A Highly Sensitive Ratiometric Fluorescent Probe for the Detection of

Sep 8, 2014 - Mitochondria-Targeted Ratiometric Fluorescent Probe Based on Diketopyrrolopyrrole for Detecting and Imaging of Endogenous Superoxide ...
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A Highly Sensitive Ratiometric Fluorescent Probe for the Detection of Cytoplasmic and Nuclear Hydrogen Peroxide Ying Wen,† Keyin Liu,† Huiran Yang,† Yi Li,† Haichuang Lan,† Yi Liu,† Xinyu Zhang,*,‡ and Tao Yi*,† †

Department of Chemistry and Concerted Innovation Center of Chemistry for Energy Materials, Fudan University, Shanghai 200433, China ‡ Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200433, China S Supporting Information *

ABSTRACT: As a marker for oxidative stress and a second messenger in signal transduction, hydrogen peroxide (H2O2) plays an important role in living systems. It is thus critical to monitor the changes in H2O2 in cells and tissues. Here, we developed a highly sensitive and versatile ratiometric H2O2 fluorescent probe (NP1) based on 1,8-naphthalimide and boric acid ester. In response to H2O2, the ratio of its fluorescent intensities at 555 and 403 nm changed 1020-fold within 200 min. The detecting limit of NP1 toward H2O2 is estimated as 0.17 μM. It was capable of imaging endogenous H2O2 generated in live RAW 264.7 macrophages as a cellular inflammation response, and especially, it was able to detect H2O2 produced as a signaling molecule in A431 human epidermoid carcinoma cells through stimulation by epidermal growth factor. This probe contains an azide group and thus has the potential to be linked to various molecules via the click reaction. After binding to a Nuclear Localization Signal peptide, the peptide-based combination probe (pep-NP1) was successfully targeted to nuclei and was capable of ratiometrically detecting nuclear H2O2 in living cells. These results indicated that NP1 was a highly sensitive ratiometric H2O2 dye with promising biological applications. eactive oxygen species (ROS, including H2O2, O2−, and 1 O2) are endogenous metabolites that are classically known as indicators of oxidative stress. Hydrogen peroxide (H2O2) is one of the most important ROS. When H2O2 is generated at low concentrations (< 0.7 μM) in a regulated fashion, it functions as a ubiquitous intracellular second messenger.1 H2O2 can activate signaling pathways to stimulate cell proliferation,2 differentiation,3 and migration.4 However, unlike the classical second messenger Ca2+, H2O2 is an oxidant.5 Under conditions of stress or stimulation by exogenous chemicals, H2O2 can be generated aberrantly and result in oxidative stress. The resulting ROS can attack cellular structures or biomolecules such as proteins,6,7 liposomes,8 and DNA,9,10 which has been associated with aging,11 Alzheimer’s disease,12 and cancer.13 With respect to cancer, DNA is considered the most important target.14 Oxidative modifications of DNA bases, including oxidation of purines and pyrimidines, the generation of alkali labile sites, and strand breaks,13 can induce mutations and even cancer, if unrepaired in a timely manner. Several oxidative DNA modifications have been shown to be present in cancer tissues.15 Many chemical tools have been developed to detect intracellular H2O2, such as mass probes,16 proteomics probes,17 and fluorescent probes.18 Fluorescent probes have been widely used because of their nondisruptive features. Chang and

colleagues developed several fluorescent probes,18 some of which could be used to monitor H2O2 at physiological levels in vitro and in vivo and to explore the cellular mechanisms associated with H2O2.19,20 The vast majority of these probes are intensity-based turn-on fluorescent sensors.18−23 However, interpretation of the results obtained with turn-on probes could be complex.24 A change in signal does not necessarily represent a change in the amount of H2O2 generated; it could also be attributed to a change in the concentration of one of the compounds that compete for H2O2. In this regard, the fluorescent signal depends on how much probe is present, because a higher concentration of probe will compete better for H2O2 and lead to a higher signal. However, the intracellular probe concentration, which does not depend on treatment concentrations during the experimental process, is difficult to be quantified for a turn-on probe. To address this issue, ratiometric probes, which fluoresce both before and after reaction with H2O2 but with different wavelengths, have been developed. Because these probes emit fluorescence before detection of H2O2, it is easy to determine if the intracellular concentrations of probes change. Moreover,

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© XXXX American Chemical Society

Received: August 4, 2014 Accepted: September 8, 2014

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although the fluorescence intensities of the probe in the absence and presence H 2 O 2 change with the probe concentrations or time, their ratios are independent of the probe concentrations. This enables a more accurate and quantitative analysis.24,25 However, to date, small molecular probes for ratiometric detection of H2O2 have been limited, and these probes had low sensitivity and the changes in the ratios were small.26−32 Furthermore, few ratiometric probes targeted to cell nuclei have been developed. In addition, the ratiometric probes that have been developed lack versatility. In the present study, we designed and synthesized a versatile small molecular fluorescent probe for ratiometric detection of H2O2 (NP1, Scheme 1) based on a naphthalimide backbone

(PMA), and epidermal growth factor (EGF) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.). Bis(pinacolato)diboron, 4-bromo-1,8-naphthalic anhydride, 1,1′-bis(diphenylphosphino)-ferrocene [Pd(dppf)Cl2] were purchased from Alfa Aesar (Ward Hill, MA, U.S.). All organic solvents were supplied from Sinopharm Chemical Reagent Company (Shanghai, China). Propidium iodide (PI) and cell culture reagents were purchased from Invitrogen. Instruments. 1H NMR spectra were recorded with a Bruker DRX 500 spectrometer at 400 MHz. Proton chemical shifts are reported in parts per million downfield from tetramethylsilane (TMS). The high-resolution mass spectra (HR-MS) were measured on a Bruker Micro TOF II 10257 instrument. Fourier transform infrared (FTIR) spectra were collected using a Mettler Toledo anin situReactIR 45m spectrometer. UV− visible spectra were recorded on a Shimadzu UV-2550 spectrometer. Steady-state emission experiments at room temperature were measured on an Edinburgh instrument FLS-920 spectrometer with a Xe lamp as an excitation source. A Hamamatsu absolute PL quantum yield spectrometer C11347 and Hamamatsu compact fluorescence lifetime spectrometer C11367 were used for fluorescent quantum yield and lifetime measurement, respectively. HPLC analysis and separation were performed on a Waters Alliance 2695 HPLC system (Milford, MA, U.S.A.). The detector is a Waters 2996 diode array detector with the wavelength range from 210 to 400 nm. Analytical separation was carried out on an Agilent ZORBAX 80 Å StableBond SB-C18 analytical column (4.6 mm × 250 mm, 5 μm). Preparative separation was performed on a Waters SunFire C18 preparative column (10 mm × 150 mm, 5 μm). Synthesis Details of NP1. The synthesis route of NP1 is shown in Scheme S1 in the Supporting Information. Compound 1 was synthesized according to the previous report.42 Compound 2. 4-Bromo-1,8-naphthalic anhydride (500 mg, 1.81 mmol), 1 (250 mg), and triethylamine (1.5 mL) were dissolved in 20 mL of ethanol. The mixture was stirred and refluxed for 2 h. 2 was obtained by precipitation and used for the next step without further purification. Yield 95%; 1H NMR (400 MHz, DMSO) δ 8.57 (dd, J = 9.5, 8.2 Hz, 2H), 8.34 (d, J = 7.9 Hz, 1H), 8.23 (d, J = 7.9 Hz, 1H), 8.06−7.96 (m, 1H), 4.06 (t, J = 7.0 Hz, 2H), 3.38 (t, J = 6.7 Hz, 2H), 1.77−1.66 (m, 2H), 1.66−1.54 (m, 2H). NP1. 2 (700 mg, 1.88 mmol, 1 equiv), bis(pinacolato)diboron (715.2 mg, 2.81 mmol, 1.5 equiv), Pd(dppf)Cl2 (133.2 mg, 0.18 mmol), and potassium acetate (546.5 mg, 5.57 mmol, 2.9 equiv) were dissolved in 50 mL of toluene. The mixture was stirred and refluxed for 16 h. The product was purified by column chromatography with dichloromethane/petroleum ether (4:1, v/v) to give NP1 as a pale yellow powder. Yield 40%; 1H NMR (400 MHz, CDCl3) δ 9.12 (d, J = 8.4 Hz, 1H), 8.61 (d, J = 7.3 Hz, 1H), 8.57 (d, J = 7.3 Hz, 1H), 8.30 (d, J = 7.3 Hz, 1H), 7.83−7.73 (m, 1H), 4.22 (t, J = 7.2 Hz, 2H), 3.36 (t, J = 6.8 Hz, 2H), 1.90−1.79 (m, 2H), 1.73 (dt, J = 13.9, 6.8 Hz, 2H), 1.45 (s, 12H). 13C NMR (100 MHz, CDCl3) δ 25.11, 25.50, 26.64, 39.79, 51.31, 87.74, 122.60, 124.70, 127.21, 127.99, 129.96, 131.06, 135.20, 135.40, 135.89, 164.44. HRMS: calcd for C22H26BN4O4+ [MH]+, 421.2047; found, 421.2049. Preparation of pep-NP1. The peptide sequence (VQRKRQKLMP-NH2) was purchased from Shanghai Abbiochem Co., Ltd. 5-Hexynoic acid was modified to the N-

Scheme 1. Chemical Structures of NP1 and pep-NP1

and a boric acid ester H2O2 reporter, with an azide group modified at the other end of the naphthalimide. 1,8Naphthalimide, a fluorescent chromophore with excellent spectroscopic properties, was used previously as a sensor system by our group.33,34 Boric acid ester was used as the H2O2 sensor.18 NP1 showed several advantages over previous probes as follows: (1) It exhibited high selectivity and sensitivity toward H2O2 with a fluorescence ratio change of up to 1020fold. Due to its high sensitivity, NP1 was able to detect H2O2 generated as a signaling molecule in A431 human epidermoid carcinoma cells when stimulated by epidermal growth factor. (2) The azide group made the probe versatile and easily functionalized via the click reaction with targeting groups or biological molecules. Considering that oxidative DNA damage plays a critical role in the induction of tumors,14 it is important to detect H2O2 near DNA. Thus, we linked NP1 to a molecular transport agent targeted to nuclei. A Nuclear Localization Signal (NLS) peptide was used as an efficient nuclear delivery carrier. Specifically, the sequence (VQRKRQKLMP-NH2), derived from the transcription factor NF-κB, which functions to internalize NF-κB into the nucleus, was selected.35,36 NLS peptides have been shown to guide uptake and nuclear localization of several molecules, including Ru(II) polypyridyl complexes,37 nanoparticles,38 anticancer therapeutics,39 liposome protamine/ DNA lipoplex,40 and cathepsins.41 Using the NLS peptide as the transmembrane molecular cargo carrier, we achieved the delivery of the probe (the probe linked to the peptide was designated as pep-NP1; see Scheme 1) to nuclei and ratiometric detection of nuclear H2O2 in living cells.

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EXPERIMENTAL SECTION Materials. All of the starting materials were obtained from commercial suppliers and used as received. H2O2, tertbutylhydroperoxide (tBHP), hypochlorite (NaOCl), 3-(aminopropyl)-1-hydroxy-3- isopropyl-2-oxo-1-triazene (NOC5), xanthine oxidase, xanthine sodium salt, catalase, potassium superoxide, ferrous perchlorate, tris[(1-benzyl-1H-1,2,3-triazol4-yl)methyl]-amine (TBTA), phorbol-12-myristate-13-acetate B

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semiconductor laser at 405 nm provided the excitation, and luminescence emission at both the blue channel (425−465 nm) and yellow channel (550−600 nm) were collected as output signals. A photomultiplier tube (R6357 enhanced model, Hamamatsu, Japan) was used as a detector. A 60× oilimmersion objective lens and a 20× air-immersion objective lens were used. Quantization by line plots was accomplished using the software package provided by Olympus instruments. Carestream software was used to process ratiometric images. Cells shown are representative images from replicate experiments (n = 3). Cell Viability. In vitro cytotoxicity was measured by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assays on the Hela cells and RAW 264.7 macrophages. Cells were seeded into a 96-well cell culture plate at 2 × 103/well and were cultured at 37 °C with 5% CO2 for 24 h. Different concentrations of NP1 (0, 1, 2.5, 5, and 10 μM) were then added to the wells. The cells were subsequently incubated for 12 or 24 h. Thereafter, MTT (0.5 mg/mL) was added to each well and the plate was incubated for an additional 4 h. The optical density OD 490 value (Abs.) of each well was measured by means of a Tecan Infinite M200 monochromatic-based multifunction microplate reader. The following formula was used to calculate the inhibition of cell growth: Cell viability (%) = (mean of Abs. value of treatment group/mean of Abs. value of control) × 100%. Fresh Rat Hippocampal Slices. Fresh rat hippocampal slices, 60 μm-thick, were obtained from the Department of Clinical Medicine, Shanghai Medical College, Fudan University. The rat hippocampal slices were loaded with 20 μM NP1 for 30 min at 37 °C and were then washed three times with PBS for CLSM images.

terminus of the peptide (namely pep-1; HPLC and HR-MS of pep-1 were in the Supporting Information). Pep-1 stock solutions of NP1 (100 mM), TBTA (100 mM) were prepared in DMSO. Stock solutions of pep-1 (100 mM), sodium ascorbate (500 mM), and CuSO4 (500 mM) were prepared in ddH2O. Pep-1 (10 μL, 1 mM), NP1 (10 μL, 1.2 mM), TBTA (10 μL, 10 mM), sodium ascorbate (10 μL, 50 mM), and CuSO4 (10 μL, 50 mM) were added to 950 μL of DMSO/H2O (1:1). The mixture was stirred for 30 min. The reaction process was followed by HPLC (see details in the Supporting Information). Two main products were produced with the retention time of 11.25 and 12.15 min, respectively. After validation of HR-MS, the latter one was considered as the targeted product pep-NP1. HR-MS: calcd for C77H126N25O17SB2+ [M + 2H]2+, 857.9789; found, 857.9854; calcd for C77H127N25O17SB3+ [M + 3H]3+, 572.3219; found, 572.3271. The following nonlinear gradient program was used when using HPLC for the analytical separation and the preparative separation process: starting at 1 min from 0.1% pump B to 33% pump B over 11 min [pump A, ddH2O with the pH being adjusted to 2.5 with trifluoroacetic acid (TFA); pump B, acetonitrile with 0.1% (v/v) TFA at pH ∼ 2.5] at a flow rate of 1 mL/min and then at 11 min from 33% to 80% pump B over 9 min, at 21 min from 80% to 0.1% pump B over 2 min, and stopped at 22 min. In the preparative separation process, the flow rate was changed to 3 mL/min. Selectivity of NP1/pep-NP1 toward H2O2 in PBS. Various oxidants were generated according to the previous report.28 H2O2, tert-butylhydroperoxide (tBHP), and hypochlorite (NaOCl) were delivered from 30%, 70%, and 10% aqueous solutions, respectively. Superoxide (O2−) was from potassium superoxide solution. A hydroxyl radical (HO·) and tert-butoxy radical (·OtBu) were generated by the reaction of 1 mM Fe2+ with 200 μM H2O2 and tBHP, respectively. Singlet oxygen (1O2) was produced by the reaction of 1 mM OCl− with 200 μM H2O2. Nitric oxide (NO) was generated from NOC5 (10 mM stock solution in 0.1 M NaOH). Degradation of 280 μM NOC5 in aqueous solution generates 200 μM NO after 60 min. A superoxide radical (O2−·) was generated from the enzymatic reaction of xanthine oxidase and xanthine sodium salt in the presence of catalase as a scavenger for H2O2. 1 mM xanthine sodium salt and 3.5 mg/mL Catalase (from Bovine Liver, 2000−5000 units/mg solid) in H2O were prepared, respectively. A 2 mL reaction solution was prepared consisting of 60 μL of xanthine oxidase (from buttermilk, 0.3 units/mg, 11 mg protein/mL), 30 μL of catalase mix, 20 μL of xanthine sodium salt mix, and 1890 μL of PBS. The final concentrations are 0.099 units/mL xanthine oxidase, 110− 262.5 units/mL catalase, 5 μM NP1, and 100 μM xanthine sodium salt. Cell Culture. The Hela cells, RAW 264.7 macrophages, and A431 human epidermoid carcinoma cells were provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). The Hela cells and RAW 264.7 macrophages were grown in RPMI 1640 supplemented with 10% FBS (fetal bovine serum). The A431 cells were cultured in DMEM supplemented with 10% FBS. All cells were grown at 37 °C in humidified air containing 5% CO2. Confocal Laser Scanning Microscope (CLSM) Imaging. CLSM images were performed on an inverted microscope (Olympus IX81, Japan) and a confocal scanning unit (FV1000, Olympus, Japan). For the luminescence microscopy imaging, a

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RESULTS AND DISSCUSION Capability of NP1 to Detect H2O2 in the Solution. The details of synthesis and characterization of NP1 are provided in the Experimental Section and the Supporting Information. The spectroscopic properties of NP1 were assessed under in vitro physiological conditions (in PBS, pH 7.2). In the absence of H2O2, NP1 displayed one major absorption band centered at 354 nm (ε = 11 340 M−1 cm−1). This band red-shifted to 446 nm (ε = 10 820 M−1 cm−1) after the addition of 100 equiv of H2O2 (Figure 1a), suggesting that NP1 was converted to a new compound. The high-resolution mass spectra (HR-MS) data indicated that the boric acid ester was replaced by a hydroxyl group after reacting with H2O2 (Figure S1). The NP1 solution in PBS (5 μM) emitted blue fluorescence (λem = 403 nm, Φ = 6.1%, τ = 1.0 ns) in the absence of H2O2. The intensity of the blue fluorescence decreased and that of yellow fluorescence (λem = 555 nm, Φ = 8.7%, τ = 2.3 ns) increased after 200 μM H2O2 were added to the solution (Figure 1b). The shift of the emission wavelength (152 nm) of NP1 from blue to yellow in response to H2O2 was much greater than those of probes reported in the literature (RPF1 = 53 nm,29 PL1 = 65 nm,28 PN1 = 50 nm,27 SHP-Mito = 75 nm26), which might contribute to the high sensitivity of NP1 toward H2O2. The ratio of fluorescence intensity (F555/F403) varied from 0.025 in the absence of H2O2 to 25.5 after complete reaction (200 min, λex = 380 nm) with H2O2, a 1020-fold increase [the fold change in the ratio was expressed as R555/403 = (F555/F403)/(F555/F403)0, where (F555/F403) and (F555/F403)0 indicate the fluorescence intensity ratios of F 555 /F403 with and without H 2 O 2 , respectively, Figure 1c]. The R555/403 value of NP1 at 1 h C

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microscopy (CLSM). HeLa cells incubated with NP1 (5 μM) for 30 min at 37 °C showed strong fluorescence in the blue channel (425−465 nm) and weak fluorescence in the yellow channel (550−600 nm) with excitation at 405 nm. The ratio (RY/B) images were constructed by fluorescence detection at 575 ± 25 nm (yellow channel) and 445 ± 20 nm (blue channel) using the software Carestream. Treatment of NP1loaded cells with 200 μM H2O2 for 0, 15, 30, 45, and 60 min triggered an increase in RY/B from 0.3 to 2.0 (Figure 2). RY/B

Figure 1. (a) Absorption spectra of NP1 (50 μM) in the presence and absence of 50 mM H2O2 in a mixed solvent of Me2SO and PBS (5:1000, v/v) at pH 7.2. (b) Fluorescent spectral changes of NP1 (5 μM) with time after addition of H2O2 (200 μM). Insets of (a) and (b): the color and fluorescence changes of NP1 (50 μM) in the absence and presence of H2O2, respectively (λex = 365 nm). (c) The fold changes (R555/403) of NP1 (5 μM) at 0, 2, 5, 10, 15, 30, 45, 60, 90, 120, and 200 min after addition of 200 μM H2O2, λex = 380 nm, room temperature. (d) The R555/403 values of NP1 (5 μM) in response to various ROS/RNS (200 μM) at 0 to 120 min. A to J represent H2O2, NO, O2−, ·OtBu, HO·, OCl−, O2−·, tBHP, 1O2, and H2O2 with catalase, respectively.

Figure 2. CLSM ratio (RY/B) images of 5 μM NP1-loaded HeLa cells stimulated with 200 μM H2O2 for (a) 0, (b) 15, (c) 30, (d) 45, and (e) 60 min at 37 °C. (f) The overlay image of (e) and the brightfield image. The ratio (RY/B) images were generated by measuring fluorescence at a range from 575 ± 25 nm (yellow channel) to 445 ± 20 nm (blue channel). λex = 405 nm; scale bar = 20 μm.

after H2O2 addition reached 244-fold, which was also greater than those reported previously for ratiometric probes (8−75 times).26−29,32 These results suggested that NP1 was more sensitive to H2O2 than the ratiometric fluorescent probes already reported in the literature. The reaction of NP1 and H2O2 exhibited a pseudo-first-order kinetics (1 μM NP1, 1 mM H2O2, λem = 403 nm). The pseudo-first-order rate constant was 1.6 × 10−3 s−1 (Figure S2), which was similar to those reported for previous probes.18 In addition, a linear correlation between the ratio of the emission intensities (F555/F403) and the H2O2 concentration was observed with R2 being 0.9908. The detection limit of H2O2 was estimated as 0.17 μM (Figure S3), which was much lower than those of the established probes.26,30,32 To verify the selectivity of NP1 for H2O2, the fluorescence changes of NP1 were examined upon addition of H2O2 (40 equiv) and other ROS/RNS [40 equiv of nitric oxide (NO), superoxide (O2−), tert-butoxy radical (·OtBu), hydroxyl radical (HO·), hypochlorite (OCl−), superoxide radical (O2−·), tertbutylhydroperoxide (tBHP), and singlet oxygen (1O2)] in PBS for 0−120 min (Figure 1d). At 2 h, NO induced a weak response with R555/403 being 47.5, which was markedly smaller than that obtained with H2O2 (561 times). Negligible changes in the ratio (R555/403 < 6.5) were observed with other ROS (Figure 1d, inset). A recent report showed that arylboronates could react with OCl− in HEPES to yield hydroxyl derivatives much faster than H2O2.43 However, OCl− did not cause changes in the fluorescence intensity of NP1 in PBS in our experiment. The good selectivity showed that NP1 could serve as a ratiometric fluorescent probe for H2O2 with minimum interference from other ROS/RNS. In Vivo Detection of Hydrogen Peroxide with NP1. Practical applications of NP1 for the detection of H2O2 in living cells and tissues were tested by using confocal laser scanning

showed a nonlinear increase of up to 6.5 times compared to that in the absence of H2O2 (Figure S4a). Compared with the PBS solution, in the cells the fold change of the ratio more rapidly reached a constant (approximately 30 min, Figure S4a and S4b). Thus, in the cellular experiments it should be sufficient to monitor cells up to 30 min. The selectivity of NP1 toward H2O2 was also verified at the cellular level. When catalase (a specific scavenger of cellular H2O2) or N-acety-L-cysteine (NAC, a widely used antioxidant) was added to the H2O2-stimulated cells preloaded with NP1, a decrease in the ratio was observed compared to that in the absence of these agents (Figure S5). These data suggested that NP1 was capable of detecting intracellular H2O2 pools. In particular, NP1 was capable of detecting H2 O 2 at a concentration as low as 10 μM in live HeLa cells (Figure S6). Moreover, NP1 was nontoxic to both RAW 264.7 macrophages and HeLa cells during the experiments. The viabilities were estimated to be greater than 90% after 24 h in the presence of 1−10 μM NP1, as determined by the MTT assay (Figure S7). Detection of Hydrogen Peroxide with NP1 in Cells and Brain Tissues. We assessed the ability of NP1 to detect endogenous H 2 O 2 produced by exogenous compound stimulation. Phorbol-12-myristate-13-acetate (PMA) was used to induce H2O2 generation through a cellular inflammation response.44 In this experiment, RAW 264.7 macrophages loaded with NP1 (5 μM) were treated with PMA for 15 min (1 μg/mL) (Figure 3). The fluorescence signal from the yellow channel (575 ± 25 nm) was increased after PMA stimulation (Figure 3b, 3f). The merged images also reflected the differences between PMA negative (Figure 3c) and PMA positive cells (Figure 3g). The control group gave low RY/B values (0.25) (Figure 3h). Taken together, these results indicated that NP1 allowed visualization of endogenous H2O2 generation in RAW 264.7 macrophages. Based on these results, we investigated whether NP1 was sensitive enough to detect H2O2 produced at physiological signaling levels. Cellular growth factors, cytokines, and angiotensin II stimulate H2O2 generation as a cellular signal transduction molecule.45 Specifically, A431 human epidermoid carcinoma cells produce H2O2, which is utilized as a signal transducer, following epidermal growth factor (EGF) stimulation through activating NADPH-dependent oxidases.46 To examine sensitivity of NP1, we investigated if NP1 was able to detect H2O2 generated in A431 cells. Cultured A431 cells were loaded with 5 μM NP1 for 30 min at 37 °C and then were stimulated with 500 ng/mL EGF. Indeed, fluorescence signals from the yellow channel (575 ± 25 nm) intensified with stimulation time (Figure 4). The result was clearer when using

Figure 5. CLSM images of rat hippocampal slices loaded with 20 μM NP1 without/with H2O2 (1 mM). λex = 405 nm; scale bar = 200 μm.

slices were comparable to those in cells. These results suggested that the dye was a H2O2 ratiometric sensor with application potential in biological samples. Synthesis of the Nuclear Targeted Ratiometric H2O2 Sensor pep-NP1. In the design of NP1, we introduced an azide group in this molecule so that NP1 was capable of being readily linked to other molecules through the click reaction. To demonstrate this, NP1 was linked to an NLS peptide (VQRKRQKLMP-NH2) via the click reaction (the probe linked to the NLS peptide was designated as pep-NP1), since it has been observed that NP1 accumulated in the cytoplasm and was hardly taken up into nuclei (Figures 2−4). This NLS peptide was purchased from a commercial source, and its Nterminus was modified with 5-hexynoic acid (the modified peptide was designated as pep-1). The click reaction between NP-1 and pep-1 was performed by using a protocol similar to that reported before51−53 and was monitored with HPLC (see the Supporting Information for experimental details). We found that the boric acid ester moiety in NP-1 was converted to a boric acid moiety in pep-NP1 after the reaction. However, it did not affect the capability of pep-NP1 to detect H2O2. Spectroscopic Properties of pep-NP1. As expected, the spectroscopic properties of pep-NP1 under in vitro physiological conditions (in PBS, pH 7.2) were similar to those of NP1. Pep-NP1 displayed a single absorption band centered at 353 nm (ε = 5 900 M−1 cm−1) (Figure S9) and emitted blue fluorescence with the maximum at 403 nm (Figure 6a). After addition of H2O2, the fluorescence intensity at 403 nm decreased with time, and a new fluorescence peak at 551 nm appeared and its intensity correspondingly enhanced. H2O2 caused a large increase in the ratio of fluorescent intensity at 551 nm with that at 403 nm (R551/403), resulting in an R551/403

Figure 4. CLSM images of 5 μM NP1-loaded A431 cells incubated with EGF (500 ng/mL) for 0, 10, and 30 min. λex = 405 nm; scale bar = 20 μm.

the ratio RY/B. In the control group, the RY/B values were small (RY/B < 0.05), whereas treatment with EGF for 30 min resulted in a more than 2-fold increase (RY/B > 0.10). The increase in the RY/B values was suppressed by catalase (Figure S8), providing evidence that NP1 was able to detect endogenous H2O2 in A431 cells. These results indicated that NP1 was a highly sensitive ratiometric H2O2 probe that was capable of monitoring H2O2 generation as a signal molecule at the physiological level. To date, few ratiometric probes that E

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treated with 200 μM H2O2. The RY/B values in the nuclear regions increased with time (Figure 8), suggesting that pepNP1 allowed visualization of changes in localized nuclear H2O2 pools.

Figure 6. (a) Fluorescence spectral changes of pep-NP1 (5 μM) with time after addition of H2O2 (200 μM). (b) The fold change (R551/403) of pep-NP1 (5 μM) to various ROS/RNS (200 μM) at 120 min. A to J represent none, tBHP, OCl−, NO, 1O2, HO·, ·OtBu, O2−, O2−·, and H2O2, respectively.

value of 125. By contrast, superoxide anion and nitric oxide induced much weaker responses with the values being 5.7 and 1.5, respectively. Other ROS led to negligible fluorescent responses (R551/403 < 1.5) (Figure 6b). Monitoring of the H2O2 Level in Nuclei. To examine if the NLS peptide could indeed carry the probe into nuclei as designed, HeLa cells were incubated with 50 μM pep-NP1. Fluorescence in the blue channel (425−465 nm) was observed (Figure 7a). To determine the subcellular location of pep-NP1,

Figure 8. CLSM ratio (RY/B) images of 50 μM pep-NP1-loaded HeLa cells stimulated with 200 μM H2O2 for (a) 0, (b) 15, (c) 30, (d) 60, and (e) 75 min at 37 °C. (f) The overlay image of (e) and the brightfield image. λex = 405 nm; scale bar = 20 μm.

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CONCLUSION In summary, we have developed a versatile hydrogen peroxide ratiometric fluorescent probe with high sensitivity and selectivity. In the presence of H2O2, the ratiometric signal (F555/F403) of the probe changed by 1020-fold. The probe was sensitive enough to detect endogenous H2O2 generated in live RAW 264.7 macrophages through a cellular inflammation response and in A431 human epidermoid carcinoma cells as a signaling molecule. The presence of an azide group in the probe molecule gave it great potential to be linked to a variety of molecules or matrices for detection of H2O2 for various purposes, including, but not limited to, the H2O2 levels at different subcellular locations, in tissues, in the microenvironments around a specific material, etc. The potential of the probe was demonstrated through being linked to an NLS peptide, leading to a specific probe that was able to detect H2O2 in nuclei. The combination of ratiometric capability, sensitivity, specificity, and versatility makes the probe a candidate for have a lot of potential applications. Further optimization in the structure may lead to an even better probe.

Figure 7. CLSM images of HeLa cells colabeled (a−c) with pep-NP1 (50 μM)/PI (1 nM) and (e−g) with NP1 (5 μM)/ PI (1 nM) at 37 °C; (a, e) blue channel, (b, f) red channel, and (c, g) the overlay images of blue and red channels; (d, h) cross-sectional analysis along the white line in the insets (amplified images of a single cell in green squares in (c) and (g), respectively). Blue channel: 445 ± 20 nm for NP1/pep-NP1; red channel: 625 ± 25 nm for PI. λex = 405 nm; scale bar = 20 μm.

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the pep-NP1-loaded cells were further incubated with the nuclear stain propidium iodide (PI), whose emission was observed in the red channel (600−650 nm) (Figure 7b). The images of the cells showed good colocalization of the two dyes (Figure 7c), indicating that pep-NP1 specifically accumulated in nuclei. The cross-sectionsl analysis of a single cell in the blue and red channels showed a large signal ratio between the nucleus and cytoplasm, further establishing that pep-NP1 was located in the nucleus (Figure 7d). Importantly, the fluorescence observed in the blue channel indeed came from pep-NP1 rather than the interference of background fluorescence or that of PI fluorescence (Figures S10 and S11). Under the same conditions, NP1 largely accumulated in cytoplasm (Figure 7e−h). Therefore, the results indicated that the NLS peptide efficiently ferried the probe into nuclei. Pep-NP1 was then tested for its capability to respond to local changes at nuclear H2O2 levels. HeLa cells loaded with 50 μM pep-NP1 (in comparison with NP1, a higher concentration was needed to get enough probe accumulated in nuclei) were

ASSOCIATED CONTENT

S Supporting Information *

Detection mechanism of NP1, additional information, and characteristics of NP1 and pep-NP1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: (+86) 21-55664621 (T.Y.). *E-mail: [email protected]. Fax: (+86) 21-66136928 (X.Z.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the National Basic Research Program of China (2013CB733700), the China National Funds for F

dx.doi.org/10.1021/ac502909c | Anal. Chem. XXXX, XXX, XXX−XXX

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Distinguished Young Scientists (21125104), National Natural Science Foundation of China (51373039), Specialized Research Fund for the Doctoral Program of Higher Education (20120071130008), Program for Innovative Research Team in University (IRT1117), Program of Shanghai Subject Chief Scientist (12XD1405900), and Shanghai Leading Academic Discipline Project (B108) for financial support. Y.W. thanks Mr. Feng Zhou and Professor Wei Zhu from Shanghai Medical College of Fudan University for the gift of fresh rat hippocampal slices. We are grateful to Mr. Liangyu Sun from Shanghai Abbiochem Co., Ltd for his assistance in the preparation of peptides.

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