Selective and sensitive detection of hydrogen sulfide in live cells

Abstract

We report herein a fluorescence switch-on probe suitable for the detection of hydrogen sulfide (H₂S) in complex biological systems. The probe features a straightforward synthesis, high sensitivity and good selectivity. As a proof of concept, its capability for detecting biological H₂S has been illustrated by imaging H₂S in live cells.

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The wide distribution of hydrogen sulfide (H₂S) in mammals indicates its potential use in biological functions. Actually, numerous researchers have demonstrated that the toxic H₂S is indeed playing a pivotal role in the controllable regulation of various intracellular signaling pathways, and the modulation of various pathological and physiological processes in human biology.¹ H₂S is primarily synthesized from cysteine (Cys), thiocysteine, 3-mercaptopyruvate or other sulfur derivatives, under the enzymatic catalysis of cystathionine β synthetase (CBS), cystathionine γ lyase (CSE), and so on.² As a gasotransmitter, its imbalance during homeostasis is involved in the progress of diverse diseases such as atherosclerosis,³ osteoarthritis,⁴ Alzheimer’s disease,⁵ Parkinson’s disease,⁶ etc.

Due to its elusive nature, the local concentration of H₂S in the biological sample is hard to analyze. However, the precise detection of the concentration of H₂S at the site of action will lay a foundation for revealing its exact biological role in the human body as well as furthering its biomedical applications. Traditional gas-chromatography and sulfide precipitation methods are limited by their incompatibility with live samples.^7,8 Small molecule fluorescent probes, on the other hand, have significantly developed in the last decade, attributing to their high sensitivity, good selectivity and more importantly, being applicable for live organisms.⁹ In fact, numerous fluorescent probes have been devised based on specific probe–analyte reactions for the in situ detection of various biological molecules, among which some even hold promise for live animal imaging.¹⁰

It is common sense to chemists that H₂S is readily oxidized, favoring electrophilic reagents, and binding copper(II) with a high affinity. Full advantage of these chemical properties has been taken by researchers to develop reaction-based fluorescent probes for the sensitive detection of H₂S in complex biological environments. Pioneered by Chang’s group, who used an azide group which is reducible by H₂S to mask the fluorescence of rhodamine,¹¹ the azide group has been widely employed as a reaction trigger for sensing H₂S.¹² Inspired by the azide–H₂S chemistry, azo-, nitro-, and hydroxyamino-based probes were also developed.^13–15 Besides its reducibility, the nucleophilic reactivity of H₂S was also fully exploited for probe design.^16,17 Though other biothiols were widely present, the capability of H₂S for two sequential nucleophilic reactions simplifies the challenge of H₂S-selective probes, which can be constructed by installing two adjacent electrophilic functionalities, usually moderately reactive ones such as benzene aldehyde and Michael receptors, to the photo-property-adjusting part of a fluorophore.¹⁶ H₂S can also be fluorogenically detected by relying on a Cu²⁺-bound and thereby quenched fluorophore,¹⁸ attributed to the especially low solubility of CuS. Actually, there have been several elegant review papers focusing on recent research efforts towards reaction-based fluorescence sensing of H₂S in biological samples.¹⁹ Among the various reaction triggers that respond to H₂S, the azide group, though widely used, is sometimes limited by the potential hazard of photoactivation due to its photosensitivity.²⁰ The nucleophilicity-based two adjacent electrophiles should also be used with caution because they are also susceptible to strong oxidants such as reactive oxygen or nitrogen species that widely exist in biological settings.²¹ CuS precipitation-based probes, though being able to sense S²⁻ with rapid kinetics, sometimes don’t show an ideal cell permeability due to their ionic structure. Therefore, new probes employing proper sensing groups which are both orthogonal to the biological environment and can react specifically and sensitively to H₂S are still highly desirable.

To devise new probes, it is primarily important to understand chemistry of H₂S. H₂S, along with its reactivity of undergoing two sequential nucleophilic reactions, differs from other biothiols due to its small size. These structural features render it much more nucleophilic than other biothiols. Actually, the nucleophilicity of H₂S is strong enough to thiolyze dinitrophenyl ether,²² a formidable mission for other biothiols. Since the dinitrophenyl group is photostable and insensitive to oxidants, along with having an excellent fluorescence quenching capacity, its thiolysis by H₂S therefore offers a valuable opportunity for the design of H₂S-specific fluorescence switch-on probes.

Lin’s group first reported a 2,4-dinitrophenyl ether-based H₂S probe in 2012 and the probe was developed by installing this functionality into the meso position of BODIPY.²³ To bathochromic shift the emission of the probe, the π system of the fluorophore was extended by condensation of the 3-methyl group with a Fisher aldehyde. Though this probe was spectroscopically silent to the reactive oxygen/nitrogen species examined in their study, the electron-rich alkene attached to the indoline moiety may be susceptible to highly reactive oxidative species, such as peroxynitrite, etc.²⁴ Alternatively to the incorporation of the Fisher aldehyde, another strategy to red shift the emission of the probe is to fix the 2,4-dinitrophenyl group directly into the 3-position of BODIPY via a phenol linker. It has been well documented that the 3-methyl group of BODIPY readily undergoes a Knoevenagel-type reaction with electron-rich benzaldehydes to furnish red-shifted fluorophores with high yields.²⁵ Employing this reaction, we have successfully fused some specific sensing warheads to the 3-position of BODIPY and developed several highly sensitive fluorescence probes for the detection of H₂S and other biothiols.²⁶ With these considerations, we have designed the probe PS1. We envisioned that the 2,4-dinitrophenyl group, with its strong electron-withdrawing effect, could effectively quench the emission of the BODIPY core via photo-induced electron transfer (PeT).²⁷ While nucleophilic cleavage of the ether bond by H₂S would eliminate this quenching effect and thereby restore the strong emission, signaling the distribution and the concentration of H₂S.

The probe PS1 was facilely synthesized via nucleophilic substitution of 3,4-dinitrofluorobenzene by the BODIPY fluorophore 1 (Scheme 1), with the latter prepared according to literature procedures.²⁸ Its structure was carefully confirmed by NMR and HRMS analysis, and the purity was monitored by fluorescence analysis.

Scheme 1 Structure of PS1 and its synthesis.

With PS1 in hand, we first tested its fluorescence response towards H₂S under pseudo biological conditions with the presence of cetyltrimethyl ammonium bromide (CTAB) as a cationic surfactant.²³ As shown in Fig. 1, PS1 alone (5 μM) in PBS (10 mM, pH 7.4, with 5% ethanol, 37 °C) was only weakly emissive. While treating the probe with NaHS (500 μM) as the H₂S source, a time-dependent increase of the fluorescence intensity was triggered. The time-lapsed increase of the fluorescence intensity at the emission maximum (570 nm) reached a plateau in about 10 minutes, indicating the fast kinetics of the detection reaction.

Fig. 1 The fluorescence switch-on response of PS1 towards NaHS. (a) Fluorescence spectra of PS1 (5 μM) before and after treatment with NaHS (500 μM) for various times. (b) Time-dependent fluorescence intensity enhancement of PS1 at 570 nm after treatment with NaHS (500 μM). Measurements were conducted in PBS (10 mM, pH = 7.4, 5% ethanol, 37 °C) with CTAB (100 μM). λ_ex = 535 nm. Slit widths: 10 nm for excitation and 5 nm for emission.

Then, we evaluated the feasibility of the probe PS1 to quantify H₂S. For this purpose, the fluorescence responses of PS1 (5 μM) in PBS (10 mM, pH 7.4, with 5% ethanol, 37 °C) towards NaHS at various concentrations were recorded (Fig. 2a and S1†). Much to our delight, the logarithm of the fluorescence intensity difference between the maximum intensity and that recorded at a given NaHS concentration was linear for the concentration of NaHS ranging from 0–80 μM with a correction coefficient of 0.991 (Fig. 2b), implying the potential of PS1 to quantify H₂S. The detection limit was determined to be 500 nM (Fig. S2†).

Fig. 2 The fluorescence response of PS1 (5 μM) towards various concentrations of NaHS. (a) The fluorescence spectra of PS1 after treatment with various concentrations of NaHS for 20 min. (b) Plot of ln(F_max − F) versus the concentration of NaHS, where F_max is the maximum fluorescence intensity of PS1 at 570 nm after treatment with excess NaHS for 20 min, and F is the intensity at the same wavelength of PS1 in the presence of NaHS at a given concentration. Data were collected in PBS (10 mM, pH = 7.4, 5% ethanol, 37 °C) with CTAB (100 μM) as the cationic surfactant. λ_ex = 535 nm. Slit widths: 10 nm for excitation and 5 nm for emission.

Later on, the selectivity of PS1 was examined to determine if the fluorescence switch-on response of this probe was specific towards NaHS. Since biothiols are often regarded as the main interference for the detection of H₂S, as has been reported that the selectivity of some reduction-based H₂S probes do suffer from the interference from Cys and GSH,^29,30 we were especially concerned about the selectivity of PS1 towards various biothiols. It turned out that among the various biothiols tested, only NaHS could trigger the fluorescence intensity enhancement of PS1. Moreover, to further confirm the specificity of PS1 towards H₂S over the other amino acids, cations or anions which are widely present in complex biological systems, its fluorescence response towards these analytes was also studied and it turned out that none could switch on the fluorescence of PS1, indicating the high sensitivity of this probe towards H₂S. What is more, PS1 could still respond to NaHS with a dramatic fluorescence increase in the presence of other analytes, implying the potential of PS1 to detect H₂S in complex biological environments (Fig. 3).

Fig. 3 PS1 is highly selective towards H₂S over other bio-relevant molecules. (a) Fluorescence spectra of PS1 (5 μM) after treatment with various analytes (500 μM) for 20 min. (b) The fluorescence intensity of PS1 (5 μM) at 570 nm after treatment with NaHS (1), Ala (2), Cys (3), 2-mercaptoethanol (4), Gly (5), GSH (6), Hcy (7), Cu²⁺ (8), Mg²⁺ (9), Fe²⁺ (10), Ca²⁺ (11), Na⁺ (12), K⁺ (13), Zn²⁺ (14), Fe³⁺ (15), CN⁻ (16), F⁻ (17), SO₃²⁻ (18), ethanethiol (19), and H₂O₂ (20) for 20 min with or without the presence of NaHS (500 μM). Data were obtained in PBS (10 mM, pH = 7.4, 5% ethanol, 37 °C) with CTAB (100 μM) as the cationic surfactant. λ_ex = 535 nm. Slit widths: 10 nm for excitation and 5 nm for emission.

With the fluorescence response of PS1 towards NaHS carefully characterized, we finally tested its feasibility to image H₂S in live cells. For this purpose, HeLa cells were chosen as the model cell line. After loading the cells with PS1 for 15 min, the cells were washed with fresh PBS twice and then various concentrations of NaHS (0, 100, 200, 300 μM) were added to the different wells. After one hour of further incubation, the cells were washed quickly with fresh PBS twice and imaged with fluorescence microscopy. As shown in Fig. 4, intact HeLa cells showed only negligible fluorescence. While those cells treated with exogenous NaHS showed an obvious fluorescence enhancement and the increased tendency was positively related to the concentration of NaHS in the culture medium. These results, on the one hand indicate the good cell-permeability of PS1 and its capacity to image H₂S in live cells, on the other hand they suggest that endogenous H₂S in HeLa cells, if there is any, is expressed at levels below the detection limit of PS1.

Fig. 4 Imaging H₂S in HeLa cells with PS1. Intact HeLa cells were treated with PS1 (5 μM) for 30 min. After two quick washes, the cells were then treated with 0 μM (a), 100 μM (b), 200 μM (c), and 300 μM (d) of NaHS. After a further incubation of one hour, the cells were quickly washed with fresh PBS and observed under a LEICA DMI 4000B fluorescence microscope equipped with a Cy3 filter set.

Conclusions

Altogether, by employing a dinitrophenyl ether functionality as both a fluorescence quencher and a H₂S-reaction trigger, we have developed a probe for the fluorescence switch-on detection of H₂S. The probe features a fast response and good selectivity towards H₂S. It is also easily available via a one-step synthesis. Its capability to image H₂S in live cells has been demonstrated.

Acknowledgements

This work was supported by Zhejiang Provincial Natural Science Foundation of China (LY15H300003).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Synthesis and characterization of PS1. See DOI: 10.1039/c5ra11590f

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