Highly selective fluorescent and colorimetric probe for live-cell monitoring of sulphide based on bioorthogonal reaction

Abstract

H₂S is the third endogenously generated gaseous signaling compound and has also been known to involve a variety of physiological processes. To better understand its physiological and pathological functions, efficient methods for monitoring of H₂S are desired. Azide fluorogenic probes are popular because they can take place bioorthogonal reactions. In this work, by employing a fluorescein derivative as the fluorophore and an azide group as the recognition unit, we reported a new probe 5-azidofluorescein for H₂S with improved sensitivity and selectivety. The probe shows very low background fluorescence in the absence of H₂S. In the presence of H₂S, however, a significant enhancement for excited fluorescence were observed, resulting in a high sensitivity to H₂S in buffered (10 mmol/L HEPES, pH 7.0) aqueous acetonitrile solution (H₂O/CH₃CN = 1:3, v/v) with a detection limit of 0.035 μmol/L observed, much lower than the previously reported probes. All these features are favorable for direct monitoring of H₂S with satisfactory sensitivity, demonstrating its value of practical application.

Fluorogenic probes activated by bioorthogonal chemical reactions can enable biomolecule imaging in situations where it is not possible to wash away unbound probe¹. Much work has been devoted to expanding the toolbox of bioorthogonal reactions, and these efforts can be complemented by the development of fluorogenic probes². Such probes are typically endowed with a functionality that suppresses fluorescence. Its transformation during the reaction creates a new functionality that no longer quenches the fluorescence of the underlying system, resulting in a fluorescence enhancement. Such probes offer significant advantages for imaging studies in which it is not possible to wash away unreacted probe, such as real-time imaging of dynamic processes in cells or visualization of molecules in live organisms.

One of the most widely used bioorthogonal reactions is the azide−alkyne [3 + 2] cycloaddition to form a triazole³,⁴. This reaction has enabled the selective visualization of azide- or alkyne-labeled proteins, glycans, nucleic acids, and lipids⁴,⁵. Several azide-⁶,⁷,⁸,⁹ fluorogenic probes have been reported, largely based on coumarins⁶,¹⁰, naphthalimides⁸, and other systems that require UV excitation and emit blue light⁷,¹¹,¹². Such wavelengths are not ideal for biological imaging because of high levels of autofluorescence and poor tissue penetrance¹³.

An obvious improvement upon these designs would be the development of azido fluorogenic probes with longer excitation and emission wavelengths. Some attempts at achieving this goal have been made⁸,¹²,¹⁴. The utility of azide pairs in biological settings remains unclear. Thus, fluorogenic azido probes that perform well as cell-imaging reagents remain an important goal. Bertozzi reported the rational design and experimental validation of azide-functionalized fluorogenic probes based on the widely used blue-excitation/green-emission fluorescein scaffold¹⁵. In their work, they have prepared a series of azidefluorescein compounds under NaNO₂/NaN₃ condition, and the azidefluorescein was used to biological imaging in Chinese hamster ovary (CHO) cells labled with alkynylsialic acid nor H₂S.

It is well known that H₂S have been demonstrated to exert protective effects in many pathologies and physiologies¹⁶,¹⁷,¹⁸,¹⁹,²⁰,²¹,²²,²³,²⁴,²⁵,²⁶. So the discovery of these emerging biological roles of H₂S has resulted in rising interest in H₂S research. Accordingly, rapid, accurate and reliable methods for H₂S detection are in high demand, as they have potential to provide useful information for better understanding its biological functions²⁷. And simple, specific, and real-time analytic methods/sensors are highly desirable for H₂S in biological systems. In fact, it is a good choice to introduce an azido group into probes to be reduced by H₂S due to the simple synthesis, relatively good selectivity, suitable reaction time, and non-cell toxicity²⁸,²⁹,³⁰,³¹,³²,³³,³⁴.

With these considerations in mind, we also prepared 5-azidefluorescein from 5-aminofluorescein under NaNO₂/NaN₃ condition according to literature¹⁵ (Fig. 1) and tried to use this compound to detect H₂S. It is delightful that we obtained the crystal of 5-aminofluorescein and found probe can be used as a high selective and sensitive fluorescent probe for H₂S firstly. Furthermore, the probe also was applied in cell imaging.

Figure 1. Structure and thermal ellipsoids of probe are drawn at the 50% probability level.

5-aminofluorescein (0.35 g, 1 mmol), a deep-red solid, was dissolved in 10 mL 2:1 AcOH/H₂O and cooled to 0°C. To this deep red solution was added NaNO₂, a white powder (0.10 g, 1.5 mmol). After stirring for 15 minutes, the solution had turned to a light red color. NaN₃ (0.10 g, 1.5 mmol) was then carefully added (caution: gas evolution!), turning the solution to a yellow slurry. The reaction was stirred for 2 hr at 0°C. The slurry was filtered over vacuum and the solid washed with 20 mL 2 mol/L HCl and 100 mL H₂O, yielding 5-azidofluoresceinquinone (0.30 g, 80%) as a yellow solid after further drying in vacuo and characterized by NMR, ESI-MS, elemental analysis, X-ray crystal diffractometer (see Figure S1).

Reaction of probe (1 μmol/L) with Na₂S (2 μmol/L) as an aqueous sulphide source at room temperature in buffered (10 mmol/L HEPES, pH 7.0) aqueous acetonitrile solution (H₂O/CH₃CN = 1:3, v/v) yielded a time-dependent fluorescence increase, which was completed within 5 s (Supplementary Fig. S2). ΔF > 50-fold increase in the fluorescence intensity accompanied (Φ = 0.35) with a green emission at 531 nm. However, the analytes without hydrogen sulfide induced no changes in the fluorescence emission properties under the same conditions (Fig. 2a). The competing experiments indicated other analytes did not disturb the determination for sulphide (Fig. 2b). It is noted that the unprecedented speed of this probe's response and high selectivity compared with other probes³⁵,³⁶,³⁷,³⁸ suggests the possibility of quantitative detection without the need for sample pretreatment. The results reason that H₂S-mediated reduction of azides to amines would generate highly fluorescent products (Fig. 3)³⁹. H₂S-induced product was confirmed its molecular formula by electrospray ionization mass spectrometry (ESI-MS). The peak at m/z 346.42 corresponding to [5-aminofluorescein-H]⁺, was clearly observed (Supplementary Fig. S3). Further ¹HNMR spectroscopic analysis also provided the evidence for the product of 5-aminofluorescein. With addition of 2 equiv. of Na₂S (containing crystal water) to probe in DMSO-d₆ (Fig. S4), the resonance of the original proton (azidebenzene CH) at 7.28 and 7.49 ppm all shifted to upfield owing to presence of electron- pushing group NH₂ (Supplementary Fig. S4) and appeared at 6.65 ~ 6.76 ppm.

Figure 2.

(a) Fluorescence spectra of probe (1 μmol/L) with various analytes (20 μmol/L) in water:CH₃CN (1:3 v/v, HEPES buffer, pH 7.0) solutions (λ_ex = 425 nm, slit: 5 nm/5 nm), inset: a visual fluorescence change photograph for H₂S (green) and other analytes (colorless) under illumination with a 365 nm UV lamp; (b) Relative fluorescent intensity (λ_ex = 425 nm, λ_em = 531 nm) of the system. (black bar: various analytess, red bar: probe + various analytes + H₂S).

Figure 3. The proposed mechanism for the determination of H₂S.

Next, varying concentrations of Na₂S (0–2.0 μmol/L) were added to the test reaction solution. The fluorescence intensity increased linearly with the concentration of Na₂S up to 2.0 μmol/L, and, thereafter, reached a steady state (Fig. 4). The detection limit, based on the definition by IUPAC (C_DL = 3 S_b/m)⁴⁰, was found to be 0.035 μmol/L from 10 blank solutions (Supplementary Fig. S5). This probe therefore shows a high sensitivity toward sodium sulfide comparable to that of other reported S₂−chemosensors³⁵,³⁶,³⁷,³⁸ (Table 1).

Figure 4.

Fluorescence spectra of probe (1 μmol/L) in the presence of various concentrations of H₂S (0-2.0 μmol/L) in water:CH₃CN (1:3 v/v, HEPES buffer, pH 7.0) solution. (λ_ex = 425 nm, slit: 5 nm/5 nm); each spectrum is recorded 0.5 min after H₂S addition.

Table 1. A compared table about the detection limits and time course for H₂S.

Method	Analyte	Signal output	Solvent	Detection Limit (μmol/L)	Response Time	Time course in cell
Ref. 35	H2S	Fluorescence	HEPES buffer	0.08	20 min	50 min
Ref. 38	H2S	Fluorescence	PBS-DMSO(1:1, v/v, pH 7.4)	3.05	40 min	120 min
Ref. 36	H2S	Fluorescence	PBS–CH3CN (1:1, v/v, pH 7.4)	2.5	10 min	30 min
Ref. 37	H2S	Fluorescence	PIPES buffer (pH 7.4)	2.4	30 min	60 min
This work	H2S	Fluorescence	HEPES:CH3CN (1:3 v/v, pH 7.0)	0.035	10 s	30 min

We also performed absorption spectral experiments in the buffered (10 mmol/L HEPES, pH 7.0) aqueous acetonitrile solution (H₂O/CH₃CN = 1:3, v/v) containing probe (8 μmol/L) when the H₂S was added gradually. Fig. S6 showed absorbance changes of probe in the buffered (10 mmol/L HEPES, pH 7.0) aqueous acetonitrile solution (H₂O/CH₃CN = 1:3, v/v) after the addition of 4 equiv. of H₂S. The probe has no absorbance at UV-Vis area, immediately there generated an absorbance at 510 nm and the absorbance intensity enhanced with increased H₂S corresponding solution color change from colorless to yellow. The notable variation was ended after about 4 equiv. of H₂S added, relating to the H₂S-mediated reduction of 5-azidefluoresceinquinone to 5-aminofluorescein (ring-open).

Most publications suggest that the average endogenous H₂S level is in the μmol/L range³¹,³²,⁴¹, Since the detection limit of this probe was found to be 0.035 μmol/L, thus it become possible that the probe can detect H₂S level in tissue imaging. The ability of probe to detect sulphide within living cells was also evaluated by laser confocal fluorescence imaging using a Leica TCS SP5 laser scanning microscope. Imaging of sulphide substrates in HeLa cells after 30 min incubation using probe (2 μmol/L) showed weak green fluorescence (Fig. 5b). HepG2 cells incubated with 2 μmol/L probe for 30 min at 37°C, and with 4 μmol/L exogenous H₂S for another 30 min at 37°C, showed green fluorescence (Fig. 5c) (it is noted that 30 min was usually selected in cell imaging experiment). We also carried out time course experiment in the cell. Fig. 6 (left) indicated that a 15 min is enough for cell permeability (Fig. 6h) reaction and the cell can survive even if in a 45 min after H₂S was added (Fig. 6i). In addition, according to the Qian's method⁴², we employed sodium nitroprusside (SNP, a NO donor) to stimulate the production of endogenous H₂S in cells⁴³. With the addition of probe into the culture of the SNP (100 μmol/L or 200 μmol/L)-loaded cells for 20 min, a drastic increase of emission intensity (Fig. 6l, 6m), indicating the generation of endogenous H₂S within the cells. These results demonstrate that this probe is selective for sulphide and amenable for live-cell imaging.

Figure 5. Confocal fluorescence images in HepG2 cells.

(a) absence and presence of 2 μmol/L probe (b); (c) Fluorescence image of HepG2 cells incubated with 2 μmol/L probe for 30 min at 37°C and then incubated with 4 μmol/L H₂S for 30 min at 37°C; (d), (e), (f) were their brightfield images of (a), (b), (c).

Figure 6.

(a) The time dependence of confocal fluorescence images of exogenous sulphide in HepG2 cells. (h) and (i) Fluorescence image of HepG2 cells incubated with 2 μmol/L probe for 30 min at 37°C and then incubated with 4 μmol/L H₂S for 15 min and 45 min at 37°C, respectively; (j), (k) were their brightfield images of (h), (i); (b) Confocal fluorescence images of endogenous H₂S in living HepG2 cells with probe (2 μmol/L) upon excitation at 425 nm. Cells were prestimulated with SNP (100 μM) for 30 min, then incubated with probe (2 μmol/L) for 20 min (l, n). Cells were prestimulated with SNP (200 μmol/L) for 30 min, and then incubated with probe (2 μmol/L) for 20 min (m, o).

The development of innovative fluorescent imaging probes has revolutionized cell biology, allowing localization and dynamic monitoring of cellular metabolite and inorganic ion pools⁴³,⁴⁴,⁴⁵. Recently, fluorescence and/or colorimetric chemosensors for H₂S/aqueous sulphide based on some reaction mechanisms between probes and H₂S have been reported. These include the cleavage of alcoxyl(R-O) bond⁴⁵,⁴⁶,⁴⁷, the cleavage of S-O bond⁴⁷,⁴⁸,⁴⁹, copper displacement approach⁴⁹,⁵⁰,⁵¹, nucleophilic addition approach⁵,³⁸,⁵²,⁵³. A significant bottleneck in the above emerging field of H₂S/aqueous sulphide signalling is the absence of technology for effective in vivo detection and imaging. And this problem is exacerbated by fact that similar substances such as sulphide which contain SH group may mislead the detection of intracellular thiol concentration. In this study, we have successfully developed an azide-to-amine reduction chemical strategy for selective sulphide detection, which can be used to monitor sulphide generation in live cells in the presence of large excess of thiols. We show that the same chemistry can be readily adapted to different fluorescent templates for sulphide detection and imaging. The same chemistry will lead to new probes with faster response, which may help to monitor fluctuations of H₂S in situ. Further optimization and utilization of this strategy and this class of probes should dramatically accelerate future studies of H₂S in biology.

Methods

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and sodium nitroprusside were purchased from Sigma–Aldrich (St. Louis, MO). Sodium hydroxide solution (0.1 mol/L) was added to aqueous HEPES (10 mmol/L) to adjust the pH to 7.0. Anionic salts were purchased from Shanghai Experiment Reagent Co., Ltd (Shanghai, China). All other chemicals used were of analytical grade. Deionized water was used to prepare all aqueous solutions. The solutions of anions were prepared from their sodium salts.

Instruments

A pH meter (Mettler Toledo, Switzerland) was used to determine the pH. Fluorescence spectra were measured on Cary Eclipse fluorescence spectrophotometer. A PO-120 quartz cuvette (10 mm) was purchased from Shanhai Huamei Experiment Instrrument Plants, China. ESI-MS was measured with an UPLC-ESI-Q-TOF synapt G2 (Waters) instrument. The ability of probe reacting to hydrogen sulfide in the living cells was also evaluated by laser confocal fluorescence imaging using a Leica TCS SP5 laser scanning microscope.

Imaging of HepG2 cells

The HepG2 cells were grown in 1 × SPP medium (1% proteose peptone, 0.2% glucose, 0.1% yeast extract, 0.003% EDTA ferric sodium salt) at 37°C. The HepG2 were treated with 2 μmol/L of probe (methanol stock solution) in culture media for 30 min at 37°C and washed 3 times with PBS. The HepG2 cells were first incubated with 2 μmol/L of probe for 30 min at 37°C and with 20 μM exogenous H₂S for final 30 min at 37°C.

Author Contributions

F.J. and C.X. conceived the idea and directed the work. Y.B. designed experiments. J.K. performed the synthesis and in vitro tests. J.B. carried out NMR experiment. All authors contributed to data analysis and manuscript writing.

Supplementary Material

Supplementary Information

Supplementary Information