A Fluorescent Probe for Hydrogen Peroxide in Vivo ... - ACS Publications

Apr 18, 2017 - (University of Jinan), Shandong Provincial Key Laboratory of Fluorine Chemistry and Chemical Materials, University of Jinan, Jinan. 250...
10 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/ac

A Fluorescent Probe for Hydrogen Peroxide in Vivo Based on the Modulation of Intramolecular Charge Transfer Yuzhi Chen,¶,† Xiaomin Shi,¶,§ Zhengliang Lu,*,§ Xuefei Wang,*,⊥ and Zhuo Wang*,†,‡ †

College of Science, State Key Laboratory of Chemical Resource Engineering, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing 100029, China ‡ State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, China § School of Chemistry and Chemical Engineering, Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong (University of Jinan), Shandong Provincial Key Laboratory of Fluorine Chemistry and Chemical Materials, University of Jinan, Jinan 250022, China ⊥ School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: Endogenous hydrogen peroxide in vivo is related to many diseases, including cancer, diabetes, cardiovascular disease, and neurodegenerative disorders. Although many probes for detection of H2O2 have been explored, rapid response probes are still expected for in vivo application. Here, a new probe (PAM-BN-PB) was designed based on an intramolecular charge transfer (ICT) process with three parts: phenanthroimidazole, benzonitrile, and phenyl boronate. By modulation ICT process of PAM-BN-PB, H2O2 in solution systems can be detected with good selectivity. The exogenous and endogenous H2O2 in normal living cells, ischemiareperfusion injury cells, and animals all can be imaged by PAM-BN-PB.

H

time still has room to improve. A quick-response probe will be more helpful for in vivo detection. Several probes were reported to quickly sense H2O2 in minutes,44−47 but these probes were mainly applied in aqueous samples. Few probes could detect hydrogen peroxide of in vivo samples with a fast response. On the basis of the modulation of intramolecular charge transfer process, we developed a new fluorescent probe which features fast response toward H2O2 with good sensitivity and selectivity in aqueous samples and in vivo. Herein, we introduce a new chemical fluorescent probe with an intramolecular charge transfer (ICT) process. ICT probes are developed to detect various target analytes.25,39,48−50 We apply phenanthroimidazol, benzonitrile, and phenyl boronate to construct the three-component fluorescent probe PAM-BNPB for detecting H2O2. By the modulation of ICT process of PAM-BN-PB, the turn-on probe can detect H2O2 in several minutes with good sensitivity and selectivity. PAM-BN-PB has obvious enhanced fluorescence and quick response to H2O2 and is a good candidate to detect endogenous H2O2 in living cells and animals. In this paper, we demonstrate the synthesis and the working mechanism of PAM-BN-PB, the detection of

ydrogen peroxide (H2O2) is produced by the substance oxidative decomposition process in metabolism of organisms. As one of the important reactive oxygen species (ROS), H2O2 plays a significant role in the various biological progresses such as host defense, immune response, pathogen invasion, and so on.1,2 However, excessive levels of H2O2 may damage protein, DNA, and RNA of organisms, which can lead to many diseases, including cancer,3 diabetes,4 cardiovascular disease,5 and neurodegenerative disorders such as Parkinson’s disease, Alzheimer disease, and so on.6 Damage of H2O2 to neurons leads to an irreversible process.7 The ability to monitor H2O2 level change in living cells and in vivo is becoming very necessary. The detection of endogenous H2O2 would be a useful analytical method in clinical applications. Some fluorescent molecular sensors for H2O2 have been designed based on different chemical structures8−22 such as the hydrolysis of sulfonic esters by H2O2,23 the conversation of diketone to acid group,24−26 the reaction of arylboronates to phenols,18,27−37 and protein sensors.38 However, the explored probes always have some minor defects. The probes based on sulfonic ester hydrolysis also respond to other analytes besides H2O2.39 Several boronate-based probes react more slowly with H2O2 compared to other reactive species (e.g., peroxynitrite and hypochlorite).40−43 To overcome these disadvantages, some excellent sensors for H2O2 have been developed recently, and the selectivity is improved clearly.25 However, the reaction © 2017 American Chemical Society

Received: December 3, 2016 Accepted: April 18, 2017 Published: April 18, 2017 5278

DOI: 10.1021/acs.analchem.6b04810 Anal. Chem. 2017, 89, 5278−5284

Article

Analytical Chemistry

reflux until all solids were dissolved and then cooled to room temperature. Compound 3 (288 mg, 0.2 mmol) and a drop of acetic acid were added to the mixture, which was again heated to reflux for 2 h and cooled, and the crude product was filtered and washed with ethanol to afford 0.098 g of PAM-BN-PB (0.098 g, 79%) as a light yellow solid. 1H NMR (400 MHz, dDMSO) δ 12.96 (s, 1H), 8.90(t, 2H), 8.64 (d, 1H, J = 7.59 Hz,), 8.50 (d, 1H, J = 8.26 Hz,), 8.44 (d, 1H, J = 8.26 Hz), 8.08−7.42 (m, 14H), 5.54 (s, 2H), 1.28 (s, 12H). 13CNMR (125 MHz, d-DMSO) δ 156.73, 146.95, 144.33, 140.62, 135.04, 134.79, 133.31, 131.87, 129.65, 128.89, 128.18, 127.69, 127.50, 127.37, 127.29, 126.87, 125.69, 124.32, 122.37, 120.95, 119.26, 115.15, 110.29, 84.17, 71.14, 25.15. HRMS (TOF+) calcd for C41H34BN3O3 (M + H+) 628.2773, found 628.2767. Synthesis of PAM-PB. PAM-PB was synthesized by a procedure similar to that of PAM-BN-PB, and only the starting reagent was different. 1H NMR (400 MHz, d-DMSO) δ 12.90 (s, 1H), 8.91(m, 2H), 8.66 (d, 1H, J = 7.60 Hz), 8.45 (m, 2H), 7.86−7.68 (m, 11H), 7.54 (m, 2H), 7.42 (m, 2H), 5.54 (s, 2H), 1.31 (s, 12H). 13CNMR (100 MHz, d-DMSO) δ 155.62, 147.26, 140.97, 139.78, 135.06, 133.68, 133.31, 129.50, 129.29, 128.58, 128.13, 127.97, 127.67, 127.45, 127.12, 126.90, 125.81, 125.65, 124.61, 124.27, 122.41, 120.54, 114.73, 84.16, 70.45, 25.14. HRMS (TOF+) calcd for C40H35BN2O3 (M + H+) 603.2820, found 603.2815. In Vitro Cytotoxicity Examination. HeLa cells were incubated in DMEM supplemented with 10% fetal bovine serum and 1% penicillin−streptomycin at 37 °C in a humidified atmosphere of 5% CO2. To determine the cytotoxicity, HeLa cells were seeded in 96-well plates for 12 h to allow cell attachment. Then, the cells were treated with different concentrations of PAM-BN-PB ranging from 0 to 200 μM for 24 h. The viability of HeLa cells in the culture environment of PAM-BN-PB was then analyzed using MTT cytotoxicity assays. Confocal Microscopy Imaging. HeLa cells were seeded in 35 mm glass dishes at a density of 3 × 105 cells per dish in culture media. After overnight culture, HeLa cells were incubated with 10 μM PAM-BN-PB for 10 min and then washed with phosphate-buffered saline (PBS) twice. To determine the ability of PAM-BN-PB to respond to H2O2 in living biological systems, H2O2 solution (50 μM) was added into PAM-BN-PB treated cells above. Fluorescence images were acquired at different time points (0, 5, 10, 15, 20, and 30 min) to observe the fluorescence intensity change process by confocal laser scanning microscopy. Confocal Imaging during Oxygen−Glucose Deprivation/Reperfusion (OGD/R). HeLa cells were seeded in 35 mm glass dishes at a density of 3 × 105 cells per dish in culture media. After overnight culture, HeLa cells were washed twice with PBS and then cultured with glucose-free DMEM containing sodium hydrosulfite (0.3 mmol/L) to remove residual oxygen. The dishes were placed into an airtight AnaroPouch bag, and the cells were incubated in this condition for 5 h to produce OGD environment. After this, the HeLa cells were incubated again in high-glucose DMEM at 37 °C in 95% air/5% CO2 for another 1 h to induce formation reperfusion injury. Then, PAM-BN-PB (10 μM) was added into the dishes to record the existing H2O2, and the dishes were incubated for 10 min. Fluorescence images were acquired by confocal microscopy. Animal Models and in Vivo Imaging. Before imaging, the Kunming mice were fasted for 12 h to avoid the possible

exogenous and endogenous H2O2 in cells, and simulated H2O2 detection in animals.

■

EXPERIMENTAL SECTION Materials and Instruments. All solvents were purified using standard methods. All starting materials came from commercial suppliers and were used directly. 1H and 13C NMR were performed on a 400 MHz/100 MHz Bruker Advance DRX 400 spectrometer and a 500 MHz/125 MHz Bruker AVIII 500 spectrometer. TOF-MS was measured by Autoflex III MALDI-TOF-MS. High resolution mass measurements were carried out on a Waters-Q-TOF-Premier (ESI) or a Shimadzu LCMS-IT-TOF (ESI). UV−vis absorption spectra were measured on a Shimadzu UV-2100 spectrophotometer. Fluorescence spectra were obtained on an F-380 spectra fluorophotometer. 3-(4, 5-Dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and NAC were purchased from KeyGEN BioTECH. Rotenone was from Aladdin Industrial Corporation. Chloral hydrate was from Macklin (Shanghai China). The MGC AnaeroPouch, oxygen indicator, and anaerobic bag were purchased from Mitsubishi Gas Chemical Co. (Tokyo, Japan). All the cells were purchased from China Infrastructure of Cell Line Resources. All of the cells were grown in DMEM high glucose medium (HyClone) supplemented with 10% heatinactivated fetal bovine serum (TBD, Tianjin), 1% penicillin, and streptomycin (HyClone) under a 5% CO2 humidified atmosphere at 37 °C. Male Kunming mice (20 g) were purchased from the Academy of Military Medical Sciences. The fluorescence images of cells were taken using a confocal laser scanning microscope (Leica Microsystems) with an objective lens (63× ) and excitation at 405 nm. In vivo imaging was performed on an IVIS Spectrum Imaging System. The absorbance was measured using a PerkinElmer Multimode plate reader in the MTT assay. Synthesis of Compound 2a. Compound 2a was synthesized according to the reported procedure.51 Dry paraformaldehyde (6.6 g) was added to a mixture of 4′hydroxybiphenyl-4-carbonitrile (3.1 g, 16 mmol), triethyl-amine (8.4 mL, 61 mmol), and anhydrous MgCl2 (2.3 g, 24 mmol) in dry acetonitrile (50 mL). The mixture was heated to reflux for 6 h and then cooled to room temperature, acidified with 1 M HCl, and extracted with ethyl acetate (3 × 20 mL). The combined organic layer was washed with water and dried over MgSO 4 . The crude material was purified by column chromatography to give 1.95 g of the title compound as a white solid in 55% yield. Synthesis of Compound 3a. Compound 3a was synthesized by mixing compound 2 (100 mg, 0.45 mmol) and 2-(4-bromobenzyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (133 mg, 0.45 mmol) in acetone with anhydrous K2CO3, and the mixture was refluxed at 50 °C overnight. The crude material was purified by column chromatography in 85% yield. 1H NMR (400 MHz, CDCl3) δ 10.63 (s, 1H), 8.13 (d, 1H, J = 2.4 Hz), 7.88 (d, 2H, J = 8.0 Hz), 7.76−7.67 (m, 5H), 7.48(d, 2H, J = 7.6 Hz), 7.15 (d, 1H, J = 8.8 Hz), 5.32 (s, 2H), 1.377 (s, 12H). 13 C NMR (100 MHz, CDCl3) δ 189.25, 161.16, 143.87, 138.67, 135.27, 134.19, 132.72, 131.43, 130.29, 128.00, 127.22, 127.02, 126.44, 125.45, 118.82, 113.93, 110.97, 83.98, 70.71, 24.88. Synthesis of PAM-BN-PB. A mixture of 9,10-phenanthrenequinone (42 mg, 0.2 mmol) and ammonium acetate (0.15 g, 2 mmol) was suspended in a solution of ethanol (15 mL) and dichloromethane (1.5 mL). The suspension was heated to 5279

DOI: 10.1021/acs.analchem.6b04810 Anal. Chem. 2017, 89, 5278−5284

Article

Analytical Chemistry

Scheme 2. Synthesis Route of PAM-BN-PB and PAM-PBa

impact of food fluorescence. The mice were divided into four groups. The first group was given an injection of PAM-BN-PB (20 μM, 100 μL) into the peritoneal cavity as the negative control. The second group was given an injection of rotenone (2.5 mM, 100 μL, thus 5 mg/kg of animal weight) into the peritoneal cavity followed by injection of PAM-BN-PB (20 μM, 100 μL) at the same region after 1 h. The third group was successively treated with rotenone (2.5 mM, 100 μL) for 1 h, NAC (20 mM, 100 μL, thus 16 mg/kg of animal weight) for 1 h, and PAM-BN-PB (20 μM, 100 μL) at the same region. The fourth group was given an injection of NAC (20 mM. 100 μL) for 1 h and then treated with PAM-BN-PB (20 μM, 100 μL) at the same region. Before imaging, the mice were anesthetized with 4% chloral hydrate (15 mL/kg) by intraperitoneal injection. The mice in vivo imaging was then acquired using the IVIS Spectrum system (excitation wavelength range is from 415 to 760 nm).

a

(i) Anhydrous MgCl2, (HCHO)n, Et3N/anhydrous CH3CN; (ii) 2(4-bromobenzyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, K2CO3/acetone; (iii) 9,10-phenanthrenedione, ammonium acetate/CH3CH2OH.

■

Compound 1a refluxed with dry paraformaldehyde in anhydrous CH3CN to get compound 2a. To introduce phenyl boronate group for sensing H2O2, compound 2a reacted with 2(4-bromobenzyl)-4,4, 5,5-tetramethyl-1,3,2-dioxaborolane in acetone to obtain compound 3a. Then, PAM-BN-PB was constructed by the reaction of compound 3a with 9,10phenanthrenedione. PAM-BN-PB contains a phenyl boronate group, which is sensitive to H2O2. At the other end of PAM-BN-PB, benzonitrile as an electron-withdrawing part modulates the ICT process to induce the enhancement of fluorescence. PAMBN-PB shows weak fluorescence because of the quench process between PAM and the benzonitrile group. To identify the proposed mechanism, we characterized PAM-BN-PB without H2O2 by TOF-MS and found m/z 628.3, which was the massto-charge ration of PAM-BN-PB. After addition of H2O2 in PAM-BN-PB, we found m/z 412.2, which was the mass-tocharge ratio of PAM-BN (Scheme 1, Figure S2). The mass spectra that identified the sensing reaction mechanism of PAMBN-PB with H2O2 was correct. Spectra Properties and Sensing Behavior of PAM-BNPB in the Solution Sample. While H2O2 was present, the reaction of PAM-BN-PB and H2O2 happened and resulted the dissociation of the phenyl boronate group from PAM-BN-PB with the recovery of the fluorescence. The polarity of the solvents affected the fluorescence of ICT probes. We optimized the solvent condition before the detection experiments. We recorded the fluorescence intensities of PAM-BN-PB in the absence and present of H2O2 in THF, CH3CN, CH3OH, DMF, and DMSO (Figure S3). In these solvents, PAM-BN-PB had a different response to H2O2. In DMSO, PAM-BN-PB showed the most clear enhancement degree of fluorescence intensity. To apply PAM-BN-PB in biological samples, we investigated the aqueous solubility of PAM-BN-PB. With the aid of DMSO (10%, volume ration) in aqueous solution, PAM-BN-PB can be used to sense H2O2 both in solution and biological samples. We investigated the spectra behavior of PAM-BN-PB with the addition of H2O2 in the mixed DMSO/H2O (1/9, PBS buffer). When the concentration of PAM-BN-PB was under 20 μM, the relationship of absorption and the concentration was linear (Figure S4). We recorded the UV−vis spectra of PAM-BN-PB with and without H2O2 in the optimized solvent (Figure S5). Compared with those of the reported PAM sensor,42 the UV− vis spectra moved to a longer wavelength. The addition of hydrogen peroxide did not cause the shift of peaks, while the absorption intensity increased in some degree. The fluorescent spectra of PAM-BN-PB (10 μM) were recorded with the

RESULTS AND DISCUSSION Sensing Mechanism and Synthesis of PAM-BN-PB. The previously reported probes with phenanthroimidazole show good fluorescence behavior with the ICT process. Here, we adjust the fluorescence of phenanthroimidazole from “off” to “on” and move the excitation wavelength beyond 400 nm by using pull and push groups, which will be more suitable for the biological imaging. Usually, the excitation wavelength of phenanthroimidazole is shorter than 400 nm.51,52 Benzonitrile as an electron-withdrawing group is linked with phenanthroimidazole (PAM) to quench the fluorescence because of the ICT process. We designed the probe with arylboronate, which acts as a recognition unit for H2O2. By the reaction of PAMBN-PB with H2O2, arylboronate is changed to a phenolic group, which is a good electron donor and inhibits the ICT process between PAM and benzonitrile with the recovery of fluorescence. By modulation of ICT processes, the fluorescence of PAM-BN-PB is changed from “off” to “on” by H2O2. (Scheme 1) This mechanism was identified by the frontiers Scheme 1. Sensing Mechanism of PAM-BN-PB for H2O2

molecular orbital profiles of PAM-BN-PB and PAM-BN (Figure S1). HOMO of PAM-BN-PB localized on the PAM moiety and LUMO on BN, which implied a typical ICT process. In contrast, for PAM-BN, both HOMO and LUMO were located on the PAM moiety. Therefore, it is reasonable to attribute the observed fluorescence to the native excited sate of PAM, but the ICT performs a nonradiative deactivation.27 PAM-BN-PB was synthesized as shown in Scheme 2. The synthesis of PAM-BN-PB started from compound 1a. 5280

DOI: 10.1021/acs.analchem.6b04810 Anal. Chem. 2017, 89, 5278−5284

Article

Analytical Chemistry

which were 40.8 and 2.12 M−1min−1, individually. The conversions of PAM-BN-PB and PAM-PB were 94 and 92%, separately (Supporting Information). For the following biological analysis, we checked pH effect of PAM-BN-PB in the detection of H2O2. When the range of pH was from 5 to 10, PAM-BN-PB showed good response to hydrogen peroxide (Figure S8). Before analyzing the biological samples, we checked the selectivity of PAM-BN-PB. In the following experiments, we tested some interferences which could exist in the biological samples. Some ROS and reactive molecules (glutathione, GSH; vitamin C, Vc) were examined by adding then into PAM-BNPB. We set the concentration of these interferences as 200 μM. The fluorescence of PAM-BN-PB hardly changed in the presence of these interferences, including peroxyacetic acid (CH3COOOH), hydroxyl radical (·OH), tert-butylhydroperoxide (TBHP), tert-butoxy radical (·OtBu), superoxide (O2−), and hypochlorite (ClO−). Peroxynitrite (ONOO−) can proceed in a reaction similar to that of arylboronate,40 which may also possibly induce the fluorescence recovery of PAMBN-PB. We added peroxynitrite (200 μM) into the solution of PAM-BN-PB. Compared with that in H2O2, PAM-BN-PB has a weaker response with ONOO−. The amount of ONOO− in living cells and in vivo is relative to some diseases. In normal cells and organisms, the interference of ONOO− can be ignored.52 GSH and Vc also cannot induce the fluorescence recovery of PAM-BN-PB (Figure 2).

concentration range of H2O2 from 0 to 200 μM in DMSO/ H2O (1/9, PBS buffer) excited at 410 nm (Figure 1). Without

Figure 1. (a) Fluorescence response (λ = 410 nm) of 10 μM PAMBN-PB to different concentrations of H2O2 (0−200 μM) in PBS buffer (10% DMSO) at room temperature for 8 min. (b) Linear relationship from fluorescence titration.

H2O2, PAM-BN-PB showed weak fluorescence around 480 nm. With the addition of H2O2, the fluorescent intensity increased clearly. When the amount of H2O2 reached 200 μM, the fluorescent intensity reached the largest value. PAM-BN-PB and H2O2 had good linear relationship from 0 to 150 μM with an excellent linear correlation (R2 = 0.999). The limitation of detection (LOD) was calculated to be 148 nM based on S/N = 3.25 We recorded the reaction dynamic curves of PAM-BN-PB with different concentrations of H2O2 from 0 to 15 min (Figure S6). The concentrations of H2O2 were set as 0, 100, and 200 μM separately, and when the time lapsed, the reaction of PAMBN-PB and H2O2 ceased in 8 min. Compared with those of the reported probes,8 PAM-BN-PB showed faster response to H2O2. Generally, the reaction of arylboronate and H2O2 gets to the reaction equilibrium point in an hour. PAM-BN-PB has a shorter reaction time mainly due to the pull-and-push groups in the molecular structure. Benzonitrile is a strong electron withdrawing group, which quickens the nucleophilic reaction between phenyl boronate and H2O2. Then, PAM-BN-PB can sense H2O2 within 10 min, and the quick response time gives a feasible analysis application in vivo. To elucidate nitrile group effect, the other molecule without a nitrile group (PAM-PB) was synthesized with a similar procedure (Scheme 2). We recorded the dynamic curve of PAM-PB (Figure S7). The reaction of PAM-PB and H2O2 reached an equivalent point in 20 min, so the reaction rate of PAM-PB was clearly lower than that of PAM-BN-PB. We calculated the second order rate constants of PAM-BN-PB with H2O2 and PAM-PB with H2O2,

Figure 2. Fluorescence response of 10 μM PAM-BN-PB to various interferences, including CH3COOOH, TBHP, ClO−, O2−, ·OH, · OtBu, glutathione (GSH), vitamin C (Vc), and ONOO-; the concentration of these interferences was 200 μM.

Detection of Exogenous and Endogenous H2O2 in Cells. From the above data, PAM-BN-PB has quick response with H2O2 with good selectivity. Before sensing H2O2 in living cells and animals, we checked the biocompatibility of PAM-BNPB by MTT assay kits. We checked the cytotoxicity of PAMBN-PB at different concentrations (10, 50, 100, and 200 μM). Even when the concentration of PAM-BN-PB reached up to 200 μM, the cell viability was still more than 90% after 24 h. (Figure S9) PAM-BN-PB showed good biocompatibility and could be used in cells and animals safely. Then, we checked the response time of PAM-BN-PB in biological samples. First, we applied PAM-BN-PB to image exogenous H2O2 in HeLa cells. HeLa cells were incubated with 10 μM PAM-BN-PB for 10 min and washed with phosphate-buffered saline, and then H2O2 solution was added into the cells. We took the fluorescent images of the cells at 0, 5, 10, 15, 20, and 30 min individually. In 5281

DOI: 10.1021/acs.analchem.6b04810 Anal. Chem. 2017, 89, 5278−5284

Article

Analytical Chemistry Figure 3, in 15 min, the fluorescence image of HeLa cells reached the biggest intensity. PAM-BN-PB can detect H2O2 in

Figure 4. Confocal images of H2O2 taken during OGD/R in HeLa cells. (a) Control PAM-BN-PB (10 μM, 10 min). (b) HeLa cells incubated in OGD environment for 5 h followed by 1 h reperfusion and then incubated with 10 μM PAM-BN-PB for 10 min. Scale bar is 25 μm.

using an IVIS Spectrum system. In Figure 5, the mice injected with PAM-BN-PB showed lower fluorescence intensity (Figure

Figure 3. Fluorescence images of HeLa cells. HeLa cells were incubated with 10 μM PAM-BN-PB for 10 min, washed with PBS, and then incubated with H2O2 (50 μM). Fluorescence images were acquired at different time points (0, 5, 10, 15, 20, and 30 min) to observe the fluorescence intensity change by confocal laser scanning microscopy. Scale bar is 10 μm.

living cells less slowly compared with the aqueous samples, while it still shows a faster response than other reported probes. PAM-BN-PB can detect exogenous H2O2 successfully, which gives the possibility to detect endogenous H2O2 in living cells. Rotenone was employed as a stimulator to trigger the upregulation of intracellular H2O2.53 We treated HeLa cells with rotenone, and the fluorescence images were taken and are shown in Figure S10, which indicated that the fluorescence increased obviously with the treatment of rotenone in cells. NAcetylcystein (NAC) was added to the cell culture medium to eliminate H2O2 as an antioxidant. In Figure S6c, the fluorescence enhancement was inhibited by NAC. Therefore, PAM-BN-PB is able to detect endogenous H2O2 effectively. Imaging H2O2 during OGD/R Process in Cells. H2O2 is an important member of the ROS family and plays a crucial role in the regulation of a wide variety of biological processes. When the tissue or organ is in the ischemia status, ROS will create oxidative damage, resulting in cell apoptosis and other physiological reactions.54−56 We used HeLa cells to set an ischemia model by the glucose-free DMEM containing sodium hydrosulfite. HeLa cells were exposed to OGD/R environment.57 PAM-BN-PB was used to measure the endogenous H2O2 during OGD/R. In Figure 4, an obvious enhancement of fluorescence intensity could be obtained compared with that of the control group (Figure 4a). This result further confirmed that PAM-BN-PB could track endogenously produced H2O2 in living cells. Imaging H2O2 in Vivo. The imaging experiment was performed to examine the ability of PAM-BN-PB to respond to H2O2 in vivo. There were four groups of mice in this experiment. The first group was injected with PAM-BN-PB into the peritoneal cavity as the negative control. The second group was injected with rotenone first, followed by injection of PAMBN-PB at the same region after 1 h. The third group was successively treated with rotenone, NAC, and PAM-BN-PB at the same region. The fourth group was injected with NAC first, followed by injection of PAM-BN-PB at the same region after 1 h as a control experiment. The mice imaging was then acquired

Figure 5. In vivo fluorescence imaging. (a) Only PAM-BN-PB was injected as the negative control. (b) Mice were injected with rotenone first, followed by injection of PAM-BN-PB after 1 h. (c) Rotenone and NAC were injected into the mice in turn and then PAM-BN-PB was injected into the same region. (d) Mice were injected with NAC first, followed by injection of PAM-BN-PB after 1 h. (e) Relative fluorescence intensities of a−d.

5a). As a comparison, the fluorescence intensity of the second group showed an obvious enhancement, which was pretreated with rotenone before the injection of PAM-BN-PB (Figure 5b). Rotenone could induce the production of H2O2. The fluorescence intensity (Figure 5c) showed significant reduction compared with that of the other two groups, which was attributed to the inhibitory action of NAC. In Figure 5d, the fluorescence intensity of PAM-BN-PB was very weak because NAC was a reductant to remove oxygen species in the mice. The quantitative analysis of fluorescence intensity is shown in Figure 5e. The fluorescence intensity of the second group was significantly higher than those of the other three groups. The above experimental results show that PAM-BN-PB can successfully indicate the presence of H2O2 in vivo.

■

CONCLUSION In sum, a three-component chemical probe (PAM-BN-PB) was explored to sense hydrogen peroxide in vitro and in vivo. The working mechanism of PAM-BN-PB is based on the modulation of the ICT process by reaction of the phenyl boronate group and H2O2. Compared with those of the reported probes, PAM-BN-PB had a good response to H2O2 not only in vitro but also in vivo. In the ischemia cell model, the probe can detect the upregulated H2O2 with the enhancement of fluorescent intensity. In the animal experiment, PAM-BN-PB can image H2O2 in the peritoneal cavity of mice. Actually, many hydrogen peroxide probes have been reported. The advantage 5282

DOI: 10.1021/acs.analchem.6b04810 Anal. Chem. 2017, 89, 5278−5284

Article

Analytical Chemistry

(8) Brewer, T. F.; Garcia, F. J.; Onak, C. S.; Carroll, K. S.; Chang, C. J. Annu. Rev. Biochem. 2015, 84, 765−90. (9) Xu, W.; Zeng, Z.; Jiang, J.-H.; Chang, Y.-T.; Yuan, L. Angew. Chem., Int. Ed. 2016, 55 (44), 13658−13699. (10) Zhang, Y.; Fu, Y.-Y.; Zhu, D.-F.; Xu, J.-Q.; He, Q.-G.; Cheng, J.G. Chin. Chem. Lett. 2016, 27, 1429−1436. (11) Chen, X.; Wang, F.; Hyun, J. Y.; Wei, T.; Qiang, J.; Ren, X.; Shin, I.; Yoon, J. Chem. Soc. Rev. 2016, 45, 2976−3016. (12) Zamojc, K.; Zdrowowicz, M.; Jacewicz, D.; Wyrzykowski, D.; Chmurzynski, L. Crit. Rev. Anal. Chem. 2016, 46, 171−200. (13) Li, Q.; Chen, P.; Fan, Y.; Wang, X.; Xu, K.; Li, L.; Tang, B. Anal. Chem. 2016, 88, 8610−8616. (14) Zhang, R.; Zhao, J.; Han, G.; Liu, Z.; Liu, C.; Zhang, C.; Liu, B.; Jiang, C.; Liu, R.; Zhao, T.; Han, M.-Y.; Zhang, Z. J. Am. Chem. Soc. 2016, 138, 3769−3778. (15) Zhou, W.; Cao, Y.; Sui, D.; Lu, C. Angew. Chem., Int. Ed. 2016, 55, 4236−4241. (16) Song, Z.; Kwok, R. T. K.; Ding, D.; Nie, H.; Lam, J. W. Y.; Liu, B.; Tang, B. Z. Chem. Commun. 2016, 52, 10076−10079. (17) Liu, G. J.; Long, Z.; Lv, H. J.; Li, C. Y.; Xing, G. W. Chem. Commun. 2016, 52, 10233−6. (18) Xu, J.; Zhai, J.; Xu, Y.; Zhu, J.; Qin, Y.; Jiang, D. Analyst 2016, 141, 141. (19) Wen, Y.; Liu, K.; Yang, H.; Liu, Y.; Chen, L.; Liu, Z.; Huang, C.; Yi, T. Anal. Chem. 2015, 87, 10579−10584. (20) Narayanaswamy, N.; Narra, S.; Nair, R. R.; Saini, D. K.; Kondaiah, P.; Govindaraju, T. Chem. Sci. 2016, 7, 2832−2841. (21) Xiao, H.; Li, P.; Hu, X.; Shi, X.; Zhang, W.; Tang, B. Chem. Sci. 2016, 7, 6153−6159. (22) Jing, J.; Zhang, J.-L. Chem. Sci. 2013, 4 (7), 2947−2952. (23) Maeda, H.; Fukuyasu, Y.; Yoshida, S.; Fukuda, M.; Saeki, K.; Matsuno, H.; Yamauchi, Y.; Yoshida, K.; Hirata, K.; Miyamoto, K. Angew. Chem., Int. Ed. 2004, 43, 2389−2391. (24) Zhang, K. M.; Dou, W.; Li, P. X.; Shen, R.; Ru, J. X.; Liu, W.; Cui, Y. M.; Chen, C. Y.; Liu, W. S.; Bai, D. C. Biosens. Bioelectron. 2015, 64, 542−6. (25) Xie, X.; Yang, X.; Wu, T.; Li, Y.; Li, M.; Tan, Q.; Wang, X.; Tang, B. Anal. Chem. 2016, 88, 8019−25. (26) Dong, B.; Song, X.; Kong, X.; Wang, C.; Tang, Y.; Liu, Y.; Lin, W. Adv. Mater. 2016, 28, 8755−8759. (27) Kim, D.; Kim, G.; Nam, S.-J.; Yin, J.; Yoon, J. Sci. Rep. 2015, 5, 8488. (28) Yang, Y. C.; Tseng, W. L. Anal. Chem. 2016, 88, 5355−62. (29) Xu, J.; Zhang, Y.; Yu, H.; Gao, X.; Shao, S. Anal. Chem. 2016, 88, 1455−61. (30) Liu, J.; Ren, J.; Bao, X.; Gao, W.; Wu, C.; Zhao, Y. Anal. Chem. 2016, 88, 5865−70. (31) Liu, K.; Shang, H.; Kong, X.; Ren, M.; Wang, J. Y.; Liu, Y.; Lin, W. Biomaterials 2016, 100, 162−71. (32) Xu, J.; Li, Q.; Yue, Y.; Guo, Y.; Shao, S. Biosens. Bioelectron. 2014, 56, 58−63. (33) Ren, M.; Deng, B.; Wang, J. Y.; Kong, X.; Liu, Z. R.; Zhou, K.; He, L.; Lin, W. Biosens. Bioelectron. 2016, 79, 237−43. (34) Han, Z.; Liang, X.; Ren, X.; Shang, L.; Yin, Z. Chem. - Asian J. 2016, 11, 818−22. (35) Dickinson, B. C.; Chang, C. J. J. Am. Chem. Soc. 2008, 130, 9638−9639. (36) Miller, E. W.; Tulyanthan, O.; Isacoff, E. Y.; Chang, C. J. Nat. Chem. Biol. 2007, 3, 263−7. (37) Zhu, B.; Jiang, H.; Guo, B.; Shao, C.; Wu, H.; Du, B.; Wei, Q. Sens. Actuators, B 2013, 186, 681−686. (38) Warren, E. A.; Netterfield, T. S.; Sarkar, S.; Kemp, M. L.; Payne, C. K. Sci. Rep. 2015, 5, 16929. (39) Li, X.; Gao, X.; Shi, W.; Ma, H. Chem. Rev. 2014, 114, 590−659. (40) Zhou, J.; Li, Y.; Shen, J.; Li, Q.; Wang, R.; Xu, Y.; Qian, X. RSC Adv. 2014, 4, 51589−51592. (41) Reverte, M.; Vaissiere, A.; Boisguerin, P.; Vasseur, J.-J.; Smietana, M. Acs Sensors 2016, 1 (8), 970−974.

of PAM-BN-PB focuses on the rapid response to H2O2 in minutes in vivo. Generally, detection of H2O2 in solution samples is quick. However, detection in vivo usually requires more time. We designed PAM-BN-PB with a nitrile group to structure an ICT probe, and the sensing mechanism is supported by theoretical analysis. Because the fluorescence of PAM-BN-PB is blue, it is not a perfect probe to use in vivo. In the future, we are planning to move the emission wavelength close to red by introduction of other push-and-pull groups in the probe.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b04810. Supplementary data, experimental details, and characterization figures of compounds (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]; Fax: +86-0164434898. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Zhuo Wang: 0000-0002-2858-7646 Author Contributions ¶

Y.C. and X.S. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Dr. Yingyi Liu for designing the TOC figure. We thank the microscopy imaging department in the Beijing Laboratory of Biomedical Materials. We thank support from the Natural Science Foundation of China (Grants 21101074 and 21575032), Research Foundation for Advanced Talents of Beijing University of Chemical Technology, Shandong Provincial Natural Science Foundation of China (Grant ZR2013BQ009), the Doctor’s Foundation of University of Jinan (Grant XBS1320), Open Ground from State Key Laboratory of Chemo/Biosensing and Chemometrics Hunan University, the Fundamental Research Funds for the Central Universities (Grants PYBZ1707 and buctrc201607), and the Open Ground from Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, CAS.

■

REFERENCES

(1) Rossi, F.; Zatti, M. Experientia 1964, 20, 21−23. (2) Babior, B. M.; Kipnes, R. S.; Curnutte, J. T. J. Clin. Invest. 1973, 52, 741−744. (3) Radeke, H. H.; Meier, B.; Topley, N.; Flöge, J.; Habermehl, G. G.; Resch, K. Kidney Int. 1990, 37, 767−775. (4) Houstis, N.; Rosen, E. D.; Lander, E. S. Nature 2006, 440 (7086), 944−948. (5) Burgoyne, J. R.; Oka, S.-i.; Ale-Agha, N.; Eaton, P. Antioxid. Redox Signaling 2013, 18, 1042−1052. (6) Lin, M. T.; Beal, M. F. Nature 2006, 443, 787−795. (7) Mann, H.; McCoy, M. T.; Subramaniam, J.; Van Remmen, H.; Cadet, J. L. Brain Res. 1997, 770, 163−168. 5283

DOI: 10.1021/acs.analchem.6b04810 Anal. Chem. 2017, 89, 5278−5284

Article

Analytical Chemistry (42) Xu, S.-Y.; Sun, X.-L.; Ge, H.-B.; Arrowsmith, R. L.; Fossey, J. S.; Pascu, S. I.; Jiang, Y.-B.; James, T. D. Org. Biomol. Chem. 2015, 13, 4143−4148. (43) Wu, X.; Chen, X.-X.; Song, B.-N.; Huang, Y.-J.; Ouyang, W.-J.; Li, Z.; James, T. D.; Jiang, Y.-B. Chem. Commun. 2014, 50, 13987− 13989. (44) Ren, M.; Deng, B.; Wang, J.-Y; Kong, X.; Liu, Z.-R.; Zhou, K.; He, L.; Lin, W. Biosens. Bioelectron. 2016, 79, 237−243. (45) Liu, C.; Shao, C.; Wu, H.; Guo, B.; Zhu, B.; Zhang, X. RSC Adv. 2014, 4, 16055−16061. (46) Hitomi, Y.; Takeyasu, T.; Kodera, M. Chem. Commun. 2013, 49, 9929−9931. (47) Li, G.; Zhu, D.; Liu, Q.; Xue, L.; Jiang, H. Org. Lett. 2013, 15, 924−927. (48) Manjare, S. T.; Kim, Y.; Churchill, D. G. Acc. Chem. Res. 2014, 47, 2985−2998. (49) Xiao, H.; Li, J.; Zhao, J.; Yin, G.; Quan, Y.; Wang, J.; Wang, R. J. Mater. Chem. B 2015, 3, 1633−1638. (50) Liu, C.; Shen, Y.; Yin, P.; Li, L.; Liu, M.; Zhang, Y.; Li, H.; Yao, S. Anal. Biochem. 2014, 465, 172−178. (51) Fan, C.; Huang, X.; Black, C. A.; Shen, X.; Qi, J.; Yi, Y.; Lu, Z.; Nie, Y.; Sun, G. RSC Adv. 2015, 5, 70302−70308. (52) Song, K. C.; Kim, H.; Lee, K. M.; Lee, Y. S.; Do, Y.; Lee, M. H. Sens. Actuators, B 2013, 176, 850−857. (53) Mansouri, A.; Muller, F. L.; Liu, Y.; Ng, R.; Faulkner, J.; Hamilton, M.; Richardson, A.; Huang, T.-T.; Epstein, C. J.; Van Remmen, H. Mech. Ageing Dev. 2006, 127, 298−306. (54) Pacher, P.; Beckman, S. J.; Liaudet, L. Physiol. Rev. 2007, 87, 315. (55) Braunersreuther, V.; Mach, F.; Montecucco, F. Curr. Signal Transduction Ther. 2012, 7, 89−95. (56) Li, L.; Tan, J.; Miao, Y.; Lei, P.; Zhang, Q. Cell. Mol. Neurobiol. 2015, 35, 615−621. (57) Widgerow, A. D. Ann. Plast. Surg. 2014, 72 (2), 253−260.

5284

DOI: 10.1021/acs.analchem.6b04810 Anal. Chem. 2017, 89, 5278−5284