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A coumarin-based two-photon probe for hydrogen peroxide
Biosensors and Bioelectronics ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Short communication
A coumarin-based two-photon probe for hydrogen peroxide Kai-Ming Zhang a, Wei Dou a, Peng-Xuan Li a, Rong Shen b, Jia-Xi Ru a, Wei Liu a, Yu-Mei Cui a, Chun-Yang Chen a, Wei-Sheng Liu a,n, De-Cheng Bai b,nn a Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China b Key Laboratory of Preclinical Study for New Drugs of Gansu Province and Operative Surgery Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 26 June 2014 Received in revised form 2 September 2014 Accepted 22 September 2014
A new fluorescence probe was developed for hydrogen peroxide (H2O2) detection based on donor-excited photo induced electron transfer (D-PET) mechanism, together with the benzil as a quenching and recognizing moiety. The benzil could convert to benzoic anhydride via a Baeyer–Villiger type reaction in the presence of H2O2, followed by hydrolysis of benzoicanhydride to give benzoic acid, and the fluorophore released. The probe was synthesized by a 6-step procedure starting from 4-(diethylamino) salicylaldehyde. A density functional theory (DFT) calculation was performed to demonstrate that the benzil was a fluorescence quencher. The probe was evaluated in both one-photon and two-photon mode, and it exhibited high selectivity toward H2O2 over other reactive oxygen species and high sensitivity with a detection limit of 0.09 μM. Furthermore, the probe was successfully applied to cell imaging of intracellular H2O2 levels with one-photon microscopy and two-photon microscopy. The superior properties of the probe made it of great potential use in more chemical and biological researches. & 2014 Elsevier B.V. All rights reserved.
Keywords: Hydrogen peroxide Probe Coumarin Two-photon Cell imaging
1. Introduction Reactive oxygen species (ROS) including hydrogen peroxide (H2O2), hydroxyl (OH), singlet oxygen (1O2), superoxide (O2 ), peroxyl radical (ROO) and hypochlorous acid (HOCl) are endogenously generated from oxygen metabolism in biological systems (Dickinson and Chang, 2011; D'Autreaux and Toledano, 2007; Stadtman, 2006). H2O2, a major member of ROS, plays an important role in normal cellular growth and proliferation (Rhee, 2006; Kamata et al., 2005; Finkel and Holbrook, 2000; Veal et al., 2007; Harman, 1981; Stone and Yang, 2006). However, over production of H2O2 can cause many diseases, such as inflammatory disease, cardiovascular disease, Alzheimer's disease and cancer (Barnham et al., 2004; Mattson, 2004; Lin and Beal, 2006; Andersen, 2004; Ohshima et al., 2003; Shah and Channon, 2004). Therefore, it is necessary to develop sensitive and selective methods for the detection of H2O2 in living cells. As a useful analytical tool, fluorescent spectroscopy is of broad interest in bioanalysis. In recent years, many fluorescence probes for H2O2 have been developed (Li et al., 2013; Chen et al., 2011; Hyman and Franz, 2012; Kim et al., 2010; Lo and Chu, 2003; Chang n
Corresponding author. Fax: þ 86 931 8912582. Corresponding author. E-mail addresses: [email protected] (W.-S. Liu), [email protected] (D.-C. Bai).
nn
http://dx.doi.org/10.1016/j.bios.2014.09.073 0956-5663/& 2014 Elsevier B.V. All rights reserved.
et al., 2004; Miller et al., 2005; Dickinson et al., 2010; Albers et al., 2008; Du et al., 2008; Setsukinai et al., 2003; Maeda et al., 2004; Lippert et al., 2011; Karton-Lifshin et al., 2011; Zhu et al., 2013; Soh et al., 2005; Xu et al., 2005; Hu et al., 2014; Qian et al., 2012). For example, dichlorodihydrofluorescein derivatives are widely utilized as fluorescent probes for measuring H2O2 (Setsukinai et al., 2003). However, they have poor selectivity and undergo autoxidation upon exposure to excitation light. Another reported H2O2 probes based on arylsulfonyl (Maeda et al., 2004) and boronate ester (Lippert et al., 2011; Karton-Lifshin et al., 2011; Hyman and Franz, 2012) deprotection mechanism show high selectivity toward H2O2 over other ROS, but their response rates are not entirely satisfactory for biological research and clinical needs. Very recently, Negano et al. reported a turn-on H2O2 fluorescent probe NBNF based on a Baeyer–Villiger type reaction of the benzil moiety (Abo et al., 2011). As a quencher and specific response group to H2O2, the benzil was first applied to design H2O2 probe in his work. Moreover, this probe has higher sensitivity and enough selectivity. However, NBZF was evaluated using one-photon excitation (OPE) with relatively short excitation wavelength (490 nm), which limits its further application in tissue imaging. While two-photon excitation (TPE) has attracted much attention in bioanalysis due to its minimum interference from photobleaching and photodamage (Kim and Cho, 2009; Masanta et al., 2012). So it is still significative to extend a new fluorescent probe with TPM using benzil as a recognition moiety of H2O2. We selected
K.-M. Zhang et al. / Biosensors and Bioelectronics ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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density of 104 cells per well in culture media, then incubated with 10 μM probe 1 solution at 37 °C. Before imaging measurement, cells were washed 3 times with phosphate buffered saline (PBS).
Scheme 1. Mechanism of H2O2 sensing by probe 1.
coumarin as a two-photon fluorophore scaffold, because a coumarin-based fluorophore 6-hydroxycoumarin-4-ylme thanol has been reported to possess TPE properties (Furuta et al., 1999). Herein, we reported a TPE probe 1 for H2O2. The synthesized coumarin derivative 1 was almost non-fluorescent (quantum yield in H2O/CH3CN¼ 8: 2, Ф ¼0.0202), indicating that the benzil acted as a fluorescence quencher because of the donor-excited photo induced electron transfer (D-PET) process (Abo et al., 2011). However, compound 1 could convert to 2 in the presence of H2O2 (Scheme 1). Owing to the break of benzil's structure, the DPET process did not occur and the strong fluorescence of coumarin was emitted (quantum yield in H2O/CH3CN¼ 8: 2, Ф ¼ 0.0641).
2. Experimental section 2.1. Materials and instruments All reagents were purchased from commercial providers and used without further purification. All solvents were used after distillation or dehydration. 1H NMR and 13C NMR were recorded in CDCl3 at 25 °C on a Bruker DRX400 spectrometer, 400 MHz for 1H and 100 MHz for 13C, respectively. Mass spectra (ESI) were taken on a Bruker micrOTOF-Q II mass spectrometer. UV–visible spectra were measured using an Analytik Jena SPECORD 50 PLUS. Fluorescence spectra with both one-photon excitation (OPE) and twophoton excitation (TPE) were carried out using the Hitachi F-4500 fluorescence spectrometer and Edinburgh Instrument FLS920, respectively. Conventional fluorescence imaging were performed using a Leica DMI 4000B microscope with DFC420 C camera and a mercury lamp (short ARC) with a BP 450–490 excitation filter and a LP 515 emission filter. Two-photon fluorescence microscopy imaging were captured using Olympus FV1000 laser scanning confocal and multiphoton microscopes (set at wavelength 900 nm and PMT; Gain, 746 V) with 100 oil immersion objective lens. The signals were collected in a 12 bit unsigned 1024 1024 pixels at 8.0 us/ pixel sampling speed. 2.2. Absorption and fluorescence spectroscopy Probe 1 was dissolved in DMF for a stock solution (1 mM). H2O2 was from dilution of 30% solution in water. NaClO was from dilution of 10% solution in water. Tert-butyl hydroperoxide was from dilution of 70% solution in water. NO was produced from sodium nitroferricyanide (SNP). O2- was from KO2 in anhydrous DMSO. 1O2 was produced by mixing H2O2 and 100-exceed of NaClO. OH was produced by Fenton reaction, mixing H2O2 and 10-exceed (NH4)2Fe (SO4)2. Test solutions (10 μM) were prepared by displacing 20 μL of the stock solution into a 2-mL mixture of 0.1 M PBS and CH3CN (8:2, v/v) at pH 7.4.
3. Results and discussion 3.1. Calculation based on density functional theory (DFT) In order to understand the effect of the benzil structure on photophysical properties, the calculation based on density functional theory (DFT) for 1 was performed (Fig. 1). According to the comparison between the highest occupied molecular orbitals (HOMO) and the lowest unoccupied molecular orbitals (LUMO), it came to a conclusion that the electron density from the coumarin moiety moved to the benzil moiety upon the excitation. As a result, the fluorescence of coumarin was quenched. 3.2. Synthesis and characterization The synthesis of the probe 1 began with compound 3 in a straight forward manner by a 6-step procedure. The starting material 3 could convert to 4 according to a routine coumarin formation procedure. The compound 7 was synthesized by using the intermediate 4 as reaction material undergoing bromination, reaction of Sonogashira with trimethylsilyl acetylene (TMSA) and deprotection with K2CO3 in CH3OH. The compound 8 was afforded through the reaction of Sonogashira of 7 with 1-bromo-4-nitrobenzene. At last, the desired target probe 1 was obtained by oxidation reaction of 8 with DMSO catalyzed by PdCl2. The detail characterization data and experimental procedure were shown in the ESI. Scheme 2 3.3. UV–vis absorption and fluorescence spectra of 1 towards H2O2 We examined the optical properties of probe 1 in PBS buffer (0.1 M, pH 7.4, 20% CH3CN). UV–vis spectra of 1 (10 μM) exhibited an absorptions maximum at λabs ¼475 nm. After treatment with 10 equiv. of H2O2 at 37 °C, the absorption at 475 nm obviously declined, whereas a new absorption peak was present at 360 nm (Fig. 2a). Accordingly, the emission at 505 nm apparently appeared upon excitation at 380 nm, and the fluorescence intensity increased rapidly. Finally approximately 75-fold fluorescence enhancement after 60 min was observed (Fig. 2b). The sharp increase in the fluorescence intensity could even be perceived by the naked eye under the excitation of ultraviolet lamp (365 nm) (Scheme 1). This result also supported the idea that benzil moiety could be a strong fluorescence quenching. Moreover, this large fluorescence off–on response was extremely desirable for sensitive detection. Furthermore, upon addition of different concentrations of H2O2, the fluorescence intensity increased linearly with the concentration of H2O2 from 10 μM up to 100 μM (Fig. 2c). Thus, the detection limit (3s/slope) was calculated to be 0.09 μM (Fig. S1). We then evaluated the ability of probe 1 to H2O2 in a two-photon mode. Upon TP excitation at 760 nm, the emission spectra were similar to those obtained in one-photon mode within 60 min (Fig. 2d). Subsequently, the TP absorption cross action spectra of the resulting solution (Fig. S2) exhibited δmax values of 164.7 GM
2.3. Cell culture and cell imaging MKN-45 gastric cancer cells and SMMC-7721 liver cancer cells were cultured for 48 h in culture media (Dulbecco's Modified Eagle's Medium, High Glucose) with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator which was provided with 5% CO2 and 95% air. Both of them were cultured in 12-well plate at a
Fig. 1. The HOMO and LUMO orbitals of probe 1.
K.-M. Zhang et al. / Biosensors and Bioelectronics ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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also lead to small fluorescence enhancement. As shown in Fig. 3, the relative enhancement of fluorescence caused by TBHP was 5-fold, which was much lower than 75-fold caused by H2O2. Moreover, the concentration of lipid hydroperoxides in plasma and erythrocytes of healthy human subjects was about 1 μM and below (Abo et al., 2011), which would have little effect on probe 1 for H2O2 detection in vivo. These results demonstrated that probe 1 could detect H2O2 both qualitatively and quantitatively in complex biological systems. 3.5. Cell imaging
Scheme 2. Design and synthesis of the probe 1.
at 760 nm, indicating probe 1 was potentially useful to imaging in deep tissue. 3.4. Selectivity studies Next, parallel experiments for specificity of probe 1 towards H2O2 over other ROS were carried out. The probe barely showed any fluorescence enhancement within 60 min in response to superoxide (O2-), hydroxyl radical (OH), hypochlorite ( OCl), nitric oxide (NO), singlet oxygen (1O2), while H2O2 could trigger obvious fluorescent intensity increments. Because of having a hydroperoxide structure, tertbutyl hydroperoxide (TBHP) could
Based on the positive results shown above, we studied the feasibility of probe 1 to detect H2O2 in live cells with OPM and TPM. First we evaluated the cell imaging in OPM mode. MKN-45 gastric cancer cells showed no intracellular background fluorescence (Fig. 4a). However, pretreatment with H2O2 (100 μM) for 30 min then incubation with probe 1 (10 μM) for 15 min, MKN-45 cells showed strong fluorescence (Fig. S3). Notably, only incubation with probe 1 for 15 min, MKN-45 cells showed strong levels of fluorescence intensity (Fig. 4b). The fluorescence grew brighter when the incubation time increased to 30 min (Fig. 4c). The enhancement of fluorescence intensity could be caused by endogenous H2O2 production from MKN-45 cells. This also afforded an additional evidence that probe 1 was sensitive to H2O2. To confirm that the fluorescence signals from the reaction of probe 1 with H2O2 in cells, MKN-45 cells were incubated with 10 mM NAC (N-acetyl-L-cysteine, a common inhibitor of ROS) for 60 min to
Fig. 2. (a) Changes in the absorption spectra of probe 1 in the presence of 10 equiv. of H2O2. (b) Time-dependent fluorescence spectral changes of probe 1 with 10 equiv. of H2O2 (λex ¼380 nm, steady excitation). (c) Fluorescent emission spectra of probe 1 (10 μM) in the presence of 1–10 equiv. of H2O2. Data were acquired at 505 nm. (d) Timedependent fluorescence spectral changes of probe 1 with 10 equiv. of H2O2 (λex ¼ 760 nm, two-photon excitation).
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K.-M. Zhang et al. / Biosensors and Bioelectronics ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 3. (a) Responses of probe 1 (10 μM) to 10 equiv. of H2O2 and other ROS species after 60 min (λex ¼ 380 nm, PBS/CH3CN ¼ 8:2, pH ¼7.4, 37 °C). (b) Data were acquired at 505 nm. 1: Free probe; 2: O2 ; 3: NO; 4: 1O2; 5: –OCl; 6: OH; 7: TBHP; and 8: H2O2.
fluorescence when they were treated with probe 1 (10 μM) for 30 min upon TP excitation at 900 nm (Fig. 5). In addition, the potential toxicity of 1 to cells was evaluated by MTT assay (Fig. S4). The absorbance of cells displayed that the cells viability was more than 80% upon treatment with 10 μM 1 at 37 °C for 6 h, indicating the low cytotoxicity and good biocompatibility of the probe. Taken together, probe 1 was capable to measure endogenous H2O2 in live cells.
4. Conclusions
Fig. 4. Images of H2O2 detection in MKN-45 gastric cancer cells using probe 1 (10 μM) at 37 °C. (a) Control, (b) fluorescence image of cells incubated with probe 1 for 15 min, (c) fluorescence image of cells incubated with probe 1 for 30 min, and (d) fluorescence image of cells incubated with NAC for 1 h and then loaded with 10 μM probe 1 for 30 min.
In conclusion, we have developed a new fluorescence probe for hydrogen peroxide and its application for cell-imaging with OPM and TPM was described. The probe displays high selectivity and high sensitivity with a detection limit of 0.09 μM, and shows a significant TP cross-section in response to H2O2. More importantly, this probe can sense intracellular H2O2 levels. The targetability of the probe is in progress in our laboratory.
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Grant no. 91122007) and the Specialized Research Fund for the Doctoral Program of Higher Education (Grant no. 20110211130002).
Appendix A. Suplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.09.073.
References Fig. 5. Confocal fluorescence images in SMMC-7721 liver cancer cells using probe 1 (10 μM) at 37 °C in a TPM mode. (a) Control and (b) confocal fluorescence images of cells incubated with probe 1 for 30 min.
remove the endogenous intracellular reactive oxygen, and then loaded with 10 μM probe 1 for 30 min; as a result, we observed little fluorescence (Fig. 4d). Furthermore, SMMC-7721 liver cancer cells were utilized to imaging with TPM. Similar to that observed in OPM mode, SMMC-7721 liver cancer cells showed strong
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Masanta, G., Heo, C., Lim, C., Bae, S., Cho, B., Kim, H., 2012. Chem. Commun. 48, 3518–3520. Mattson, M., 2004. Nature 430, 631–639. Miller, E., Albers, A., Pralle, A., Isacoff, E., Chang, C., 2005. J. Am. Chem. Soc. 127, 16652–16659. Ohshima, H., Tatemichi, M., Sawa, T., 2003. Arch. Biochem. Biophys. 417, 3–11. Qian, Y., Xue, L., Hu, D., Li, G., Jiang, H., 2012. Dyes Pigm. 95, 373–376. Rhee, S., 2006. Science 312, 1882–1883. Setsukinai, K., Urano, Y., Kakinuma, K., Majima, H., Nagano, T., 2003. Biol. Chem. 278, 3170–3175. Shah, A., Channon, K., 2004. Heart 90, 486–487. Soh, N., Sakawaki, O., Makihara, K., Odo, Y., Fukaminato, T., Kawai, T., Irie, M., Imato, T., 2005. Bioorg. Med. Chem. 13, 1131–1139. Stadtman, E., 2006. Free Radic. Res. 40, 1250–1258. Stone, J., Yang, S., 2006. Antioxid. Redox Signal. 8, 243–270. Veal, E., Day, A., Morgan, B., 2007. Mol. Cell 26, 1–14. Xu, K., Tang, B., Huang, H., Yang, G., Chen, Z., Li, P., An, L., 2005. Chem. Commun., 5974–5976. Zhu, B., Jiang, H., Guo, B., Shao, C., Wua, H., Dua, B., Wei, Q., 2013. Sens. Actuators B 186, 681–686.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
A coumarin-based two-photon probe for hydrogen peroxide Kai-Ming Zhang a, Wei Dou a, Peng-Xuan Li a, Rong Shen b, Jia-Xi Ru a, Wei Liu a, Yu-Mei Cui a, Chun-Yang Chen a, Wei-Sheng Liu a,n, De-Cheng Bai b,nn a Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China b Key Laboratory of Preclinical Study for New Drugs of Gansu Province and Operative Surgery Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 26 June 2014 Received in revised form 2 September 2014 Accepted 22 September 2014
A new fluorescence probe was developed for hydrogen peroxide (H2O2) detection based on donor-excited photo induced electron transfer (D-PET) mechanism, together with the benzil as a quenching and recognizing moiety. The benzil could convert to benzoic anhydride via a Baeyer–Villiger type reaction in the presence of H2O2, followed by hydrolysis of benzoicanhydride to give benzoic acid, and the fluorophore released. The probe was synthesized by a 6-step procedure starting from 4-(diethylamino) salicylaldehyde. A density functional theory (DFT) calculation was performed to demonstrate that the benzil was a fluorescence quencher. The probe was evaluated in both one-photon and two-photon mode, and it exhibited high selectivity toward H2O2 over other reactive oxygen species and high sensitivity with a detection limit of 0.09 μM. Furthermore, the probe was successfully applied to cell imaging of intracellular H2O2 levels with one-photon microscopy and two-photon microscopy. The superior properties of the probe made it of great potential use in more chemical and biological researches. & 2014 Elsevier B.V. All rights reserved.
Keywords: Hydrogen peroxide Probe Coumarin Two-photon Cell imaging
1. Introduction Reactive oxygen species (ROS) including hydrogen peroxide (H2O2), hydroxyl (OH), singlet oxygen (1O2), superoxide (O2 ), peroxyl radical (ROO) and hypochlorous acid (HOCl) are endogenously generated from oxygen metabolism in biological systems (Dickinson and Chang, 2011; D'Autreaux and Toledano, 2007; Stadtman, 2006). H2O2, a major member of ROS, plays an important role in normal cellular growth and proliferation (Rhee, 2006; Kamata et al., 2005; Finkel and Holbrook, 2000; Veal et al., 2007; Harman, 1981; Stone and Yang, 2006). However, over production of H2O2 can cause many diseases, such as inflammatory disease, cardiovascular disease, Alzheimer's disease and cancer (Barnham et al., 2004; Mattson, 2004; Lin and Beal, 2006; Andersen, 2004; Ohshima et al., 2003; Shah and Channon, 2004). Therefore, it is necessary to develop sensitive and selective methods for the detection of H2O2 in living cells. As a useful analytical tool, fluorescent spectroscopy is of broad interest in bioanalysis. In recent years, many fluorescence probes for H2O2 have been developed (Li et al., 2013; Chen et al., 2011; Hyman and Franz, 2012; Kim et al., 2010; Lo and Chu, 2003; Chang n
Corresponding author. Fax: þ 86 931 8912582. Corresponding author. E-mail addresses: [email protected] (W.-S. Liu), [email protected] (D.-C. Bai).
nn
http://dx.doi.org/10.1016/j.bios.2014.09.073 0956-5663/& 2014 Elsevier B.V. All rights reserved.
et al., 2004; Miller et al., 2005; Dickinson et al., 2010; Albers et al., 2008; Du et al., 2008; Setsukinai et al., 2003; Maeda et al., 2004; Lippert et al., 2011; Karton-Lifshin et al., 2011; Zhu et al., 2013; Soh et al., 2005; Xu et al., 2005; Hu et al., 2014; Qian et al., 2012). For example, dichlorodihydrofluorescein derivatives are widely utilized as fluorescent probes for measuring H2O2 (Setsukinai et al., 2003). However, they have poor selectivity and undergo autoxidation upon exposure to excitation light. Another reported H2O2 probes based on arylsulfonyl (Maeda et al., 2004) and boronate ester (Lippert et al., 2011; Karton-Lifshin et al., 2011; Hyman and Franz, 2012) deprotection mechanism show high selectivity toward H2O2 over other ROS, but their response rates are not entirely satisfactory for biological research and clinical needs. Very recently, Negano et al. reported a turn-on H2O2 fluorescent probe NBNF based on a Baeyer–Villiger type reaction of the benzil moiety (Abo et al., 2011). As a quencher and specific response group to H2O2, the benzil was first applied to design H2O2 probe in his work. Moreover, this probe has higher sensitivity and enough selectivity. However, NBZF was evaluated using one-photon excitation (OPE) with relatively short excitation wavelength (490 nm), which limits its further application in tissue imaging. While two-photon excitation (TPE) has attracted much attention in bioanalysis due to its minimum interference from photobleaching and photodamage (Kim and Cho, 2009; Masanta et al., 2012). So it is still significative to extend a new fluorescent probe with TPM using benzil as a recognition moiety of H2O2. We selected
K.-M. Zhang et al. / Biosensors and Bioelectronics ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
density of 104 cells per well in culture media, then incubated with 10 μM probe 1 solution at 37 °C. Before imaging measurement, cells were washed 3 times with phosphate buffered saline (PBS).
Scheme 1. Mechanism of H2O2 sensing by probe 1.
coumarin as a two-photon fluorophore scaffold, because a coumarin-based fluorophore 6-hydroxycoumarin-4-ylme thanol has been reported to possess TPE properties (Furuta et al., 1999). Herein, we reported a TPE probe 1 for H2O2. The synthesized coumarin derivative 1 was almost non-fluorescent (quantum yield in H2O/CH3CN¼ 8: 2, Ф ¼0.0202), indicating that the benzil acted as a fluorescence quencher because of the donor-excited photo induced electron transfer (D-PET) process (Abo et al., 2011). However, compound 1 could convert to 2 in the presence of H2O2 (Scheme 1). Owing to the break of benzil's structure, the DPET process did not occur and the strong fluorescence of coumarin was emitted (quantum yield in H2O/CH3CN¼ 8: 2, Ф ¼ 0.0641).
2. Experimental section 2.1. Materials and instruments All reagents were purchased from commercial providers and used without further purification. All solvents were used after distillation or dehydration. 1H NMR and 13C NMR were recorded in CDCl3 at 25 °C on a Bruker DRX400 spectrometer, 400 MHz for 1H and 100 MHz for 13C, respectively. Mass spectra (ESI) were taken on a Bruker micrOTOF-Q II mass spectrometer. UV–visible spectra were measured using an Analytik Jena SPECORD 50 PLUS. Fluorescence spectra with both one-photon excitation (OPE) and twophoton excitation (TPE) were carried out using the Hitachi F-4500 fluorescence spectrometer and Edinburgh Instrument FLS920, respectively. Conventional fluorescence imaging were performed using a Leica DMI 4000B microscope with DFC420 C camera and a mercury lamp (short ARC) with a BP 450–490 excitation filter and a LP 515 emission filter. Two-photon fluorescence microscopy imaging were captured using Olympus FV1000 laser scanning confocal and multiphoton microscopes (set at wavelength 900 nm and PMT; Gain, 746 V) with 100 oil immersion objective lens. The signals were collected in a 12 bit unsigned 1024 1024 pixels at 8.0 us/ pixel sampling speed. 2.2. Absorption and fluorescence spectroscopy Probe 1 was dissolved in DMF for a stock solution (1 mM). H2O2 was from dilution of 30% solution in water. NaClO was from dilution of 10% solution in water. Tert-butyl hydroperoxide was from dilution of 70% solution in water. NO was produced from sodium nitroferricyanide (SNP). O2- was from KO2 in anhydrous DMSO. 1O2 was produced by mixing H2O2 and 100-exceed of NaClO. OH was produced by Fenton reaction, mixing H2O2 and 10-exceed (NH4)2Fe (SO4)2. Test solutions (10 μM) were prepared by displacing 20 μL of the stock solution into a 2-mL mixture of 0.1 M PBS and CH3CN (8:2, v/v) at pH 7.4.
3. Results and discussion 3.1. Calculation based on density functional theory (DFT) In order to understand the effect of the benzil structure on photophysical properties, the calculation based on density functional theory (DFT) for 1 was performed (Fig. 1). According to the comparison between the highest occupied molecular orbitals (HOMO) and the lowest unoccupied molecular orbitals (LUMO), it came to a conclusion that the electron density from the coumarin moiety moved to the benzil moiety upon the excitation. As a result, the fluorescence of coumarin was quenched. 3.2. Synthesis and characterization The synthesis of the probe 1 began with compound 3 in a straight forward manner by a 6-step procedure. The starting material 3 could convert to 4 according to a routine coumarin formation procedure. The compound 7 was synthesized by using the intermediate 4 as reaction material undergoing bromination, reaction of Sonogashira with trimethylsilyl acetylene (TMSA) and deprotection with K2CO3 in CH3OH. The compound 8 was afforded through the reaction of Sonogashira of 7 with 1-bromo-4-nitrobenzene. At last, the desired target probe 1 was obtained by oxidation reaction of 8 with DMSO catalyzed by PdCl2. The detail characterization data and experimental procedure were shown in the ESI. Scheme 2 3.3. UV–vis absorption and fluorescence spectra of 1 towards H2O2 We examined the optical properties of probe 1 in PBS buffer (0.1 M, pH 7.4, 20% CH3CN). UV–vis spectra of 1 (10 μM) exhibited an absorptions maximum at λabs ¼475 nm. After treatment with 10 equiv. of H2O2 at 37 °C, the absorption at 475 nm obviously declined, whereas a new absorption peak was present at 360 nm (Fig. 2a). Accordingly, the emission at 505 nm apparently appeared upon excitation at 380 nm, and the fluorescence intensity increased rapidly. Finally approximately 75-fold fluorescence enhancement after 60 min was observed (Fig. 2b). The sharp increase in the fluorescence intensity could even be perceived by the naked eye under the excitation of ultraviolet lamp (365 nm) (Scheme 1). This result also supported the idea that benzil moiety could be a strong fluorescence quenching. Moreover, this large fluorescence off–on response was extremely desirable for sensitive detection. Furthermore, upon addition of different concentrations of H2O2, the fluorescence intensity increased linearly with the concentration of H2O2 from 10 μM up to 100 μM (Fig. 2c). Thus, the detection limit (3s/slope) was calculated to be 0.09 μM (Fig. S1). We then evaluated the ability of probe 1 to H2O2 in a two-photon mode. Upon TP excitation at 760 nm, the emission spectra were similar to those obtained in one-photon mode within 60 min (Fig. 2d). Subsequently, the TP absorption cross action spectra of the resulting solution (Fig. S2) exhibited δmax values of 164.7 GM
2.3. Cell culture and cell imaging MKN-45 gastric cancer cells and SMMC-7721 liver cancer cells were cultured for 48 h in culture media (Dulbecco's Modified Eagle's Medium, High Glucose) with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator which was provided with 5% CO2 and 95% air. Both of them were cultured in 12-well plate at a
Fig. 1. The HOMO and LUMO orbitals of probe 1.
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also lead to small fluorescence enhancement. As shown in Fig. 3, the relative enhancement of fluorescence caused by TBHP was 5-fold, which was much lower than 75-fold caused by H2O2. Moreover, the concentration of lipid hydroperoxides in plasma and erythrocytes of healthy human subjects was about 1 μM and below (Abo et al., 2011), which would have little effect on probe 1 for H2O2 detection in vivo. These results demonstrated that probe 1 could detect H2O2 both qualitatively and quantitatively in complex biological systems. 3.5. Cell imaging
Scheme 2. Design and synthesis of the probe 1.
at 760 nm, indicating probe 1 was potentially useful to imaging in deep tissue. 3.4. Selectivity studies Next, parallel experiments for specificity of probe 1 towards H2O2 over other ROS were carried out. The probe barely showed any fluorescence enhancement within 60 min in response to superoxide (O2-), hydroxyl radical (OH), hypochlorite ( OCl), nitric oxide (NO), singlet oxygen (1O2), while H2O2 could trigger obvious fluorescent intensity increments. Because of having a hydroperoxide structure, tertbutyl hydroperoxide (TBHP) could
Based on the positive results shown above, we studied the feasibility of probe 1 to detect H2O2 in live cells with OPM and TPM. First we evaluated the cell imaging in OPM mode. MKN-45 gastric cancer cells showed no intracellular background fluorescence (Fig. 4a). However, pretreatment with H2O2 (100 μM) for 30 min then incubation with probe 1 (10 μM) for 15 min, MKN-45 cells showed strong fluorescence (Fig. S3). Notably, only incubation with probe 1 for 15 min, MKN-45 cells showed strong levels of fluorescence intensity (Fig. 4b). The fluorescence grew brighter when the incubation time increased to 30 min (Fig. 4c). The enhancement of fluorescence intensity could be caused by endogenous H2O2 production from MKN-45 cells. This also afforded an additional evidence that probe 1 was sensitive to H2O2. To confirm that the fluorescence signals from the reaction of probe 1 with H2O2 in cells, MKN-45 cells were incubated with 10 mM NAC (N-acetyl-L-cysteine, a common inhibitor of ROS) for 60 min to
Fig. 2. (a) Changes in the absorption spectra of probe 1 in the presence of 10 equiv. of H2O2. (b) Time-dependent fluorescence spectral changes of probe 1 with 10 equiv. of H2O2 (λex ¼380 nm, steady excitation). (c) Fluorescent emission spectra of probe 1 (10 μM) in the presence of 1–10 equiv. of H2O2. Data were acquired at 505 nm. (d) Timedependent fluorescence spectral changes of probe 1 with 10 equiv. of H2O2 (λex ¼ 760 nm, two-photon excitation).
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Fig. 3. (a) Responses of probe 1 (10 μM) to 10 equiv. of H2O2 and other ROS species after 60 min (λex ¼ 380 nm, PBS/CH3CN ¼ 8:2, pH ¼7.4, 37 °C). (b) Data were acquired at 505 nm. 1: Free probe; 2: O2 ; 3: NO; 4: 1O2; 5: –OCl; 6: OH; 7: TBHP; and 8: H2O2.
fluorescence when they were treated with probe 1 (10 μM) for 30 min upon TP excitation at 900 nm (Fig. 5). In addition, the potential toxicity of 1 to cells was evaluated by MTT assay (Fig. S4). The absorbance of cells displayed that the cells viability was more than 80% upon treatment with 10 μM 1 at 37 °C for 6 h, indicating the low cytotoxicity and good biocompatibility of the probe. Taken together, probe 1 was capable to measure endogenous H2O2 in live cells.
4. Conclusions
Fig. 4. Images of H2O2 detection in MKN-45 gastric cancer cells using probe 1 (10 μM) at 37 °C. (a) Control, (b) fluorescence image of cells incubated with probe 1 for 15 min, (c) fluorescence image of cells incubated with probe 1 for 30 min, and (d) fluorescence image of cells incubated with NAC for 1 h and then loaded with 10 μM probe 1 for 30 min.
In conclusion, we have developed a new fluorescence probe for hydrogen peroxide and its application for cell-imaging with OPM and TPM was described. The probe displays high selectivity and high sensitivity with a detection limit of 0.09 μM, and shows a significant TP cross-section in response to H2O2. More importantly, this probe can sense intracellular H2O2 levels. The targetability of the probe is in progress in our laboratory.
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Grant no. 91122007) and the Specialized Research Fund for the Doctoral Program of Higher Education (Grant no. 20110211130002).
Appendix A. Suplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.09.073.
References Fig. 5. Confocal fluorescence images in SMMC-7721 liver cancer cells using probe 1 (10 μM) at 37 °C in a TPM mode. (a) Control and (b) confocal fluorescence images of cells incubated with probe 1 for 30 min.
remove the endogenous intracellular reactive oxygen, and then loaded with 10 μM probe 1 for 30 min; as a result, we observed little fluorescence (Fig. 4d). Furthermore, SMMC-7721 liver cancer cells were utilized to imaging with TPM. Similar to that observed in OPM mode, SMMC-7721 liver cancer cells showed strong
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