A fast-response, highly sensitive and selective fluorescent probe for the ratiometric imaging of hydrogen peroxide with a 100 nm red-shifted emission

Abstract

Recently, growing attention has been paid to the accurate determination of hydrogen peroxide (H₂O₂) for elucidating its detailed biological function in physiology and pathology. A fluorescence method with the help of a fluorescent probe is the preferred technique for in situ visualization of biologically important species in vivo, even in single living cells. In the present manuscript, we developed a simple, fast response and highly selective fluorescent probe (1) with a receptor of the boronate moiety for the ratiometric imaging of H₂O₂ in living cells. Probe 1 could quantifiably detect H₂O₂ in the range of 18–540 μM by a ratiometric fluorescence spectroscopy method with a detection limit of 4 μM. Importantly, probe 1 exhibited 81 nm red-shifted absorption spectra accompanied by the color changes from colorless to yellow, and 100 nm red-shifted emission spectra upon addition of H₂O₂. Thus, 1 can serve as a “naked-eye” probe for H₂O₂. Preliminary bioimaging application and low cytotoxicity investigations further demonstrated that the proposed probe would be of great benefit to biomedical researchers for investigating the detailed biological function of H₂O₂ in biological systems.

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1. Introduction

Hydrogen peroxide (H₂O₂), a major reactive oxygen species (ROS) produced in living matrixes, is attracting growing attention for its diverse functions in physiology and pathology.^1–4 Hydrogen peroxide is a newly recognized second messenger in cellular signal transduction and can indicate the intracellular oxidant levels.^5–7 A regular level of H₂O₂ plays important roles in physiology, aging, and disease in living organisms.^8,9 Abnormal production or accumulation in cellular levels of H₂O₂ has been linked to DNA damage, mutation, and genetic instability.^6,10 Thus, the development of accurate assays for H₂O₂ is of great importance for elucidating the detailed biological function of H₂O₂ in living systems.

Fluorescence technique with the help of fluorescent probe is the preferred method for in situ visualization of biologically important species in living systems owing to its various advantages such as simplicity, sensitivity, non-invasiveness, and good biological compatibility.^11,12 Despite advances in the development of fluorescent probes for H₂O₂, the ratiometric fluorescent probes for H₂O₂ are rather few even such probes can eliminate numerous ambiguities resulting from the localization of the probe, changes of environment around the probes, excitation and emission efficiency.^4,13–18 Additionally, the ratiometric fluorescent probe should exhibit a large emission wavelength shift (above 80 nm) for enhancing resolution in the ratiometric bioimaging.^4,19,20 As far as we know, only three such probes for H₂O₂ have been reported so far. One example reported by Jiang et al. showed a large bathochromic shift of 105 nm in the recognition of H₂O₂ and this property can be achieved only in the presence of surfactant solution.¹⁸ Kumar et al. presented a ratiometric fluorescent probe exhibited an 82 nm blue-shifted emission in the presence of H₂O₂.²¹ This probe is prone to be interfered with pH for it employs the twisted intramolecular charge transfer (TICT) mechanism based the charge transfer from the nitrogen atom of the dimethylamino moiety (donor) to the imino moiety (acceptor). Very recently, we developed a ratiometric fluorescent probe for H₂O₂ with a large emission shift of 132 nm and this probe possesses a defect of shorter excitation wavelength (λ_ex = 380 nm).⁴ Although the three fluorescent probes have made great progress in the ratiometric determination of H₂O₂, more effective probes with fast-response, high sensitivity and selectivity, and practicability, especially ratiometric imaging in living cells still need to be developed.

Recently, based on the internal charge transfer (ICT) mechanism, we developed a series of ratiometric fluorescent probes employing diversified fluorophores.^22–24 For example, 4-hydroxynaphthalimide was selected firstly as the fluorescence reporter group in the design of ratiometric fluorescent probes.²⁴ A propargyl protecting 4-hydroxynaphthalimide exhibited 73 nm red-shifted emission spectra upon addition of palladium species, and thus, 4-hydroxynaphthalimide is a potential fluorophore in the construction of ratiometric fluorescent probes with a large emission wavelength shift. In continuation of our research work, we herein designed and synthesized an ICT-based ratiometric fluorescent probe (Scheme 1, 1) for H₂O₂ with apparent properties such as fast-response (within 10 min), high sensitivity (a detection limit of 4 μM) and selectivity, a large emission shift of 100 nm, visual and ratiometric detection, and ratiometric imaging in living cells.

Scheme 1 The synthesis of probe 1.

During the development of fluorescent probes for H₂O₂, Chang et al. pioneered a novel boronate-based fluorescent probe for the high selective determination of H₂O₂.^25,26 A receptor of boronate moiety was widely adopted in the design of probes for H₂O₂.^6,12 This strategy inspired us to design highly selective fluorescent probes for H₂O₂ by the adoption of boronate moiety. To achieve the ratiometric measurement, 4-hydroxynaphthalimide was selected as the fluorescence reporter group for it possesses excellent ICT structure and desirable photophysical properties.²⁴ We hypothesize that the reaction of probe 1 with H₂O₂ triggers the cleavage of a boronate-based protecting group, and as a result, restores the green fluorescence of 4-hydroxynaphthalimide 2. The reaction mechanism of 1 with H₂O₂ is shown in Scheme 2.

Scheme 2 The reaction mechanism of probe 1 with H₂O₂.

2. Experimental

2.1 Materials and instrumentations

4-Hydroxy-1,8-naphthalimide was prepared according our previous work.²⁴ All other chemicals used in this paper were obtained from commercial suppliers and used without further purification. Silica gel (200–300 mesh, Qingdao Haiyang Chemical Co.) was used for column chromatography. ¹H-NMR was recorded on a Bruker AV-400 spectrometer with chemical shifts reported as ppm (in CDCl₃, TMS as internal standard). Electrospray ionization (ESI) mass spectra were measured with an LC-MS 2010A (Shimadzu) instrument. Absorption spectra were recorded on UV-3101PC spectrophotometer. Fluorescence emission spectra were measured on Perkin-Elmer Model LS-55. All pH measurements were made with a Sartorius basic pH-meter PB-10.

2.2 Synthesis of probe 1

To a solution of 4-hydroxy-1,8-naphthalimide 2 (269 mg, 1 mmol) in CH₃CN (30 mL) were added K₂CO₃ (276 mg, 2 mmol) and 4-(bromomethyl)benzene boronic acid pinacol ester (297 mg, 1 mmol). The reaction mixture was heated to reflux overnight. After removal of solvent, the residues were purified by silica gel column chromatography using dichloromethane as eluent to afford pure product. ¹H-NMR (400 MHz, CDCl₃) δ (*10⁻⁶): 0.971 (t, J = 7.4 Hz, 3H), 1.358 (s, 12H), 1.415–1.472 (m, 2H), 1.669–1.745 (m, 2H), 4.167 (t, J = 7.4 Hz, 2H), 5.389 (s, 2H), 7.093 (d, J = 8.4 Hz, 1H), 7.519 (d, J = 8.0 Hz, 2H), 7.709 (t, J = 8.0 Hz, 1H), 7.882 (d, J = 8.0 Hz, 2H), 8.526 (d, J = 8.4 Hz, 1H), 8.604–8.627 (m, 2H). ESI-MS calcd for C₂₉H₃₃BNO₅ [M + H]⁺ 486.2, found 486.2.

2.3 Determination of the detection limit

The detection limit was calculated based on the fluorescence titration. In the absence of H₂O₂, the fluorescence emission spectrum of probe 1 was measured by five times and the standard deviation of blank measurement was achieved. To gain the slope, the ratio of the fluorescence intensity at 546 nm to the fluorescence intensity at 446 nm (F₅₄₆/F₄₄₆) was plotted as a concentration of H₂O₂. So the detection limit was calculated with the following equation:

Detection limit = 3σ/k.

where σ is the standard deviation of blank measurement, k is the slope between the fluorescence intensity ratio versus H₂O₂ concentration.

2.4 Cell culture and fluorescence imaging

RAW264.7 macrophage cells (gifted from the center of cells, Peking Union Medical College) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 U mL⁻¹ of penicillin and 100 μg mL⁻¹ streptomycin at 37 °C under a humidified atmosphere containing 5% CO₂. RAW264.7 macrophage cells were seeded in a 96-well plate at a density of 10⁴ cells per well in culture media. After 24 h, they were incubated with 5 μM probe 1 in culture media for 20 min at 37 °C. Fluorescence imaging of living RAW264.7 macrophage cells was observed under an Olympus FV1000 confocal fluorescence microscope with excitation wavelength fixed at 405 nm. Then, the cells were incubated with 100 μM H₂O₂ for another 15 min and fluorescence imaging of living RAW264.7 macrophage cells was also observed under confocal fluorescence microscope with excitation wavelength fixed at 405 nm.

2.5 Cytotoxicity assays

RAW264.7 macrophage cells were cultured in culture media (DMEM) in an atmosphere of 5% CO₂ and 95% air at 37 °C. The cells were seeded into 96-well plates at a density of 3 × 10³ cells per well in culture media, then 0, 5, 10, 25 and 50 μM (final concentration) probe 1 were added respectively. Next, the cells were incubated at 37 °C in an atmosphere of 5% CO₂ and 95% air for 1 h. Finally, 20 μL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg mL⁻¹) was added and were cultured for another 4 h, respectively.

3. Results and discussion

3.1 Characteristic spectrum

It is reported that the appropriate balance between hydrophilicity and lipophilicity of probe is beneficial to the cell permeability and bioimaging in living systems. So, the determination of H₂O₂ with probe 1 was investigated under a mixture of ethanol and water (5 : 5, v/v) solution containing phosphate buffered saline (PBS) (5 mM, pH 7.4).

Firstly, we assessed the spectroscopic properties of free probe 1 under the above analytical conditions. As shown in Fig. 1a and d, the solution of free probe 1 showed one major absorption peak at around 372 nm and fluorescence emission peak at around 446 nm. However, upon addition of H₂O₂ (500 μM), the maximum absorption peak exhibited an about 81 nm red shift and the color of the solution turned from colorless to yellow, and the maximum emission peak underwent a red shift to 546 nm. Thus, probe 1 can serve as a “naked-eye” indicator for H₂O₂. The considerable changes of absorption and fluorescence spectra might be ascribed to the cleavage of a boronate-based protecting group and the coinstantaneous production of stronger electron-push capacity of oxygen anion. These spectra of the reaction solution are in good agreement with ones of 4-hydroxy-1,8-naphthalimide reported by us.²⁴ The results implied that the cleavage of boronate protecting 4-phenylmethanol moiety was induced by H₂O₂.

Fig. 1 Absorption and fluorescence responses of 1 (5 μM) toward different concentrations of H₂O₂ (final concentration: 0, 18, 36, 54, 72, 90, 108, 126, 144, 162, 180, 216, 252, 288, 324, 360, 450, 540 μM) in PBS (5 mM, pH 7.4) solution (ethanol–water = 5 : 5, v/v). (a) Absorption spectra of 1 in the presence of increasing concentrations of H₂O₂, the inset shows the color changes of 1 in the absence and presence of H₂O₂; (b) absorbance ratio (A₄₅₃/A₃₇₂) of 1 versus increasing concentrations of H₂O₂; (c) the photographs of the solution of probe 1 (10 μM) in the presence of different concentrations of H₂O₂ (from left to right: 0, 20, 50, 100, 150, and 500 μM); (d) fluorescence spectra of 1 in the presence of increasing concentrations of H₂O₂; (e) fluorescence intensity ratio (F₅₄₆/F₄₄₆) of 1 versus increasing concentrations of H₂O₂. Each spectrum was obtained after H₂O₂ addition at 25 °C for 10 min. Excitation wavelength = 410 nm, excitation and emission slit widths = 15 nm and 6 nm. Error bar = RSD (n = 5).

Then, to demonstrate the recognition mechanism of probe 1 for H₂O₂, the reaction of probe 1 and H₂O₂ was carried out under the above-mentioned analytical conditions. The green fluorescent reaction product was obtained and characterized to be compound 2 by ¹H-NMR and ESI-MS. Therefore, combined with the previous conclusions by other groups,^6,12 a possible mechanism was proposed as shown in Scheme 2.

3.2 Effects of reaction time on sensing H₂O₂

Response time is a fundamental parameter for most reaction-based probes. Then, the time required for the reaction of probe 1 and H₂O₂ at 25 °C was investigated. As shown in Fig. 2, the fluorescence intensity at 446 nm decreases with reaction time, and synchronously the fluorescence intensity at 546 nm increases with reaction time. The ratio of fluorescence intensities (F₄₄₆/F₅₄₆) decreases with reaction time and then levels off at reaction time greater than about 10 min. The result showed that the rate of the reaction of probe 1 with H₂O₂ is superior to those reported boronate-based fluorescent probes without additional reagents.^4,6,12 That is to say, our proposed probe could provide a rapid analytical method for the detection of H₂O₂.

Fig. 2 The fluorescence spectra of probe 1 (5 μM) incubated with H₂O₂ (500 μM) in PBS (5 mM, pH 7.4) solution (ethanol–water = 5 : 5, v/v) for 0–40 min at 25 °C, the inset shows the fluorescence intensity ratio (F₅₄₆/F₄₄₆) of probe 1 vs. increasing time. Excitation wavelength = 410 nm, excitation and emission slit widths = 15 nm and 6 nm.

3.3 Quantification of H₂O₂

In the absorption spectrum, the subsequent addition of H₂O₂ to the solution of probe 1 resulted in a gradual decrease of the maximum absorption peak centered at around 372 nm and a progressive increase of the maximum absorption peak centered at around 453 nm, and a well-defined isosbestic point at 402 nm is observed (Fig. 1a). Additionally, a good linearity between the absorbance ratio (A₄₅₃/A₃₇₂) and the concentrations of H₂O₂ in the range of 18 to 540 μM exhibited the determination of H₂O₂ would be carried out by the ratiometric measurement (Fig. 1b). Additionally, the detection limit by the naked-eye was estimated to be about 50 μM (Fig. 1c). In the fluorescence emission spectrum, the subsequent addition of H₂O₂ to the solution of probe 1 resulted in a gradual decrease of fluorescence peak centered at around 446 nm and a progressive increase of fluorescence peak centered at around 546 nm (Fig. 1d). In addition, a well-defined isoemission point at 520 nm is also observed (Fig. 1d). The production of isosbestic point and isoemission point distinctly demonstrated that a new species 4-hydroxy-1,8-naphthalimide 2 came into being. Moreover, there was a good linearity between the fluorescence intensity ratio (F₅₄₆/F₄₄₆) and the concentrations of H₂O₂ in the range of 18 to 540 μM with a detection limit of 4 μM (Fig. 1e). These results demonstrated that probe 1 could detect H₂O₂ qualitatively and quantitatively by ratiometric absorption and fluorescence methods with excellent sensitivity.

3.4 Selectivity to H₂O₂

Then we examined the selectivity of probe 1 toward H₂O₂ under the above-mentioned analytical conditions. As shown in Fig. 3c, nearly no fluorescence intensity changes were observed in the presence of biological metal ions, anions, and other ROS including ClO⁻, O₂⁻, ˙OH, TBHP, ¹O₂, NO, and ˙O^tBu (Fig. 3c), which is ascribed to the adoption of a H₂O₂-specific receptor of boronate moiety. In addition, the effects of interference of the above-mentioned other analytes on monitoring H₂O₂ were investigated (Fig. 3d). The similar results were obtained in the absorption spectra (Fig. 3a and b). Thus, these results demonstrated that probe 1 possesses high selectivity toward H₂O₂ when present with other analytes.

Fig. 3 (a) The absorption responses of probe 1 (5 μM) toward various analytes (200 μM), (b) the absorption responses of probe 1 (5 μM) toward H₂O₂ (200 μM) in the presence of other analytes (200 μM), and all data represent the absorbance ratio (A₄₅₃/A₃₇₂) of probe 1; (c) the fluorescence responses of probe 1 (5 μM) toward various analytes (200 μM), (d) the fluorescence responses of probe 1 (5 μM) toward H₂O₂ (200 μM) in the presence of other analytes (200 μM), and all data represent the fluorescence intensity ratio (F₅₄₆/F₄₄₆) of probe 1. Conditions: each spectrum was obtained after various analytes addition in PBS (5 mM, pH 7.4) solution (ethanol–water = 5 : 5, v/v) at 25 °C for 10 min. Excitation wavelength = 410 nm, excitation and emission slit widths = 15 nm and 6 nm. Error bar = RSD (n = 5).

3.5 Bioimaging and cytotoxicity investigation

Next, we attempted to apply probe 1 for the ratiometric fluorescence imaging of H₂O₂ in living systems. Confocal fluorescence imaging in living RAW264.7 macrophage cells was carried out (Fig. 4.). An intense intracellular fluorescence of the cells incubated with probe 1 (5 μM) for 20 min demonstrated that probe 1 is cell-permeable (Fig. 4b and c). And then, 100 μM H₂O₂ was added to the above-mentioned cells for another 15 min. As expected, distinct changes of ratiometric fluorescence responses generated from green channel and blue channel in living cells were observed (Fig. 4d and h). These results revealed that probe 1 could be used for the ratiometric fluorescent imaging of H₂O₂ in living matrixes.

Fig. 4 Confocal fluorescence images of living Murine RAW264.7 macrophage cells: the cells were incubated with probe 1 (5 μM) for 20 min (a) bright-field transmission image, (b) blue channel at 460 ± 30 nm, (c) green channel at 560 ± 30 nm, and (d) ratio image generated from (c) and (b); cells pretreated with probe 1 were further incubated with H₂O₂ (100 μM) for 15 min (e) bright-field transmission image, (f) blue channel at 460 ± 30 nm, (g) green channel at 560 ± 30 nm, and (h) ratio image generated from (g) and (f). Incubation was performed at 37 °C under a humidified atmosphere containing 5% CO₂.

Further, to estimate cytotoxicity of probe 1, we performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in RAW264.7 macrophage cells with 5, 10, 25 and 50 μM probe 1 for 1 h, respectively. The experiment results were shown in Fig. 5. The obtained results exhibited the absorbances at 490 nm were in good agreement with the control experiment. This conclusion implied that our proposed probe was of low toxicity to cultured cells under the experimental conditions at the concentration of 5 μM for 35 min.

Fig. 5 Cytotoxicity assays of probe 1 at different concentrations for RAW264.7 macrophage cells.

4. Conclusions

In conclusion, we have presented the design, synthesis and properties of an ICT-based ratiometric fluorescent probe 1 for H₂O₂ with a new design platform of benzyl protecting 4-hydroxynaphthalimide. Probe 1 exhibits high H₂O₂-selectivity over various analytes, which is ascribed to the adoption of a boronate moiety. In addition, probe 1 displays an 81 nm red-shift of absorption spectra and the color changes from colorless to yellow upon addition of H₂O₂, and thus can serve as a “naked-eye” probe for H₂O₂. Importantly, probe 1 can detect H₂O₂ quantitatively by ratiometric fluorescence method with a 100 nm red-shifted emission with excellent sensitivity. We highlight the simplicity of the design and synthesis, yet its combined properties, such as high specificity and sensitivity, fast response, visual and ratiometric fluorescent determination with a large red-shifted emission and ratiometric bioimaging in living cells, and anticipate that this probe would be of great benefit to biological researchers for investigating the function of H₂O₂ in living systems. Our proposed strategy by modulation of the benzyl protecting 4-hydroxynaphthalimide provides a promising methodology for the design of ratiometric fluorescent probe with a large emission shift.

Acknowledgements

We gratefully acknowledge financial support from the National Nature Science Foundation of China (no. 21275018, 21107029 and 21203008), Outstanding Young Scientists Award Fund of Shandong Province (BS2013HZ007), Postdoctoral Science Foundation of China (2013M541953), Research Fund for the Doctoral Program of Higher Education of China (RFDP) (no. 20121101110049) and the 111 Project (B07012) for financial support.

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