A novel FRET-based ratiometric fluorescent probe for highly sensitive detection of hydrogen sulfide

Abstract

A novel FRET-based ratiometric fluorescent probe H₂S-CR for the quantitative detection of H₂S was designed and synthesized. It exhibits a response time of 20 min, a considerable fluorescence signal enhancement (15 fold), and an extremely low detection limit (19 nM). It can be successfully applied to imaging H₂S in living cells.

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As a member of the reactive sulfur species (RSS) family, hydrogen sulfide (H₂S) has been regarded for a long time as a toxic pollutant with the typical smell of unpleasant rotten eggs. However, more recent studies on endogenous H₂S have challenged this traditional view of H₂S as a toxin and suggested that H₂S is the third member of the gasotransmitter family, in addition to nitric oxide (NO) and carbon monoxide (CO), existing in the human body and other biological systems.¹ Furthermore, H₂S at physiological concentration appears to be involved in various physiological processes, including regulation of cell growth,² stimulation of angiogenesis,³ modulation of neuronal transmission,⁴ the anti-inflammation effect, etc.⁵ Concurrently, abnormal H₂S levels are engaged in diseases such as Alzheimer's disease,⁶ Down's syndrome,⁷ gastric mucosal injury,⁸ diabetes,⁹ liver cirrhosis, and etc.¹⁰ Therefore, highly sensitive and selective detection of H₂S and direct indication of its concentration in living systems are crucial for the better understanding of its related physiological and pathological functions.

In the past decades, the detection of H₂S has attracted a wide research interest, and some strategies including metal-induced sulfide precipitation,¹¹ colorimetric assays,¹² electrochemical analysis¹³ and gas chromatograph¹⁴ have been developed. Among them, fluorescence-based detection with a fluorescent probe is extremely attractive because of its simplicity, real-time imaging, and nondestructive detection of intracellular biomolecules.^15–22 Recently, a number of fluorescent probes for H₂S have been constructed using the special properties of H₂S, such as its dual nucleophilicity,¹⁵ good reducing property toward azides,¹⁶ nitros¹⁷ and hydroxyl amines,¹⁸ high binding affinity with copper and zinc ions,¹⁹ efficient thiolysis of dinitrophenyl ether²⁰ as well as specific Michael addition reaction towards unsaturated double bonds.²¹ Besides, as far as we know, the probes utilizing the unique dual nucleophilic character of H₂S exhibited superior selective advantage because the potential interference from bio-thiols can be well excluded.¹⁵ Usually, such fluorescent probes contain a potential fluorescent group and a specific H₂S trap group with two nucleophilic reaction sites.

Although these probes utilizing the unique dual nucleophilic character of H₂S are innovative and effective, some improvements are still needed. One of the main concern using this strategy is how to avoid the probe consumption by biothiols,^15b which would otherwise lead to high probe loading and low sensitivity. Another concern is that most of these probes exhibit a response to H₂S with changes only in fluorescence intensity at a single wavelength,^15a,c,d and single increase or decrease emission detection is sometimes problematic for precise fluorometric analysis because fluorescence intensity can be affected by variables such as excitation intensity variations, environmental factors, light scattering, probe concentrations, and etc. Moreover, the development of novel fluorescent probes for H₂S detection with better optical properties and higher sensitivity is of pressing need. Herein, we reported the design and synthesize of a novel ratiometric fluorescence probe H₂S-CR (Scheme 1) based on a H₂S induced Michael addition–cyclization cascade reaction and the FRET modulated fluorescence process. It exhibits a response time of 20 min, a considerable fluorescent signal enhancement (15-fold), and an extremely low detection limit (19 nM) toward H₂S. Moreover, we successfully applied it to image the change of H₂S level in living cells.

Scheme 1 The proposed mechanism of ratiometric fluorescent probe H₂S-CR for H₂S detection.

As shown in Scheme 2, probe H₂S-CR can be conveniently prepared from 4-(diethylamino)-2-hydroxybenzaldehyde by a four-step procedure under mild conditions with a good yield. The coumarin donor building block 7 and rhodamine/fluorescein acceptor building block 8 were synthesized by a reported synthetic procedure.²³ The FRET dyad CR was prepared by the condensation of carboxyl compound 7 with amide 8. Finally, we transformed the compound CR to probe H₂S-CR. The structural characterization of the probe was characterized by standard ¹H NMR, ¹³C NMR, HRMS, and etc. (Fig. S3†).

Scheme 2 The synthesis of the probe H₂S-CR.

We first evaluated the effect of buffer solution to the fluorescence of the probe. As shown in Fig. S5,† the probe could work well in phosphate buffered solutions. The photophysical properties of the probe (10 μM) were investigated under simulated physiological conditions (30 mM, pH 7.4, 1 : 9 v/v CH₃CN–PBS). Prior to reaction with NaHS, probe presented a fluorescence maximum at 470 nm (Fig. 1A) with a corresponding major absorption band centered at 408 nm (Fig. 1B) (Φ = 0.132). With the addition of NaHS (80 μM) into the solution of probe, the fluorescence emission intensity at 470 nm gradually decreased within 12 min, along with a time-dependent fluorescence emission intensity increase centred at 541 nm (Fig. 1A) (Φ = 0.100). Simultaneously, there emerged a new absorption peak at 511 nm, accompanied by a dramatic change in the probe solution from colorless to bright orange. Furthermore, we performed the time-dependent fluorescent spectra studies. As shown in Fig. 2, although 80 μM NaHS could cause the reaction to be completed within 12 min, the low concentrations of NaHS needed the longer reaction time (20 min) to reach the fluorescence intensity ratio (I₅₄₁/I₄₇₀) saturation. Thus, the time point after the addition of NaHS was selected to be 20 min in the subsequent experiments.

Fig. 1 (A) Fluorescence responses of 10 μM probe to 80 μM NaHS. (B) Time-dependent absorption spectra of probe (10 μM) in the presence of 80 μM NaHS. Conditions: excitation wavelength is 414 nm, acetonitrile–PBS buffer solution (30 mM, pH 7.4, 1 : 9 v/v) at 25 °C.

Fig. 2 Time-dependent fluorescence intensity changes of probe (10 μM) at 541 nm upon addition of varied concentrations of NaHS. Conditions: excitation wavelength is 414 nm, acetonitrile–PBS buffer solution (30 mM, pH 7.4, 1 : 9 v/v) at 25 °C.

To gain insight into potential of H₂S-CR as a probe for H₂S, a titration experiment was performed under simulated physiological conditions. Accordingly, as shown in Fig. 3A, upon excitation at 414 nm, the fluorescence intensity around 470 nm decreased along with the incremental addition of NaHS, and simultaneously a new emission band around 541 nm gradually increased. The fluorescence intensity could reach saturation when the addition amount of NaHS reached 9 equiv. of the probe (Fig. 3B). At this amount, the ratio of the fluorescence emission intensities at 541 and 470 nm (I₅₄₁/I₄₇₀) exhibited a drastic change from 0.15 in the absence of NaHS to 2.3 after complete conversion, a 15-fold enhancement in the I₅₄₁/I₄₇₀ ratios. This suggested that the excitation energy of the coumarin donor is efficiently transferred to the rhodamine/fluorescein acceptor. The intramolecular energy transfer efficiency from the coumarin donor to the rhodamine/fluorescein acceptor in H₂S-CR was calculated to be 70.7% (in ESI†). The detection limit (S/N = 3) of the ratiometric probe was determined to be 19 nM (in ESI†). Additionally, a standard curve between emission intensity ratio (I₅₄₁/I₄₇₀) and NaHS concentration was set up. To our satisfaction, as shown in Fig. 3B, a good linearity between the fluorescence intensity ratio (I₅₄₁/I₄₇₀) and the NaHS concentration in the range of 0–50 μM was observed, suggesting that H₂S-CR is potentially useful for quantitative determination of NaHS.

Fig. 3 (A) Fluorescence response of 10 μM probe in the presence of 0–10 equiv. of NaHS. (B) Fluorescence ratio (I₅₄₁/I₄₇₀) of probe (10 μM) in the presence of 0–10 equiv. of NaHS. Conditions: excitation wavelength is 414 nm, acetonitrile–PBS buffer solution (30 mM, pH 7.4, 1 : 9 v/v) at 25 °C for 30 min.

Based on the well-established dual nucleophilicity mechanism, the mechanism has been proved by reaction between H₂S-CR and NaHS, and reaction product coumarin–rhodamine/fluorescein and cyclization product were confirmed by HPLC-MS experiments (Fig. S6†).

To study the specificity of H₂S-CR towards NaHS, an important test was performed to determine whether biological species other than NaHS could potentially introduce signal response (Fig. 4). As expected, H₂S-CR was considerably inert to the common cations and anions, such as Na⁺, Mg²⁺, Ca²⁺, Zn²⁺, Cu²⁺, Cu⁺, K⁺, Fe³⁺, and Fe²⁺ (4 mM for each) (Fig. 4A); F⁻, Cl⁻, I⁻, AcO⁻, N₃⁻, CO₃²⁻, NO₃⁻, NO₂⁻, SO₄²⁻, and HCO₃⁻ (4 mM for each) (Fig. 4A). The results revealed that the NaHS-induced ratiometric fluorescence response is hardly affected in response to reactive oxygen/nitrogen species (ROS/RNS), such as H₂O₂, NO, ROO˙, ˙O²⁻, ClO⁻, ˙OH, ¹O₂, and CNOO⁻ (4 mM for each) (Fig. 4B), reducing condition, such as sodium ascorbate (4 mM) (Fig. 4B), and non-thiol amino acids, such as Lys, Ser, Glu, Pro, Phe, Arg, Thr and Asn (4 mM for each) (Fig. 4B). Moreover, CN⁻ (4 mM) (Fig. 4A), reactive sulfur species (SO₃²⁻, HSO₃⁻, and S₂O₃²⁻ (0.8 mM for each)) (Fig. 4B) and biothiols (GSH, Cys, and Hcy (0.8 mM for each)) (Fig. 4B) underwent limited fluorescence response. However, the fluorescence ratio (I₅₄₁/I₄₇₀) increase was far weaker than that caused by NaHS. By contrast, NaHS induced a robust increase in the fluorescence intensity ratio (I₅₄₁/I₄₇₀), and only the probe solution turned from colorless to bright orange when treated by NaHS in the selective experiment. High selectivity toward H₂S in the presence of other competitive species is a very important feature to evaluate the performance of the fluorescent probe. Therefore, the competition experiments were also conducted when CN⁻/biothiols/(reactive sulfur species) and NaHS co-existed in the system. To our delighted, when NaHS and these competitive species coexisted, almost the same I₅₄₁/I₄₇₀ signal enhancement was observed as that only treated by NaHS (Fig. S7†). Taken together, H₂S-CR can selectively respond to NaHS independently of negligible disturbance from the interference of other biological species, and it can serve as a “naked-eye” probe for colorimetric detection of H₂S (Fig. S7†).

Fig. 4 Fluorescence ratio (I₅₄₁/I₄₇₀) of 10 μM probe in an acetonitrile–PBS buffer solution (30 mM, pH 7.4, 1 : 9 v/v) towards hydrosulfide and potential interferences for 30 min. Bars represent fluorescence ratio (I₅₄₁/I₄₇₀) to each compound. (A) The fluorescence emission of probe spiked with selected cations, such as Na⁺, Mg²⁺, Ca²⁺, Zn²⁺, Cu²⁺, Cu⁺, K⁺, Fe³⁺, and Fe²⁺ (4 mM for each) and selected anions, such as F⁻, Cl⁻, I⁻, AcO⁻, N₃⁻, CN⁻, CO₃²⁻, NO₃⁻, NO₂⁻, SO₄²⁻, and HCO₃⁻ (4 mM for each). (B) Fluorescence responses of probe supplemented with reactive oxide species, such as H₂O₂, ROO˙, ˙O²⁻, ClO⁻, ˙OH, and ¹O₂ (4 mM for each), reactive nitrogen species, such as NO and CNOO⁻ (4 mM for each), reactive sulfur species, such as SO₃²⁻, HSO₃⁻, and S₂O₃²⁻ (0.8 mM for each), sodium ascorbate (4 mM) non-thiol amino acids, such as Lys, Ser, Glu, Pro, Phe, Arg, Thr and Asn (4 mM for each) and biothiols, such as GSH, Cys, and Hcy (0.8 mM for each).

To verify whether the probe is suitable for the physiological detection, we evaluated the effect of pH on the fluorescence of the probe. As shown in Fig. S8,† in the absence of NaHS, almost no change in fluorescence ratio (I₅₄₁/I₄₇₀) was observed in the free probe over a wide pH range of 2–11 indicating excellent pH stability. Furthermore, upon treatment with NaHS, the maximal fluorescence ratio (I₅₄₁/I₄₇₀) displayed constant in the pH range of 6–11. Thus, the observation that H₂S-CR had the maximal sensing response at physiological pH, suggested that H₂S-CR is promising for biological applications.

Having demonstrated the selectivity and sensitivity of H₂S-CR for H₂S in vitro, we next evaluated the potential utility of H₂S-CR as a probe for H₂S within living cells (Fig. 5). H9C2 cells were incubated with 10 μM H₂S-CR for 20 min at 37 °C. After washed three times with physiological saline to remove the remaining probes, the cells were then incubated with buffer containing different concentrations of NaHS (10, 20, 30, 40 and 80 μM) for 30 min. As for the control experiment, the cells untreated with NaHS were examined. The optical imaging was carried out by a fluorescent inverted microscope. A faint fluorescence was observed in the control experiments, and the lever changes were depended on the concentration of NaHS (Fig. 5). It is worth noting that the inverted fluorescence images grew brighter as the concentrations of NaHS increased from 10 to 80 μM (Fig. 5A–E). These results demonstrated that H₂S-CR is cell membrane permeable and has potential in visualizing H₂S levels change of living cells.

Fig. 5 Fluorescence response of the probe with increasing concentrations of NaHS in living H9C2 cells. The cells were pre-treated with the probe (10 μM) for 20 min, and then incubated with NaHS (A) 80 μM, (B) 40 μM, (C) 30 μM, (D) 20 μM, (E) 10 μM, (F) 0 μM, for 30 min.

Conclusions

In conclusion, with recognition of the biological significance of H₂S, we have developed a unique FRET-based ratiometric fluorescent probe H₂S-CR. Based on a H₂S induced Michael addition–cyclization cascade reaction, the probe exhibited a high selectivity and sensitivity for H₂S over other biologically relevant species, a 15-fold fluorescent signal enhancement, and an obvious colour change from colourless to bright orange. Moreover, H₂S-CR can detect H₂S quantitatively with a low detection limit up to 19 nM. Fluorescent inverted microscope images indicated that this probe can detect the level changes of H₂S in living cells. In addition, this ratiometric fluorescence probe has the potential to be a useful tool for the fast and real-time detection of H₂S in more types of biological samples.

Acknowledgements

We are grateful to National Natural Science Foundation of China (81271634), Doctoral Fund of Ministry of Education of China (no. 20120162110070), the Fundamental Research Funds for the Central Universities, Hunan Provincial Natural Science Foundation of China (12JJ1012) and Hunan Provincial Innovation Foundation for Postgraduate (CX2014B120).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra16578k

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