Pyrene Derivative Emitting Red or near-Infrared Light with Monomer

Research Article www.acsami.org

Pyrene Derivative Emitting Red or near-Infrared Light with Monomer/Excimer Conversion and Its Application to Ratiometric Detection of Hypochlorite Yinglong Wu, Jun Wang, Fang Zeng,* Shuailing Huang, Jing Huang, Huiting Xie, Changmin Yu, and Shuizhu Wu* College of Materials Science & Engineering, State Key Laboratory of Luminescent Materials & Devices, South China University of Technology, Guangzhou 510640, China S Supporting Information *

ABSTRACT: Fluorescent sensors are attractive and versatile tools for both chemical sensing and biological imaging. Herein, a novel pyrene derivative fluorophore, Py-Cy, possessing the monomer/excimer conversion feature, was synthesized; and the design rationale for this fluorophore is combination of extending conjugation length and incorporating donor−π−acceptor structure. The positively charged PyCy shows quite good water solubility and exhibits absorption in the visible-light range, and its monomer and excimer emit red light and near-infrared light respectively, which is extremely beneficial for biosensing or bioimaging. To explore the potential utilization of this new fluorophore, we choose hypochlorite as a model analyte, which can break the double bond in the molecular structure, thereby generating the water-insoluble pyrenecarboxaldehyde; this process correspondingly leads to fluorescence changes and thus affords the ratiometric fluorescent detection of hypochlorite in real samples and cell imaging. The approach offers new insights for designing other fluorophores which emit red or near-infrared light and for devising technically simple ratiometric fluorescent sensors. KEYWORDS: fluorescent sensor, red-light emission, pyrene derivative, hypochlorite, cell imaging

■

fluorescence signals at different wavelengths followed by the calculation of their intensity ratio, is an elegant approach for achieving ratiometric sensing by simply using a single fluorophore molecule and is aimed to overcome unfavorable effects of the turn-on mode sensing. In recent years, some new pyrene-derivative chromophores emitting red fluorescence and none-pyrene chromophores with excimer-forming capability have been synthesized.29,30 For example, Ramaiah’s group created a red fluorescence sensor based on pyrene and Cy fluorophore and used it in the detection of cyanide.30 However, for most pyrene-based fluorophores, the excitation wavelength is still in the range of ultraviolet band and their emission wavelength is relatively short in visible-light band, which severely restricts their applications, especially in biological imaging, because short-wavelength light is harmful to organisms (in particular, UV light) and weak in tissue penetration. Hence, it is our primary interest to develop pyrene-based fluorophores that retain the excimer-forming feature and emit red or nearinfrared light, so as to offer new approaches for achieving technically simple ratiometric fluorescent sensing or bioimaging applications.

INTRODUCTION Fluorescent sensors are attractive and versatile tools for both chemical sensing and biological imaging because of their rapid response, high sensitivity, and technical simplicity.1−7 To a large extent, an excellent sensing system depends on the nature of chromophores themselves. Therefore, it is essential for fluorophores to have a good luminescent property, and the development of new fluorophores with satisfactory photophysical properties is highly desirable. Until now, researchers have synthesized and utilized many chromophores or their derivatives in sensing systems, such as tetraphenylethylene,8−10 BODIPY,11 rhodamine,12 fluorescein,13 and fluorescent polymers.14 Pyrene is also used frequently as fluorophore with a unique luminescence pattern.15,16 In dilute solutions (molecularly dissolved state), upon UV excitation, emission from the pyrene monomer (λ ≈ 380−410 nm) can be observed; at increased concentrations (aggregated state), the red-shifted emission band appears (λ ≈ 450−500 nm), which is attributed to the excimer formed when an excited-state molecule is brought into close proximity with another ground-state molecule. Thanks to the predictability of the two luminescence signals, pyrene moieties have been introduced in the design of numerous ratiometric sensors as reporters and other functional materials.17−28 The ratiometric mode of pyrene-involved sensing, which involves the synchronous measurement of two © 2015 American Chemical Society

Received: November 15, 2015 Accepted: December 24, 2015 Published: December 24, 2015 1511

DOI: 10.1021/acsami.5b11023 ACS Appl. Mater. Interfaces 2016, 8, 1511−1519

Research Article

ACS Applied Materials & Interfaces

Co., Ltd. Phorbol myristate acetate (PMA) was purchased from Abcam. RAW254.7 (murine macrophage cells) and Hela (human cervical cancer cell) were purchased from KeyGen Biology Co. Ltd. (Nanjing, China). Water used herein was the triple-distilled water upon being further treated with ion exchange columns and a Milli-Q water purification system. Dichloromethane, methanol, and other solvents were analytically pure reagents and distilled before use. Synthesis of 1,3,3-Trimethyl-2-(2-(pyren-1-yl)vinyl)-3Hindol-1-ium (Py-Cy). 1,2,3,3-Tetramethyl-3H-indolium iodide (260 mg, 0.864 mmol) was added into a two-neck round-bottom flask and dissolved with methanol (20 mL). Subsequently, 1-pyrenecarboxaldehyde (200 mg 0.864 mmol) was dissolved in tetrahydrofuran (5 mL) and then was added to the flask. The reaction mixture was refluxed at 70 °C for 12 h. The solvent was then evaporated in vacuo and the resulting solid was dissolved in CH2Cl2. The organic layer was separated and washed three times with water and dried over anhydrous Na2SO4. The crude product was purified on a silica-gel column using CH2Cl2 /MeOH (v/v 1:50, Rf = 0.50) as eluent. For a higher fluorescence quantum yield, then the dark red solid product was dissolved in DMF (10 mL) and NaBF4 (0.95 g, 8.64 mmol) was added in one portion to the solution. The suspension was stirred for another 3 h in the dark at room temperature. Afterward, the suspension was filtered with Celite, and the filtrate was condensed to dryness under reduced pressure. The residue was recrystallized from ethanol three times. The product Py-Cy was finally obtained in 65% yield. 1H NMR (600 MHz, CDCl3, δ ppm): 9.15−9.25 (d, J = 18 Hz, 1H), 9.05−9.15 (d, J = 8.4 Hz, 1H), 8.25−8.35 (d, J = 9 Hz, 1H), 8.15−8.25 (m, 4H), 8.05−8.15 (m, 2H), 7.95−8.02 (m, 2H), 7.52−7.63 (m, 4H), 4.40− 4.55 (s, 3H), 1.91−2.03 (s, 6H). MS (ESI): m/z 385.54 [M]+. Measurements. 1H NMR spectra were measured with Bruker Avance 600 MHz NMR Spectrometer. Mass spectra were obtained by using Bruker Esquire HCT Plus mass spectrometer and Thermo DSQ II mass spectrometer. High-resolution mass spectrum were obtained by using Bruker Agilent 1260/maXis impact. UV−vis spectra were measured on a Hitachi U-3010 UV−vis spectrophotometer. Fluorescence spectra were measured by using a Hitachi F-4600 fluorescence spectrophotometer. The fluorescence lifetime data were obtained by using an Edinburgh Instrument FLS920 fluorescence spectrometer. HPLC data were acquired from Agilent 1260 Infinity liquid chromatograph (stationary phase, Eclipse plus C18, 4.8 × 100 mm; solvents, acetonitrile/methanol/water = 25:70:5). Fluorescence quantum yield was measured using a HAMAMATSU C11347-11 Absolutely Photoluminescence Quantum Yield Measurement System. Fluorescence images were obtained using an Olympus IX 71 with a DP72 color CCD. Preparation of RNS and ROS. Various RNS and ROS including ClO−, ·OH, H2O2, 1O2, NO2−, NO3−, ONOO−, NO, t-BuOOH, and O2− were prepared as follows: Hypochlorite (ClO−) was prepared from sodium hypochlorite;38 the concentration of hypochlorite (ClO−) was determined by using an extinction coefficient of 350 M−1cm−1 (292 nm) at pH 12.0. The hydroxyl radical (·OH) was generated by Fenton reaction between H2O2 and FeII(EDTA) quantitatively, and FeII(EDTA) concentrations represent ·OH concentrations.39 The concentration of the commercially available stock H2O2 solution was estimated by optical absorbance at 240 nm.40 Singlet oxygen (1O2) was generated by the addition of NaClO to H2O2 according to the literature report.41 The source of NO2− and NO3− was from NaNO2 and NaNO3.36 Nitric oxide (NO) was generated from diethylamine NONOate.42 Peroxynitrite (ONOO −) was prepared according to the reported method;43 the concentration of peroxynitrite was estimated by using an extinction coefficient of 1670 M−1cm−1 (302 nm). Superoxide (O2−) was prepared from KO2.44 TBuOOH and CuOOH was obtained from Aladdin and was diluted to the required concentration. Fluorometric Analysis. Fluorescence spectra were recorded with the excitation at 430 nm. The assay was prepared by dissolving Py-Cy in DMSO and then diluting it with PBS buffer under stirring for fluorescence or absorbance spectra measurement (the content of DMSO in the final test solutions is 5% v/v). The fluorescence change of Py-Cy upon the addition of varied amounts of ClO− in 100 mM

Some methods have been employed to red shift the excitation bands, emission bands, or both of fluorophores. For example, extending conjugation length or introducing donor−π−acceptor (D−π−A) electronic structure into the chromophore molecules,31 are two good ways. In this study, inspired by Ramiah’s work,30 we utilized Knoevenagel reaction to realize the extension of conjugation length and the introduction of D−π−A electronic structure, and then incorporated BF4− into the molecule to avoid the original quenching effect of I−, thereby successfully producing a Py-Cybased sensor with much higher quantum yield, which could potentiate its utilization in biological field, as shown in Figure 1.

Figure 1. Comparison of structure and fluorescence of pyrene (Py) and Py-Cy.

In addition, we introduced a positive charge in the molecule; this not only greatly improves the fluorophore’s water solubility which is conducive to applications in the aqueous biological milieu, but also ensures electrostatic interactions with negative analytes. Toward this end, we chose hypochlorite (ClO−) as a model analyte to explore the potential utilization of Py-Cy in ratiometric fluorescent sensing and bioimaging, because, on one hand, hypochlorite can break the double bond and disrupt the conjugated structure of Py-Cy; on the other hand, it is of great importance to establish a ratiometric sensor for detecting hypochlorite in real samples and cell imaging, because hypochlorite, as one of the important reactive oxygen species (ROS), is involved in numerous diseases,32−36 such as neurodegenerative diseases including Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, amyotrophic lateral sclerosis, atherosclerosis, inflammatory diseases, cerebral ischemia, and cancer. Also, when the content of hypochlorite is excessive in real samples, such as running water or aquaculture water, it would cause serious damages to organisms, especially to aquatic organisms such as fishes.37

■

EXPERIMENTAL METHODS

Reagents and Materials. 1-Pyrenecarboxaldehyde, 1,2,3,3tetramethyl-3H-indolium iodide, piperidine, Sodium tetrafluoroborate (NaBF4), 4-aminobenzoic hydrazide (4-ABAH), methanol (MeOH for HPLC), dimethyl sulfoxide (DMSO for HPLC), and tetrahydrofuran (THF for HPLC) were purchased from Sigma-Aldrich and used as received. Tert-butylhydroperoxide (t-BuOOH, 70%), cumene hydroperoxide (CuOOH), KO2, NaNO2, NaNO3, Na2SO4, hydroxylammonium chloride, sodium hypochlorite (NaClO, 14.5% available chlorine), and hydrogen peroxide solution (30%) were purchased from Aladdin. FeII(EDTA) was obtained from Jinan Great Chemical 1512


Research Article


Figure 2. (A) Absorption and (B) fluorescence emission spectra of Py-Cy (20 μM) in DMSO/water mixture with different water fractions under 508 nm excitation. additional 2 h at 37 °C under 5% CO2 respectively. Then, the culture dishes were washed with PBS buffer; glass slides were taken out and finally imaged on an Olympus IX71 inverted fluorescence microscope equipped with a DP72 color CCD. To confirm that the intracellular fluorescence was associated only with ClO− generation, an MPO inhibitor, 4-aminobenzoic acid hydrazide (4-ABAH), and a hypochlorite scavenger, taurine, were used. The Raw 264.7 cells were pretreated with 100 μM 4-ABAH for 20 min, and then stimulated with 2 mg/mL PMA for 30 min. After that, the cells were incubated with 10 μM Py-Cy for 2 h. Also, after being stimulated with 2.0 mg/mL PMA, the cells were treated with 5 mg/mL taurine, and then incubated with 10 μM Py-Cy for 2 h. Prior to imaging, all cells were rinsed three times with PBS buffer.

PBS (pH = 7.4) containing 5% DMSO was recorded after 70 min of mixing. For time-course experiments, selectivity experiments, ClO− and other RNS/ROS were added into the probe solution [100 mM PBS (pH = 7.4) containing 5% DMSO in the final test solutions]. For quantum yield and fluorescence lifetime measurements, Py-Cy was diluted to 20 μM in pure DMSO for monomer and in PBS buffer (pH 7.4) containing 5% DMSO for excimer. Hypochlorite Detection in Real Samples. The aquaculture water was obtained from a local fishpond: the water was centrifuged for 20 min at 10000 rpm. Then the supernatant was collected as soon as possible and storied at 2−8 °C for use. For ClO− detection in aquaculture water, the fluorescence measurements were conducted for samples containing Py-Cy (final concentration: 10 μM) with or without ClO− addition in 100 mM PBS buffer (pH 7.4) containing 5% DMSO at 25 °C (for the samples with added ClO−, the measurements were conducted after 70 min of mixing). The fluorescence intensity ratios I470/I613 were calculated. The final concentration of the aquaculture water in the test solution is 10-fold diluted. With the same procedures, running water was 5-fold diluted for measurements. Cell Viability Assay. To examine the cytotoxicity of Py-Cy, we incubated RAW254.7 (murine macrophage cells) and Hela (human cervical cancer cell) in DMEM medium and added 10% fetal bovine serum (FBS). The cells were maintained at 37 °C with 5% CO2 and grown for 24 h. After removal of the medium with PBS buffer, the cells were treated with Py-Cy and incubated for additional 24 h. The evaluation result of cytotoxicity for Py-Cy against the cells was acquired by MTT assay in compliance with ISO 10993-5. In these experiments, for each independent experiment, the assays were performed in eight replicates. And the estimation of cell viability was expressed by the statistical mean and standard deviation. Cell Incubation and Imaging. For imaging exogenous ClO− in live cells, two cell lines, namely Hela (human cervical cancer cell) and Raw 264.7 murine macrophages, were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (Invitrogen). The two kinds of cells were passed on polylysine-coated cell culture glass slides inside the 30 mm glass culture dishes and allowed to adhere for 24 h, respectively. Subsequently, the cells were washed with RPMI1640, and incubated in RPMI1640 medium with ClO− (0, 10, and 50 μM, respectively) at 37 °C under 5% CO2 for 30 min, and then incubated with Py-Cy with a final concentration of 10 μM for another 2 h. Then, glass slides were taken out; the cells were washed with PBS buffer for three times and finally imaged on an Olympus IX71 inverted fluorescence microscope equipped with a DP72 color CCD. For imaging endogenously generated ClO− in Raw 264.7 macrophage cells (endogenous generation of ClO− was induced by the stimulants, phorbol myristate acetate (PMA)), the cells were passed on polylysine-coated cell culture glass slides inside the 30 mm glass culture dishes and allowed to adhere for 24 h. Afterward, the Raw 264.7 macrophage cells were pretreated with PMA (2.0 mg/mL) for 4 h and then incubated with Py-Cy (final concentration: 10 μM) for

■

RESULTS AND DISCUSSION First the photophysical properties of Py-Cy were investigated. The absorption spectrum of Py-Cy is shown in Figure 2A and Figure S5; and it is clear that, compared to pyrene, the maximum absorption wavelength of Py-Cy has red-shifted to 508 nm. In addition, the maximum absorption peak just decreased with increase of water content, and without significant wavelength shift, which accounts for excimer formation. In Figure 2B, we can see the monomer emission of Py-Cy (dissolved in DMSO) is in the range of 550−650 nm. While upon addition of water, the poor solvent for Py-Cy, PyCy excimers gradually form and the excimer emission at 750− 790 nm (in the near-infrared region) shows a gradual enhancement with the increasing water fraction. In addition, we also measured the fluorescence quantum yields of the monomer/excimer of Py-Cy, as shown in Table S1. The fluorescence quantum yield of the monomer reaches 16.9% in DMSO, while the excimer quantum yield of Py-Cy in PBS buffer (pH 7.4) containing 5% DMSO is 2.96%. Furthermore, fluorescence lifetimes were tested. In Figure S6, the lifetime of the monomer (measured at 610 nm) was 0.56 ns. And the excimer lifetime measured at 767 nm was 0.59 ns. To investigate the practicability of Py-Cy, we chose hypochlorite (ClO−) as a model analyte. Because of the existence of the positive charge in Py-Cy, the probe (Py-Cy) has good water solubility. In the PBS buffer (pH 7.4) containing 5% DMSO, the probe molecules are in the molecularly dissolved state as the monomers, thus the probe solution shows the monomer emission of Py-Cy in the range of 550−650 nm. While in the presence of hypochlorite, since hypochlorite can break the double bond in the molecular structure, damage the conjugated structure of Py-Cy, and 1513


Research Article


Figure 3. Schematic illustration for ratiometric fluorescent response of Py-Cy to hypochlorite and its response mechanism. Photographs were taken under hand-held UV light (365 nm) or ambient light.

Figure 4. Fluorescence spectra (A) in the presence of different hypochlorite concentrations at 70 min upon the addition of hypochlorite to 10 μM Py-Cy in PBS buffer (pH 7.4) containing 5% DMSO and fluorescence intensity ratio as a function of hypochlorite concentration (B). Timedependent emission spectra (C) and fluorescence intensity as a function of time (D) in the presence of hypochlorite 120 μM. Excitation wavelength: 430 nm.

consequently generate the water-insoluble 1-pyrenecarboxaldehyde (Py-CHO, which has the same monomer/excimer photophysical properties as pyrene) that readily forms excimer

in the aqueous media and emit the excimer emission of pyrene in the range of 450−500 nm, and thus, this process will lead to obvious fluorescence changes at two different wavelengths, by 1514


Research Article

ACS Applied Materials & Interfaces this way ratiometric fluorescent detection for hypochlorite can be realized. The schematic illustration for the ratiometric fluorescent detection of hypochlorite is shown in Figure. 3. And 430 nm was chosen as the excitation wavelength of Py-Cy in order to simultaneously monitor the fluorescence changes of the probe and the product resulting from the reaction between the probe and hypochlorite, namely pyrenecarboxaldehyde (PyCHO). The fluorescence spectra were periodically recorded during incubation of Py-Cy with hypochlorite at 25 °C, and the results are shown in Figure. 4. In the absence of hypochlorite, the probe shows red emission centering around 613 nm. Upon addition of hypochlorite and with increased incubation time, the blue emission intensity of Py-CHO centering around 470 nm gradually increases over the incubating time from 0 to 70 min. Meanwhile, the emission of Py-Cy at 613 nm shows a gradual decrease. After 70 min, the fluorescence intensity ratio I470/I613 levels off, as shown in Figure 4D. Also, we measured the fluorescent spectra of the Py-Cy in the absence or presence of hypochlorite at different concentrations, as shown in Figure 4A. It is clear that, as the hypochlorite level is increased, the emission of Py-Cy decreases, while that of the Py-CHO enhances; therefore, this sensing system can detect the hypochlorite level in a ratiometric way. And the detection limit is determined as 0.35 μM, as shown in Figure S7. The absorption band’s change of Py-Cy in the presence of hypochlorite was also measured as shown in Figure S8. To study the selectivity of Py-Cy for ClO−, various potential interfering species (ONOO−, ·OH, 1O2, H2O2, KO2, NaNO2, NaNO3, NO, t-BuOOH, CuOOH) were examined in parallel under the same conditions. The results are shown in Figure S9. It is clear that the fluorescence intensity ratio I470/I613 induced by ClO− is much higher than that induced by other ROS or RNS. The above results indicate that the Py-Cy is a rather sensitive and selective probe for ClO− detection, and it can discriminate ClO− from other ROS or RNS. The results definitely support that Py-Cy is a quite good probe for specific detection of ClO−. Moreover, we also tested the probe’s detection of ClO− in the presence of various ROS or RNS, and the results are shown in Figure. S6B. It is evident that the coexistence of these species has a negligible interfering effect on ClO− sensing. Proposed Response Mechanism. On the basis of the fluorescent response of Py-Cy toward hypochlorite, the response mechanism is proposed as follows: due to the strong oxidizing ability of hypochlorite, ClO− first oxidize the double bond between Py moiety and Cy moiety, which results in the formation of an epoxy group within 5 min. The epoxy derivative of Py-Cy, 1,3,3-trimethyl-2-(3-(pyren-1-yl)oxiran-2yl)-3H-indol-1-ium, named Py-O-Cy, exhibits weak fluorescence and poor chemical stability. Afterward, Py-O-Cy gradually hydrolyzes into Py-CHO and Cy-CHO under such mild test environment within 70 min. Py-CHO has similar photophysical properties as pyrene, and can form excimers immediately after Py-O-Cy hydrolyzing into Py-CHO. Therefore, the changes of fluorescent intensity at 613 and 470 nm can reflect the response of Py-Cy toward hypochlorite, as shown in Figure 3. To examine and confirm the proposed response mechanism, 1 H NMR and MS measurements were conducted. Py-Cy was mixed with hypochlorite in phosphate buffer for 70 min. Then, the reaction products were purified by column chromatography and dried out before 1H NMR measurement. The 1H NMR results are shown in Figures S10 and S11. It is obvious that, the

H protons at 7.55 ppm of the double bond in Py-Cy disappear. Conversely, a new H proton peak appears at 10.5 ppm, which belongs to the aldehyde group of the product Py-CHO. In silica column Cy-CHO is extremely unstable and easy to decompose, so we could neither obtain the purified compound nor perform NMR measurement for it. However, we could use MS and HPLC measurements to characterize it as described in the following, because nonpurified samples can also be used for these measurements. Hence, we further conducted MS measurements to prove the mechanism. Hypochlorite was added to Py-Cy in phosphate buffer and reacted for 5 or 70 min. The reaction mixture was directly used for MS measurement without any purification and the results are shown in Figures S12−S14. As can been seen from Figure S12, a single peak at 402.6 m/z corresponds to the intermediate product Py-O-Cy. In Figures S13 and S14, the molecule weight peaks at 187.15 m/z and 230 m/z correspond to Cy-CHO and Py-CHO respectively. On the basis of the 1H NMR and MS measurements, it is clear that, our proposed response mechanism is reasonable. To further prove the detection mechanism, we also carried out high performance liquid chromatography (HPLC) measurements for Py-Cy in the absence and presence of hypochlorite. Py-Cy, the product resulting from the reaction between Py-Cy with hypochlorite for 5 and 70 min, and pure Py-CHO (control) purchased from Sigma-Aldrich, were measured, respectively. As can be seen from Figure 5A,B, for the solution containing Py-Cy only, a single peak at 7.5 min can be observed from both 508 nm channel and 380 nm channel. When 120 μM of ClO− was added to the Py-Cy solution (10 μM), and the mixture was kept stirring for 5 min, a single peak at 1.9 min appears, corresponding to the intermediate product Py-O-Cy. When we lengthened reaction to 70 min, a peak at 1.5 min for Py-CHO and a peak at 2.1 min for Cy-CHO can be clearly seen in Figure 5F. Therefore, HPLC measurements not only verify the proposed mechanism, but also provide another method for detection of hypochlorite. Also, we conducted a fluorescence spectral control trial, the results were shown in Figure S15. Compared with the control spectrum, which was obtained by mixing pure Py-CHO with hypochlorite in PBS containing 5% DMSO for 70 min, the final fluorescent spectrum of Py-Cy with hypochlorite was almost consistent, which proved our proposed mechanism. Hypochlorite Detection in Real Samples. To evaluate practical applications of Py-Cy to detect ClO− in real samples, the probe was applied to measuring hypochlorite in running water and aquaculture water. The two real samples were diluted 5-fold and 10-fold, respectively, for the measurements. And the determined hypochlorite levels are listed in Table S2 and Table S3. The endogenous (originally existing) ClO− in diluted running water was 8.52 μM, determined by Py-Cy herein using the calibration curve (Figure S7) as standard, and the endogenous ClO− levels for the undiluted running water sample is calculated as 42.60 μM. For aquaculture water, endogenous ClO− concentration was 6.38 and 63.80 μM, respectively, for the diluted and undiluted sample. Furthermore, the result was also compared with the iodometric method.45 The endogenous hypochlorite concentration determined by the iodometric method was 42.35 μM for running water and 65.20 μM for aquaculture water, which are close to those determined by the probe, Py-Cy. For the iodometric assay, the detection process requires complex multiple procedures; while for the fluorescent probe herein, the detection process is quite 1515


Research Article


Next, we evaluated the intracellular hypochlorite imaging capability of Py-Cy in live cells. Py-Cy was used to trace exogenous hypochlorite levels in the two cell linesHeLa (human cervical cancer cell) and Raw 264.7 murine macrophage cells. To monitor the reaction process of Py-Cy with intracellular ClO−, a time-course assay was first performed. Initially, red fluorescence can be detected in the cells without ClO− loading as shown in Figure S17a. Upon ClO− loading and after incubation for 1 h, blue fluorescence signal can be observed inside the cells, indicating the internalization of Py-Cy and its reaction with the preloaded ClO− as shown in Figure S17b. In Figure S17c, as the incubation time is prolonged to 2h, a greatly intensified blue fluorescence than before can be observed. These results suggest that the reaction proceeds more thoroughly with increased reaction time. Figure 6 and Figure

Figure 6. Fluorescence images of Hela cells incubated with Py-Cy before (a) and after (b, c, e, f) being treated with ClO−. The cells were incubated with Py-Cy (10 μM) (a, d) in the absence and in the presence of (b, e) 10 μM and (c, f) 50 μM ClO− for 2h. Figure 5. HPLC data of Py-Cy, addition of ClO− and upon reaction for 5 or 70 min, and pure Py-CHO. (A and B) HPLC of Py-Cy in the absence of ClO−. (C and D) HPLC of Py-Cy (10 μM) upon addition of ClO− (120 μM) for 5 min. (E and F) HPLC of Py-Cy (10 μM) upon addition of ClO− (120 μM) for 70 min. (G and H) HPLC of PyCHO as a control. Graphs A, C, E, and G use data from the 508 nm channel; graphs B, D, F, and H use data from the 380 nm channel. Solvent ratio was methanol/acetonitrile/water = 70/25/5.

S18 display the fluorescent images respectively for the two cell lines incubated with Py-Cy before and after being treated with two different hypochlorite levels. The cells incubated with PyCy for 2 h at 37 °C without ClO− treatment display intracellular red fluorescence (Figures 6a, S18a). In contrast, when the live cells were pretreated with 10 or 50 μM of ClO− for 30 min at 37 °C and then loaded with Py-Cy, the intracellular fluorescence turns to blue, as shown in Figure 6b,c and Figure S18b,c. These results indicate that Py-Cy has reacted with hypochlorite to generate Py-CHO in the presence of ClO− inside live cells. On the other hand, the cells treated with 50 μM of ClO− show stronger blue fluorescence than the one treated with 10 μM of ClO−, proving that Py-Cy can track the ClO− level changes inside live cells. Therefore, the probe is demonstrated to be cell membrane permeable and can monitor intracellular ClO− with ratiometric fluorescence signals as designed. Encouraged by the above promising results, we next proceeded to assess the feasibility of Py-Cy to image endogenously generated ClO− inside live cells. It is known that phorbol myristate acetate (PMA) can induce a respiratory burst by activating NADPH oxidase to generate O2− that subsequently converts to H2O2, and H2O2 is then converted into hypochlorite by the MPO enzymes released by the macrophage cell.46 In this experiment, we employed PMA to induce endogenous hypochlorite in RAW 264.7 cells. As shown in Figure 7a, the RAW 264.7 cells incubated only with Py-Cy exhibit red fluorescence. However, when incubated with 2 mg/mL PMA and Py-Cy, the cells display bright blue

convenient and could be employed as a one-step straightforward assay. In addition, the recovery of added known amounts of ClO− in the real samples is in general more than 95% by the probe herein, which suggests the accuracy and reliability of the probe for hypochlorite determination. Furthermore, the precision of the assay system was also investigated (Tables S2 and S3), which was determined using the relative standard deviation. It is obvious that the fluorescent probe herein have a quite good precision. Fluorescence Imaging of Exogenous and Endogenous Hypochlorite in Live Cells. For intracellular imaging applications, cytotoxicity of Py-Cy is an important consideration. The cytotoxicity was evaluated using Hela and RAW 254.7 cell line by MTT assay in compliance with ISO 10993-5. The results are shown in Figure S16, and Py-Cy shows little cytotoxicity for cells. Notably, even at the high concentration of 10 μM, the viability of two cell line is still more than 90%. This result indicates that Py-Cy has good biocompatibility and is suitable for fluorescence detection of ClO− in live cells. 1516


Research Article


emit red or near-infrared light and devising technically simple ratiometric fluorescent sensors.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b11023. Synthesis route for Py-Cy, 1H NMR and MS spectra for Py-Cy, fluorescence quantum yields and lifetimes of PyCy, absorption spectra, determination of detection limit, detection in real samples and fluorescence images. (PDF)

Figure 7. Fluorescence images of PMA-induced ClO− production in RAW 264.7 cells. (Bottom) Bright field images and (top) fluorescence images. (a, e) The cells incubated with 10 μM Py-Cy for 2 h without PMA treatment. (b, f) The cells treated with stimulant PMA (2.0 mg/ mL) for 30 min and then incubated with 10 μM Py-Cy for another 2h. (c, g) The cells pretreated with 100 μM 4-ABAH for 20 min, followed by stimulation with 2.0 mg/mL PMA for 30 min and then incubated with 10 μM Py-Cy for 2 h. (d, h) The cells were first stimulated with 2.0 mg/mL PMA, treated with 5 mg/mL taurine, and then incubated with 10 μM Py-Cy for 2 h.

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail:[email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We gratefully acknowledge the financial support by the National Key Basic Research Program of China (Project no. 2013CB834702), NSFC (21574044, 21474031 and 21174040), the Science and Technology Planning Project of Guangdong Province (Project No. 2014A010105009) and the Fundamental Research Funds for the Central Universities (2015ZY013).

fluorescence as shown in Figure 7b. To further prove Py-Cy can image the endogenously produced ClO−, we carried out an experiment to investigate the effect of the MPO inhibitor, 4aminobenzoic acid hydrazide (4-ABAH)47 and the hypochlorite scavenger, taurine.48 Living RAW 264.7 cells were pretreated with 100 μM 4-ABAH for 20 min, and then stimulated with 2 mg/mL PMA for 30 min. After that, the cells were incubated with 10 μM Py-Cy for 2 h. Only red fluorescence can be observed inside the cells as shown in Figure 7c, which suggests that MPO plays a vital role in the endogenous hypochlorite generation, and in the presence of the MPO inhibitor, ClO− cannot be produced by the cells. On the other hand, after being stimulated with 2.0 mg/mL PMA, the cells were treated with 5 mg/mL taurine, and then incubated with 10 μM Py-Cy for 2 h, almost no fluorescence change could be observed in Figure 7d. These results suggest that Py-Cy is capable of imaging endogenous ClO− in living RAW264.7 cells.

■

REFERENCES

(1) Sun, X. L.; Zhu, B.; Ji, D. K.; Chen, Q. B.; He, X. P.; Chen, G. R.; James, T. D. Selective Fluorescence Detection of Monosaccharides Using a Material Composite Formed between Graphene Oxide and Boronate-Based Receptors. ACS Appl. Mater. Interfaces 2014, 6, 10078−10082. (2) Zhao, C. C.; Zhang, X. L.; Li, K. B.; Zhu, S. J.; Guo, Z. Q.; Zhang, L. L.; Wang, F. Y.; Fei, Q.; Luo, S. H.; Shi, P.; Tian, H.; Zhu, W. H. Fö rster Resonance Energy Transfer Switchable Self-Assembled Micellar Nanoprobe: Ratiometric Fluorescent Trapping of Endogenous H2S Generation via Fluvastatin-Stimulated Upregulation. J. Am. Chem. Soc. 2015, 137, 8490−8498. (3) Li, Z.; He, X. Y.; Wang, Z.; Yang, R. H.; Shi, W.; Ma, H. M. In Vivo Imaging and Detection of Nitroreductase in Zebrafish by a New Near-Iinfrared Fluorescence Off−On Probe. Biosens. Bioelectron. 2015, 63, 112−116. (4) Ema, T.; Okuda, K.; Watanabe, S.; Yamasaki, T.; Minami, T.; Esipenko, N. A.; Anzenbacher, P. Selective Anion Sensing by Chiral Macrocyclic Receptors with Multiple Hydrogen-Bonding Sites. Org. Lett. 2014, 16, 1302−1305. (5) Shi, P.; Liu, Z.; Dong, K.; Ju, E. G.; Ren, J. S.; Du, Y. D.; Li, Z. Q.; Qu, X. G. A Smart “Sense-Act-Treat” System: Combining a Ratiometric pH Sensor with a Near Infrared Therapeutic Gold Nanocage. Adv. Mater. 2014, 26, 6635−6641. (6) Dickinson, B. C.; Lin, V. S.; Chang, C. J. Preparation and Use of MitoPY1 for Imaging Hydrogen Peroxide in Mitochondria of Live Cells. Nat. Protoc. 2013, 8, 1249−1259. (7) Qiu, L. P.; Zhang, T.; Jiang, J. H.; Wu, C. C.; Zhu, G. Z.; You, M. X.; Chen, X. G.; Zhang, L. Q.; Cui, C.; Yu, R. Q.; Tan, W. H. Cell Membrane-Anchored Biosensors for Real-Time Monitoring of the Cellular Microenvironment. J. Am. Chem. Soc. 2014, 136, 13090− 13093. (8) Gui, S. L.; Huang, Y. Y.; Hu, F.; Jin, Y. L.; Zhang, G. X.; Yan, L. S.; Zhang, D. Q.; Zhao, R. Fluorescence Turn-On Chemosensor for Highly Selective and Sensitive Detection and Bioimaging of Al3+ in Living Cells Based on Ion-Induced Aggregation. Anal. Chem. 2015, 87, 1470−1474.

■

CONCLUSION In summary, we have successfully developed a novel pyrene derivative fluorophore, Py-Cy, through extending conjugation length and incorporating D−π−A structure. The fluorophore exhibits very good photophysical property with its maximum absorption wavelength centering around 508 nm, which is a great improvement compared with most pyrene derivatives with ultraviolet absorption. Furthermore, Py-Cy shows an excellent monomer/excimer conversion property, compared with pyrene, Py-Cy’s monomer emission has red-shifted from 380 to 410 nm to 550−650 nm, while its excimer emission has red-shifted from 450 to 500 nm to 750−790 nm. The visible absorption and red/near-infrared emission would greatly potentiate its biorelated applications such as bioimaging and sensing. Moreover, Py-Cy has been successfully applied to detecting hypochlorite in real samples and imaging in live cell. The above results indicate that, Py-Cy features simplicity in synthesis and selective detection of hypochlorite in real samples and imaging endogenous and exogenous hypochlorite in live cells. Moreover, the probe can realize hypochlorite detection in four modes, including fluorescent method, 1H NMR method, MS method and HPLC method. The strategy herein may provide useful insights for designing other fluorophores which 1517


Research Article


pyrene excimer to DNA intercalator SYBR green I. Biosens. Bioelectron. 2015, 65, 103−107. (28) Xu, C; Zhou, R. Y.; Zhang, R. C.; Yang, L. Y.; Wang, G. J. LabelFree DNA Sequence Detection through FRET from a Fluorescent Polymer with Pyrene Excimer to SG. ACS Macro Lett. 2014, 3, 845− 848. (29) Han, G.; Kim, D.; Park, Y.; Bouffard, J.; Kim, Y. M. Excimers Beyond Pyrene: A Far-Red Optical Proximity Reporter and its Application to the Label-Free Detection of DNA. Angew. Chem., Int. Ed. 2015, 54, 3912−3916. (30) Shankar, B. H.; Jayaram, D. T.; Ramaiah, D. A Reversible Dual Mode Chemodosimeter for the Detection of Cyanide Ions in Natural Sources. Chem. - Asian J. 2014, 9, 1636−1642. (31) Zhang, X. Y.; Mao, J. Y.; Wang, D.; Li, X.; Yang, J. B.; Shen, Z. J.; Wu, W. J.; Li, J.; Ågren, H.; Hua, J. L. Comparative Study on Pyrido[3,4-b]pyrazine-Based Sensitizers by Tuning Bulky Donors for Dye-Sensitized Solar Cells. ACS Appl. Mater. Interfaces 2015, 7, 2760− 2771. (32) Zhu, H.; Fan, J. L.; Wang, J. Y.; Mu, H. Y.; Peng, X. J. An “Enhanced PET”-Based Fluorescent Probe with Ultrasensitivity for Imaging Basal and Elesclomol-Induced HClO in Cancer Cells. J. Am. Chem. Soc. 2014, 136, 12820−12823. (33) Long, L. L.; Wu, Y. J.; Wang, L.; Gong, A. H.; Hu, F. L.; Zhang, C. A Fluorescent Probe for Hypochlorite Based on the Modulation of the Unique Rotation of the N−N Single Bond in Acetohydrazide. Chem. Commun. 2015, 51, 10435−10438. (34) Reja, S. I.; Bhalla, V.; Sharma, A.; Kaur, G.; Kumar, M. A Highly Selective Fluorescent Probe for Hypochlorite and its Endogenous Imaging in Living Cells. Chem. Commun. 2014, 50, 11911−11914. (35) Hou, J. T.; Li, K.; Yang, J.; Yu, K. K.; Liao, Y. X.; Ran, Y. Z.; Liu, Y. H.; Zhou, X. D.; Yu, X. Q. A Ratiometric Fluorescent Probe for In Situ Quantification of Basal Mitochondrial Hypochlorite in Cancer Cells. Chem. Commun. 2015, 51, 6781−6784. (36) Xiao, H. D.; Xin, K.; Dou, H. F.; Yin, G.; Quan, Y. W.; Wang, R. Y. A Fast-Responsive Mitochondria-Targeted Fluorescent Probe Detecting Endogenous Hypochlorite in Living RAW 264.7 Cells and Nude Mouse. Chem. Commun. 2015, 51, 1442−1445. (37) López-Galindo, C.; Vargas-Chacoff, L.; Nebot, E.; Casanueva, J. F.; Rubio, D.; Solé, M.; Mancera, J. M. Biomarker Responses in Solea Senegalensisexposed to Sodium Hypochlorite Used as Antifouling. Chemosphere 2010, 78, 885−893. (38) Morris, J. C. The Acid Ionization Constant of HOCl from 5 to 35°. J. Phys. Chem. 1966, 70, 3798−3805. (39) Halliwell, B.; Gutteridge, J. M. C. Oxygen Free Radicals and Iron in Relation to Biology and Medicine: Some Problems and Concepts. Arch. Biochem. Biophys. 1986, 246, 501−514. (40) Long, L. L.; Zhang, D. D.; Li, X. F.; Zhang, J. F.; Zhang, C.; Zhou, L. P. A Fluorescence Ratiometric Sensor for Hypochlorite Based on a Novel Dual-Fluorophore Response Approach. Anal. Chim. Acta 2013, 775, 100−105. (41) Li, X. H.; Zhang, G. X.; Ma, H. M.; Zhang, D. Q.; Li, J.; Zhu, D. B. 4,5-Dimethylthio-4′-[2-(9-anthryloxy)ethylthio]tetrathiafulvalene, a Highly Selective and Sensitive Chemiluminescence Probe for Singlet Oxygen. J. Am. Chem. Soc. 2004, 126, 11543−11548. (42) Kim, T. I.; Park, S.; Choi, Y.; Kim, Y. A BODIPY-Based Probe for the Selective Detection of Hypochlorous Acid in Living Cells. Chem. - Asian J. 2011, 6, 1358−1361. (43) Zou, M. H.; Ullrich, V. Peroxynitrite Formed by Simultaneous Generation of Nitric Oxide and Superoxide Selectively Inhibits Bovine Aortic Prostacyclin Synthase. FEBS Lett. 1996, 382, 101−104. (44) Albers, A. E.; Okreglak, V. S.; Chang, C. J. A FRET-Based Approach to Ratiometric Fluorescence Detection of Hydrogen Peroxide. J. Am. Chem. Soc. 2006, 128, 9640−9641. (45) Vogel, A. I. A Text Book of Quantitative Chemical Analysis; Longman: London, 1989. (46) Wang, B. S.; Li, P.; Yu, F. B.; Song, P.; Sun, X. F.; Yang, S. Q.; Lou, Z. G.; Han, Z. G. A Reversible Fluorescence Probe Based on Se− BODIPY for the Redox Cycle between HClO Oxidative Stress and H2S Repair in Living Cells. Chem. Commun. 2013, 49, 1014−1016.

(9) Wu, Y. L.; Huang, S. L.; Zeng, F.; Wang, J.; Yu, C. M.; Huang, J.; Xie, H. T.; Wu, S. Z. A Ratiometric Fluorescent System for Carboxylesterase Detection with AIE Dots as FRET Donors. Chem. Commun. 2015, 51, 12791−12794. (10) Hong, Y. N.; Lam, J. W. Y.; Tang, B. Z. Aggregation-Induced Emission. Chem. Soc. Rev. 2011, 40, 5361−5388. (11) Sui, B. L.; Tang, S. M.; Liu, T. H.; Kim, B. S.; Belfield, K. D. Novel BODIPY-Based Fluorescence Turn-on Sensor for Fe3+ and Its Bioimaging Application in Living Cells. ACS Appl. Mater. Interfaces 2014, 6, 18408−18412. (12) Yuan, L.; Lin, W. Y.; Zheng, K. B.; Zhu, S. S. FRET-Based Small-Molecule Fluorescent Probes: Rational Design and Bioimaging Applications. Acc. Chem. Res. 2013, 46, 1462−1473. (13) Wang, X. D.; Stolwijk, J. A.; Lang, T.; Sperber, M.; Meier, R. J.; Wegener, J.; Wolfbeis, O. S. Ultra-Small, Highly Stable, and Sensitive Dual Nanosensors for Imaging Intracellular Oxygen and pH in Cytosol. J. Am. Chem. Soc. 2012, 134, 17011−17014. (14) Xu, X. Q.; Miao, K. S.; Chen, Y.; Fan, L. J. Preparation of Fluorescent Conjugated Polymer Fibrous Membranes for Rapid Recognition of Aromatic Solvents. ACS Appl. Mater. Interfaces 2015, 7, 7759−7766. (15) Förster, T. Excimers. Angew. Chem., Int. Ed. Engl. 1969, 8, 333− 343. (16) Winnik, F. M. Photophysics of Preassociated Pyrenes in Aqueous Polymer Solutions and in Other Organized Media. Chem. Rev. 1993, 93, 587−614. (17) Moragues, M. E.; Toscani, A.; Sancenón, F.; Martinez-Máñez, R.; White, A. J. P.; Wilton-Ely, J. D. E. T. A Chromo-Fluorogenic Synthetic “Canary” for CO Detection Based on a Pyrenylvinyl Ruthenium(II) Complex. J. Am. Chem. Soc. 2014, 136, 11930−11933. (18) Zheng, F. Y.; Guo, S. H.; Zeng, F.; Li, J.; Wu, S. Z. Ratiometric Fluorescent Probe for Alkaline Phosphatase Based on BetaineModified Polyethylenimine via Excimer/Monomer Conversion. Anal. Chem. 2014, 86, 9873−9879. (19) Hu, W. T.; Ding, L. P.; Cao, J. H.; Liu, L. L.; Wei, Y. T.; Fang, Y. Protein Binding-Induced Surfactant Aggregation Variation: A New Strategy of Developing Fluorescent Aqueous Sensor for Proteins. ACS Appl. Mater. Interfaces 2015, 7, 4728−4736. (20) Ghosh, A.; Sengupta, A.; Chattopadhyay, A.; Das, D. Lysine Triggered Ratiometric Conversion of Dynamic to Static Excimer of a Pyrene Derivative: Aggregation-Induced Emission, Nanomolar Detection and Human Breast Cancer Cell (MCF7) Imaging. Chem. Commun. 2015, 51, 11455−11458. (21) Senthamizhan, A.; Celebioglu, A.; Bayir, S.; Gorur, M.; Doganci, E.; Yilmaz, F.; Uyar, T. Highly Fluorescent Pyrene-Functional Polystyrene Copolymer Nanofibers for Enhanced Sensing Performance of TNT. ACS Appl. Mater. Interfaces 2015, 7, 21038−21046. (22) Thirupathi, P.; Park, J. Y.; Neupane, L. N.; Kishore, M. Y. L. N.; Lee, K. H. Pyrene Excimer-Based Peptidyl Chemosensors for the Sensitive Detection of Low Levels of Heparin in 100% Aqueous Solutions and Serum Samples. ACS Appl. Mater. Interfaces 2015, 7, 14243−14253. (23) Lee, H. Y.; Choi, Y. P.; Kim, S.; Yoon, T.; Guo, Z. Q.; Lee, S.; Swamy, K. M. K.; Kim, G.; Lee, J. Y.; Shin, I.; Yoon, J. Selective Homocysteine Turn-On Fluorescent Probes and Their Bioimaging Applications. Chem. Commun. 2014, 50, 6967−6969. (24) Karuppannan, K.; Chambron, J. C. Supramolecular Chemical Sensors Based on Pyrene Monomer−Excimer Dual Luminescence. Chem. - Asian J. 2011, 6, 964−984. (25) Fermi, A.; Ceroni, P.; Roy, M.; Gingras, M.; Bergamini, G. Synthesis, Characterization, and Metal Ion Coordination of a Multichromophoric Highly Luminescent Polysulfurated Pyrene. Chem. - Eur. J. 2014, 20, 10661−10668. (26) Li, W.; Wang, L.; Zhang, J. P.; Wang, H. Bis-Pyrene-based Supramolecular Aggregates with Reversibly Mechanochromic and Vapochromic Responsiveness. J. Mater. Chem. C 2014, 2, 1887−1892. (27) Zhou, R. Y.; X, C.; Dong, J.; Wang, G. J. Labeling-free fluorescent detection of DNA hybridization through FRET from 1518


Research Article

ACS Applied Materials & Interfaces (47) Kumar, S.; Patel, S.; Jyoti, A.; Keshari, R. S.; Verma, A.; Barthwal, M. K.; Dikshit, M. Nitric Oxide-Mediated Augmentation of Neutrophil Reactive Oxygen and Nitrogen Species Formation: Critical Use of Probes. Cytometry, Part A 2010, 77, 1038−1048. (48) Redmond, H. P.; Stapleton, P. P.; Neary, P.; Bouchier-Hayes, D. Immunonutrition: The Role of Taurine. Nutrition 1998, 14, 599−604.

1519


Pyrene Derivative Emitting Red or near-Infrared Light with Monomer

Recommend Documents