An extremely rapid-response fluorescent probe for hydrogen peroxide and its application in living cells

Abstract

A turn-on fluorescence probe ACF for rapid detection of H₂O₂ was constructed. The probe utilized a 2-(azidomethyl)benzoyl group as a new reaction site, which exhibited a rapid response to H₂O₂ with a 118-fold fluorescence enhancement within 5 min. The biological application of ACF was confirmed by fluorescence imaging H₂O₂ in living cells.

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Hydrogen peroxide (H₂O₂), one of the most important reactive oxygen species (ROS), has been known as a harmful metabolic product and a component of the immune response to microbial invasion for a long time.¹ However, H₂O₂ functions as an ubiquitous intracellular second messenger when it is generated at a low concentration (<0.7 μM).² It stimulates cell proliferation,³ differentiation,⁴ and migration⁵ by activating the signalling pathways. H₂O₂ might be generated aberrantly and result in oxidative stress with stimulation by exogenous chemicals. However, H₂O₂ is an oxidant unlike the classical second messenger.⁶ The resulting H₂O₂ and other ROS will attack cellular structures or biomolecules such as proteins,⁷ liposomes,⁸ and DNA,⁹ which has been correlated with aging,¹⁰ Alzheimer's disease,¹¹ and cancer.¹² Obviously, information on the location and timing of H₂O₂ generation in biological processes is important and would provide information that could help to understand the function of H₂O₂.

Currently, several chemical methods have been developed to detect intracellular H₂O₂, including mass assays,¹³ proteomics assays,¹⁴ and fluorescence-based assays.^15–21 Among these methods, fluorescence-based assays were useful because of their non-destructive features. A few fluorescent probes designed for H₂O₂ detection have been reported since 2003.^16a The first developed and most popular probes were a kind of boronate ester,¹⁶ which have occupied more than a half of all the H₂O₂ probes, including NIR, ratiometric, targetable, trappable and two-photon probes etc. Some of them have been successfully applied to monitor H₂O₂ at physiological levels in vitro and in vivo and others have been used to explore the cellular mechanisms associated with H₂O₂. Chang group contributed a lot in this field.^16b Another kind of fluorescent probes developed for H₂O₂ in the early stage contained arylsulfonyl esters as trap groups.¹⁷ After that, several kinds of probes were designed based on unique H₂O₂-responsive sites such as diphenylphosphine,¹⁸ α-diketone groups,¹⁹ metal complexes²⁰ and some chalcogen.²¹ The efforts to find novel H₂O₂-responsive sites were still in progress.

Although several H₂O₂-responsive sites have been developed and a lot of fluorescence probes have been constructed, the reaction rates and fluorescence background levels of some fluorescence probes were generally not satisfactory for biological applications. When treated with H₂O₂, most of them had a long response time which became an important and complex issue for monitoring the H₂O₂ concentration in living cells.²² Only a few of them could respond to H₂O₂ fast and selectively. It is especially important to develop rapid-response probes to monitor the H₂O₂ in biological process.

Reduced fluorescent dyes such as 2′,7′-dichlorodihydrofluorescein diacetate were commonly used as fluorescence probes for H₂O₂.²³ However, it still showed non-fluorescence if 2′,7′- dichlorodihydrofluorescein diacetate was only oxidized to 2′,7′-dichlorofluorescin diacetate.²⁴ It is obvious that the ester bond was broken during or after the oxidation. In this process, H₂O₂ as a good nucleophile,^16b might promote a nucleophilic substitution. So it is possible to construct a fluorescence probe for H₂O₂ based on breaking a special ester bond. Therefore, finding a special ester bond and linking it with a fluorophore may be a feasible strategy.

Based on this strategy, ACF was synthesized by the reaction of 2′,7′-dichlorofluorescein and 2-(azidomethyl)benzoyl acid (Scheme 1). ACF exhibited almost no fluorescence (fluorescence quantum yield: Φ = 0.0024, in CH₃OH/PBS buffer, 10 mM, pH = 7.4, 5/95, ESI†).

Scheme 1 The synthesis of probe ACF and the response of ACF to H₂O₂.

When treated with H₂O₂, ACF showed extremely rapid response. Only in 5 min, the fluorescent intensity was increased by 118-fold (Fig. 1). The fluorescent intensity increased quite fast in the first 4 min, after which the increase rate fell a little. After 10 min, the fluorescent intensity was almost linear to the time with a correlation coefficient of 0.9990. And when extended to 120 min, the fluorescent intensity even increased by 441-fold (fluorescence quantum yield: Φ = 0.6780, in CH₃OH/PBS buffer, 10 mM, pH = 7.4, 5/95, ESI†). The fluorescent intensities in the following experiments were recorded in 5 min due to the rapid-response of ACF for H₂O₂. Then the effect of pH on the fluorescence of ACF was evaluated, which showed it was stable from pH 6 to 8, even in the presence of H₂O₂ (Fig. S1, ESI†).

Fig. 1 (A) Time-dependent fluorescence spectra of ACF (5 μM) with H₂O₂ (80 eq., 400 μM) in CH₃OH/PBS buffer (10 mM, pH = 7.4, 5/95). (B) Line chart. Inset: enlarged view of time area in the first 6 min. λ_ex = 450 nm, λ_em = 527 nm. Slits: 5/5 nm.

To estimate the selectivity of probe ACF for H₂O₂, fluorescence responses to other ROS were examined. As shown in Fig. 2, significant fluorescence enhancement was observed after incubation with H₂O₂ for 5 min. Compared to H₂O₂, other ROS induced only negligible fluorescence enhancements under the same condition, including t-BuOOH, hydroxy radical, CH₃CO₃H, and so on. Considering 2-azidomethylbenzoate had been used as a H₂S trap,²⁵ H₂S was also examined. It is true that fluorescence enhancement is observed after incubation with H₂S. But the increment was far less than that of H₂O₂ (Fig. 2).

Fig. 2 (A) Fluorescence response of ACF (5 μM) incubated with ROS and H₂S (80 eq.) in CH₃OH/PBS buffer (10 mM, pH = 7.4, 5/95) for 5 min. (B) Bar graph. 1. Blank; 2. H₂O₂; 3. Na₂S; 4. tBuOO·; 5. CH₃CO₃H; 6. tBuOOH; 7. HO·; 8. NaOCl. λ_ex = 519 nm, λ_em = 527 nm. Slits: 5/5 nm.

To further estimate the selectivity of probe ACF for H₂O₂, time-dependent fluorescence changes to ROS and H₂S were recorded. As shown in Fig. 3, the fluorescence enhancements for other ROS were still negligible in 120 min. While the enhancement for H₂S in 30 min was obvious. However, it was not strong enough to obstruct the detection of H₂O₂ (the fluorescent intensity for H₂S to H₂O₂ was 1 : 9.6). What was more, the fluorescent intensity for H₂O₂ increased obviously with the time, while that for H₂S almost ceased. Thus, probe ACF showed high selectivity toward H₂O₂.

Fig. 3 Time-dependent fluorescence spectra of ACF (5 μM) with ROS and H₂S (80 eq., 400 μM) in CH₃OH/PBS buffer (10 mM, pH = 7.4, 5/95). Time points represent 30, 60, 90, and 120 min. λ_ex = 450 nm, λ_em = 527 nm. Slits: 5/5 nm.

Subsequently, we examined the reactivity of ACF towards different concentrations of H₂O₂ in CH₃OH/PBS buffer (10 mM, pH = 7.4, 5/95) at 25 °C. As expected, after incubation with H₂O₂, the fluorescence intensity of probe ACF increased gradually as the increase of the H₂O₂ amounts. Fig. 4 showed the fluorescence intensity of probe ACF increased almost linearly with the concentration of H₂O₂ in the range of 50–400 μM, and a correlation coefficient of 0.9901. Specifically, the detection limit of H₂O₂ was determined to be 6.5 nM based on the 3σ/slope method (ESI†), which was much lower than those of the reported probes. The good linearity indicated that probe ACF was able to qualitatively and quantitatively determine the level of H₂O₂.

Fig. 4 (A) Fluorescence spectra of ACF (5 μM) incubated with different concentrations of H₂O₂ (0–200 eq., 0–1.0 mM) in CH₃OH/PBS buffer (10 mM, pH = 7.4, 5/95) for 5 min. (B) Linear fitting chart. λ_ex = 450 nm, λ_em = 527 nm. Slits: 5/5 nm.

Next, we carried out competition experiments in the presence of ROS and H₂S (Fig. S2, ESI†). ACF was still able to respond to H₂O₂ with strong fluorescence enhancements in the presence of the interfering species. Moreover, the process of ACF for detection of H₂O₂ was confirmed by the HRMS-ESI spectra. The mass signal for 2′,7′-dichlorofluorescin ([M + H]⁺ calcd for C₂₀H₁₁Cl₂O₅⁺, 400.9978, found: 400.9962, Fig. S3, ESI†), was detected after probe ACF was incubated with H₂O₂. So the ester bond was possible broken by the nucleophilic substitution induced by H₂O₂. A controlled experiment showed that the probe would completely lose its effect if the azido group was instead by a hydrogen atom, which proved the importance of the azido group. Though the exact mechanism and the special effect of azido group were not clear, the in-depth mechanism study is in progress.

Encouraged by the above excellent results, we subsequently explored the potential applications of ACF for monitoring and imaging of H₂O₂ in living cells. Firstly, the cytotoxicity of ACF was evaluated using A-549 cells and Raw 264.7 cells (obtained from the College of Life Science, Nankai University, Tianjin, China; serum was purchased from Gibco) by MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Fig. S4, ESI†]. Probe ACF showed almost no cytotoxicity in 0.1–30 μM range to both of them, implying that the probe was suitable for bioimaging of H₂O₂ in living cells. Finally, we assessed the application of the probe for monitoring and imaging of H₂O₂ in living cells. HeLa cells (obtained from the College of Life Science, Nankai University, Tianjin, China) incubated with ACF (10 μM) in culture medium for 15 min at 37 °C, showed almost no fluorescence (Fig. 5B). However, if the cells were pre-treated with ACF (10 μM) for 15 min and then incubated with H₂O₂ (10 eq., 100 μM) for 15 min, strong fluorescence was observed (Fig. 5E). The obvious fluorescent enhancement indicated that probe ACF could image H₂O₂ in living cells. ACF responded to H₂O₂ only in 15 min, making the detection get close to real-time monitoring.

Fig. 5 Bright-field (A), fluorescence image (B) and the overlay (C) of (A) and (B) of HeLa cells incubated with ACF (10 μM) for 15 min. Bright-field (D), fluorescence image (E) and the overlay (F) of (D) and (E) of HeLa cells incubated with ACF (10 μM) for 15 min and washed with PBS three times. After replacement of the medium, cells were incubated with H₂O₂ (10 eq., 100 μM) for another 15 min.

Conclusions

In conclusion, aiming at finding rapid-response fluorescent probes for detection of H₂O₂, a new probe 2′,7′-dichloro-3′,6′-bis(2-(azidomethyl)benzoate)fluorescein (ACF) was developed. ACF can rapidly respond to H₂O₂ and offer highly sensitivity and selectivity by utilizing the unique chemical reactivity of the ester bonds and H₂O₂. Preliminary fluorescence imaging experiments indicate that ACF is a good fluorescent tool for rapidly monitoring H₂O₂ in living cells. We believe the novel H₂O₂ response site will be broadly used for quantitatively monitoring of H₂O₂ in biological systems. Relevant studies on this strategy and its biological applications are underway.

Acknowledgements

This work was supported by the National Science Foundation of China (21402064), the startup fund of University of Jinan, the Doctoral Fund of University of Jinan (160080304) and the Science Foundation for Post Doctorate Research from the University of Jinan (1003814).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra12984f

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