Fluorogenic sensing of H₂S in blood and living cells via reduction of aromatic dialkylamino N-oxide

Abstract

A fluorescent chemodosimeter, N-oxide of 7-diethylamino-4-methylcoumarin, was developed for H₂S detection based on a new water soluble aromatic dialkylamine N-oxide group and their selectivity and sensitivity for fluorescent detection of H₂S were evaluated in aqueous solution and blood media. Fluorescence sensing of H₂S was also achieved in MCF-7 cells.

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Hydrogen sulfide (H₂S) is a well-known toxic gas that is often present in the environment and industrial processing. Recent studies have demonstrated that endogenous H₂S in living systems is one of the gaseous transmitters, along with nitric oxide (NO) and carbon monoxide (CO) that exhibit beneficial effects in a number of pathophysiological conditions.¹ Different endogenous sulfide concentrations have been reported in the literature, suggesting large variation of the sulfide concentrations in blood due to the interference of other analytes and/or different sample processing methods.² Therefore, a direct and efficient method for sensitively and selectively probing H₂S in biological systems is highly required.

Current methods including colormetric,³ electrochemical,⁴ gas chromatography assays⁵ and metal-induced precipitation⁶ were reported in literature for H₂S detection. Due to the high sensitivity, nondestructivity, and high spatiotemporal resolution, fluorescence-based assays have exhibited widespread applications especially in biological systems. In the past few years, fluorescent probes used for fast and real time H₂S detection in biological systems were based on H₂S-triggered specific reaction including the reduction of azido,⁷ the reduction of nitro or hydroxylamine groups,⁸ the reduction of organic selenium oxide,⁹ nucleophilic reaction,¹⁰ high binding affinity towards copper ions¹¹ and efficient thiolysis of dinitrophenyl ether.¹² However, probes bearing these functional group usually have the low solubility in aqueous solution which needs organic solvents for H₂S detection. This limitation impeded their further applications in biological systems. Wu et al.¹³ reported two kinds of H₂S sensors in which chemofluorophores were respectively conjugated to poly(ethylene glycol) and carbon dots to improve water-solubility. However, this conjugation increased the size of sensors. In addition, most previous sensors always involved two or more synthetic steps. Herein, we developed a new H₂S probe bearing a dialkylamine N-oxide group that was readily synthesized in single step with available fluorophores under a mild reaction condition and maintained high solubility in aqueous solution. Moreover, detection of H₂S based on reduction of aromatic dialkylamine N-oxide group was not reported.

The rational design of this new H₂S sensitive functional group for commercially available fluorophores is depicted in Scheme 1. Two kinds of N–O functional moieties were introduced to 7-diethyl-4-methylcoumarin and resorufin, respectively. Since the formation of N–O bond consumes the electron pair of nitrogen atom, it breaks the electron conjugation of nitrogen and aromatic moiety which can quench the fluorescence of fluorophores. Upon reaction with H₂S in pH 7.4 PBS buffer, aromatic dialkylamine N-oxide was subsequently reduced and highly fluorescent species were observed. To determine the fluorescent species, the product of COU reaction with NaHS was purified and then analyzed by ¹HNMR and ¹³CNMR which were in consistent with the NMR spectra of 7-diethylamino-4-methylcoumarin (Fig. S2 and S3, ESI†). Further HRMS analysis confirmed the product to be 7-diethylamino-4-methylcoumarin (Fig. S4, ESI†). For commercially available resazurin, the addition of NaHS did not induce the change of absorbance and fluorescence spectra under physiological conditions, indicating that resazurin was more difficult to be reduced than aromatic dialkylamine N-oxide by NaHS and was not suitable for H₂S sensing (Fig. S7, ESI†).

Scheme 1 Reduction of N–O chemical bond of fluorophore for H₂S detection.

To access the applicability of H₂S sensing assay with dialkylamine N-oxide moiety, COU (5 μM) was incubated with NaHS (40 μM) in pH 7.4 solution. As shown in Fig. 1, a robust increase in fluorescence intensity was observed. Within 60 min of reaction time under above conditions, nearly 10-fold turn-on fluorescence response was observed with the probe COU.

Fig. 1 (A) Fluorescence response of COU (5 μM) after the addition of NaHS (40 μM) in different incubation time; (B) time-dependent fluorescence intensity change in the absence (solid line) and presence (dash line) of 40 μM NaHS. Time points represent 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 min. λ_ex = 360 nm, λ_em = 455 nm. Slits: 5/10 nm.

Subsequently, we assayed the sensitivity of COU for H₂S detection. Various concentrations of NaHS were added to a series of degassed PBS solutions containing the probe (100 μM), and the fluorescence intensity of the resulting reaction solutions was analyzed. Standard curve for COU in degassed PBS was gained between fluorescence signal at 455 nm and the concentrations (μM) of H₂S in 60 min incubation. The regression equation was F_{Ex/Em(360/455 nm)} = 100.496 [H₂S] + 483.612 with R² = 0.9930, suggesting a good linear relationship between turn-on fluorescence intensity of COU at 455 nm and NaHS concentration in degassed PBS buffer (Fig. 2). The detection limit is nearly down to 0.2 μM.

Fig. 2 H₂S concentration-dependent fluorescence intensity of COU (100 μM) to NaHS. The fluorescence intensity at 455 nm was recorded. Data was acquired after incubation 60 min. λ_ex = 360 nm, λ_em = 455 nm, Cut off: 420 nm, PMT: medium.

Instead of PBS buffer, we also applied this fluorescent chemodosimeter to the determination of H₂S in blood. A standard addition procedure was used in the experiment (see ESI† for the detail). A linear relationship of COU turn-on fluorescence signal at 460 nm and the concentrations (μM) of H₂S in 60 min incubation was also obtained under 25 μM H₂S in mouse blood serum and human blood plasma (Fig. 3). The regression equations were F_{Ex/Em(355/460 nm)} = 67.672 [H₂S] + 101.290 with R² = 0.9680 and F_{Ex/Em(355/460 nm)} = 65.836 [H₂S] + 66.867 with R² = 0.9922 for mouse blood serum and human blood plasma, respectively.

Fig. 3 H₂S concentration-dependent fluorescence intensity of COU (100 μM) in mouse blood serum (blue line) and human blood plasma (red line). λ_ex = 355 nm, λ_em = 460 nm; gain: 650.

To evaluate the specific selectivity of COU for H₂S, fluorescence enhancement (F/F₀) of COU incubated with relevant species including various anions (1 mM) and biothiols (5 mM, such as Cys, Hcy and GSH) was investigated. As can be shown in Fig. 4, no significant fluorescence change was found upon reaction with these species and only NaHS (40 μM) was found to induce dramatic increase in fluorescence intensity, suggesting high selectivity for H₂S over other biothiols with concentration up to 5 mM.

Fig. 4 Fluorescence enhancement (F/F₀) of COU (5 μM) in the presence of biological relevant species in pH 7.4 buffer (20 mM): (1): control, (2): Hcy (5 mM), (3): Cys (5 mM), (4): GSH (5 mM), (5): SO₄²⁻ (1 mM), (6): HCO₃²⁻ (1 mM), (7): N₃⁻ (1 mM), (8): H₂PO₄⁻ (1 mM), (9): NO₂⁻ (1 mM), (10): CO₃ ²⁻ (1 mM), (11): Br⁻ (1 mM), (12): Cl⁻ (1 mM), (13): citrate (1 mM), (14): AcO⁻ (1 mM), (15): HS⁻ (40 μM). Data was acquired after incubation 60 min. λ_ex = 360 nm, λ_em = 455 nm, Cut off: 420 nm, PMT: medium.

Cell viability indicated that no obvious toxicity was observed up to 200 μM COU (Fig. S9†). To further evaluate the feasibility of H₂S detection in living cells, MCF-7 cells were incubated with COU for 30 min, and were then treated with NaHS 250 μM for 30 min. The cells were washed to remove extracellular reagents and then visualized by fluorescence microscopy. As displayed in Fig. 5, control cells treated with only COU remained very weak fluorescence in the absence of NaHS treatment. While fluorescence intensity in MCF-7 cells became much stronger after cells were treated with NaHS for 30 min, confirming the capability of COU in sensing of H₂S in living cells.

Fig. 5 Imaging of H₂S in MCF-7 cells with COU excitated at 405 nm. MCF-7 cells were seeded on glass coverslips and then incubated with COU (100 μM) for 30 min. The cells were washed and then treated with NaHS (250 μM) for 30 min. (A) DIC image of control cells with COU. (B) Fluorescence image of control cells with COU. (C) overlay of A and B. (D) DIC image of COU-loaded cells after treatment with NaHS. (E) Fluorescence image of COU-loaded cells in the presence of NaHS. (F) Overlay of D and E. Fluorescence images were set as pseudo-color green. Scale bar: 80 μm.

In summary, a novel selective fluorescent probe based on dialkylamino N-oxide was developed for the detection of H₂S in aqueous solutions. This new H₂S sensitive functional dialkylamine N-oxide group with positive charge facilitate its high solubility in aqueous buffer and accumulation in cells. Synthesis of this kind of probe needs only one-step from commercially available fluorophore under mild conditions in the presence of m-CPBA. The new sensor COU has the high solubility in aqueous solutions with no need of other soluble functionization. Fluorescence assay indicated that linear relationships between fluorescence intensity and sulfide concentrations were obtained in buffer, blood plasma or blood serum systems. This probe with high water solubility, sensitivity and selectivity has the potential to be a useful tool for detection of sulfide in many biological systems.

Acknowledgements

This work was supported by the National Basic Research Program of China (973 Program; Grant no. 2013CB933800), the National Natural Science Foundation of China (Grant no. 21372018).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: The detailed synthesis and characterization of COU and data of fluorescence response to H₂S See DOI: 10.1039/c4ra03677h

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