16 h, interferon-γ (50 ng/ml) for 4 h, PMA (10 nM) for 30 min, and then AG (1 mM) for 16 h; (m) LPS (1 μg/ml) for 16 h, interferon-γ (50 ng/ml) for 4 h, PMA (10 nM) for 30 min, and then TEMPO (100 μM) for 16 h. The green channel (a, e, I, m) represents the fluorescence collected at 490–540 nm with an excitation wavelength at 473 nm, the red channel (b, f, j, n) represents the fluorescence collected at 575–675 nm with an excitation wavelength at 559 nm, Images (c, g, k, o) represent DIC channels (differential interference contrast), and images (d, h, I, p) represent merged images of red and green channels, respectively.
ONOO promoted oxidative cleavage of linking carbon-carbon double bond in CHCN. Finally, CHCN was successfully applied to ratiometric detection of exogenous and endogenous ONOO in
X. Zhou et al. / Biosensors and Bioelectronics 64 (2015) 285–291
living cells during phagocytic immune response. We expect that probes having the desirable properties of CHCN will find applications in living-imaging studies aimed at elucidating the roles played by ONOO in biology.
Acknowledgments This research was supported by a grant from the National Creative Research Initiative programs of the National Research Foundation of Korea (NRF) funded by the Korean Government (Ministry of Science, ICT and Future Planning) (No. 2012R1A3A2048814). J.-H.R. thanks for the Basic Science Research Program through the NRF funded by the Ministry of Education (Grant 2013R1A1A2008511), and the Bio & Medical Technology Development Program of the NRF funded by Ministry of Science, ICT and Future Planning (NRF2013M3A9D5072551).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.08.089.
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