A near-infrared BODIPY-based fluorescent probe for the detection of hydrogen sulfide in fetal bovine serum and living cells

Abstract

A near-infrared (NIR) “off–on” fluorescent probe BDP-680 was developed by taking advantage of the Michael addition reaction of unsaturated double bonds in conformationally restricted BODIPY to allow detection of H₂S. The new probe possesses a highly selective and sensitive response to H₂S. Such a NIR probe has been applied successfully to detect H₂S in 10% deproteinized fetal bovine serum (FBS). The detection limit of the probe for NaSH in 10% deproteinized FBS sample was calculated to be about 1 μM which is consistent with the value obtained from serum free solutions (0.5 μM). The probe is of low toxicity and has been successfully used to detect H₂S in living cells.

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Introduction

Hydrogen sulfide (H₂S), traditionally considered as a toxic gas, has been recognized as the third endogenously produced gasotransmitter, in addition to nitric oxide (NO) and carbon monoxide (CO).^1,2 H₂S can be endogenously produced by enzymes such as cystathionine β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulphur-transferase^3–5 in certain mammalian organs and tissues. Non-enzymatic pathways are also found to liberate H₂S from the intracellular pool of “labile” sulfur, for instance, from the “sulfane sulfur” pool (compounds containing sulfur atoms bound only to other sulfur atoms).⁶ The biological functions of H₂S have been recognized in a number of biological and pathological processes.^7–9 Research results indicate that H₂S is related to diseases such as arterial and pulmonary hypertension and Alzheimer's disease.^1,10 H₂S also performs as an antioxidant or a scavenger of reactive oxygen and nitrogen species in cells. H₂S-releasing drugs are currently being used to treat cardiovascular and inflammatory diseases.^1,10 For such a relatively new member of endogenously produced gaseous signaling molecule, our understanding of the physiological and pathological roles of H₂S is still in its infancy.^11,12 Therefore, development of accurate tools for detection and measurement of H₂S becomes important.

Conventional detection methods such as electrochemical assays, gas chromatography, colorimetric and sulfide precipitation techniques have been applied for H₂S detection.^13–18 These methods often require complicated sample processing. Recently, fluorescent probes for H₂S have drawn great attention.^19–21 Fluorescent techniques are extremely attractive due to their simplicity, high sensitivity and real-time detection. For fluorescent probes of H₂S, the design strategies are commonly based on several characteristic properties related to H₂S, namely good reducing property towards azide,^19–31 dual nucleophilicity,^32–37 efficient thiolysis of dinitrophenyl ether,^38,39 high binding affinity towards copper ions^40–42 as well as Michael addition reaction towards unsaturated double bonds.^43–46 However, near-infrared fluorescent probes to sense H₂S are still very rare. In this communication we disclose a BODIPY-based turn-on NIR fluorescent BDP-680 for discrimination of H₂S by taking advantage of Michael addition reaction to unsaturated double bonds.

BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes are known to be highly fluorescent, very stable and exceptionally insensitive to the polarity of solvents as well as to pH.⁴⁷ BODIPYs are widespreadly applied as fluorophore in probes and sensors. Near-infrared (NIR) fluorescent dyes provide many advantages over UV and visible fluorescent dyes. NIR fluorescent dyes can greatly reduce background absorption, fluorescence, light scattering, and improve the detectable sensitivity.⁴⁸ Our group has been working in design and synthesis of BODIPY-based dyes as NIR fluorescent probes.^49–53 Herein, we discovered a novel conformationally restricted NIR fluorescent BODIPY dye 1 (BDP-680) containing a meso-acrylate moiety (see Scheme 1) is capable of recognizing H₂S selectively. It was used to detect and visualize H₂S in fetal bovine serum and living cells. We anticipate that the current probe with reduced background fluorescence and improved detectable sensitivity would help the researchers detect H₂S in living systems for more insight of the role of H₂S as gasotransmitter.

Scheme 1 The synthesis of compound 1 (BDP-680) and its ORTEP diagram.

Results and discussion

Probe design and preparation

Very recently we reported that a conformationally restricted BODIPY dye 2 (see Scheme 1) containing a partially exposed aldehyde group at the meso position acts as “turn-on” fluorescent probe for the detection of homocysteine over cysteine.⁵³ By one step simple functionalization of the aldehyde group in compound 2 with ethyl cyanoacetate to form a Michael acceptor on the meso position, compound 1 (BDP-680) can be conveniently synthesized (Scheme 1). The structure of BDP-680 was confirmed by X-ray crystallographic analysis.

Sensing properties

BDP-680 absorbed broadly in far-red to NIR region with absorption maxima at 678 nm. No fluorescence of BDP-680 was noticed in organic solvent (e.g. CHCl₃), however weak fluorescence (Φ_f = 0.03, λ_em = 680 nm) of BDP-680 in MeCN/H₂O (8 : 2, v/v) was observed. Upon addition of NaSH, the absorption maximum was slightly blue-shifted to 666 nm and the full-width at half-maximum (fwhm) decreased (Fig. 1). On the other hand, slight red-shift of the emission maxima from 680 nm to 683 nm was observed and significant fluorescence enhancement up to 40-fold was observed after 0.5 h (the fluorescence quantum yield was measured to be 0.95). The normalized excitation spectra (λ_ex = 683 nm) were shown in Fig. S1 (ESI†) and very similar patterns of the excitation spectra were found both prior to and after addition of NaSH. Although the absorption and excitation spectra after addition of NaSH were closely resembled, the absorption spectrum of BDP-680 displayed broadening compared with the excitation spectrum due to aggregation of the highly conjugated system (Fig. 1 and S1, ESI†). Though close overlap of the absorption maximum (678 nm) of probe BDP-680 with the emission of newly formed product after addition of NaSH may interfere the detection, and the small stoke's shift for the conformationally restricted BODIPY dye,⁵⁴ the highly fluorescent species after addition of NaSH could still allow us to distinguish the presence of NaSH easily by using excitation wavelength apart from the emission maximum (e.g. 610 nm for detection in solutions and 633 nm for cell imaging purpose).

Fig. 1 Absorption (a) and emission spectra (b, λ_ex = 610 nm) of BDP-680 (10 μM) prior to (black curve) and after (red curve) addition of NaSH (500 μM) for 0.5 h in MeCN/H₂O (8/2, v/v; pH = 7.2) at 25 °C.

The plausible mechanism for selective reaction of BDP-680 to NaSH was shown as Scheme S1.† The unsaturated double bonds of BDP-680 reacted with NaSH through addition reaction, the acrylate moiety was destroyed and the photoinduced electron transfer (PET) process was prevented, so increment of fluorescence was displayed. We tried to monitor the reaction by HRMS and NMR, unfortunately, the expected product could not be traced presumably due to the low solubility and unstable nature of the sulfur substituted species.

To find an appropriate detection solvent system, the influence of water content on probe's sensing property was conducted (Fig. S2, ESI†). The results showed when the water content under 70%, the probe could response to NaSH with obvious fluorescence enhancement. The probe showed most fluorescence enhancement in 20% H₂O solution. So the MeCN/H₂O (8 : 2, v/v) was selected as the solvent system and the BDP-680 could be used in biological samples potentially.

Time-dependent spectra of BDP-680 were subsequently monitored in the presence of NaSH. As shown in Fig. 2, upon addition of NaSH to BDP-680, intensity of the emission band centered at 683 nm increased immediately within 1 min (12-fold fluorescence intensity enhancement) which reached the maximum 20 min later with 40-fold fluorescence increase at the end.

Fig. 2 Time-dependent changes (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30 min) of fluorescence spectra (λ_ex = 610 nm) of BDP-680 (10 μM) with NaSH (500 μM) in MeCN/H₂O (8/2, v/v; pH = 7.2) at 25 °C.

Fluorescence titration

Since BDP-680 displayed reasonably fast response to NaSH, we then carried out the studies on concentration-dependent behaviour of probe to NaSH. The responses of BDP-680 to various concentrations of NaSH were studied by fluorescence spectroscopy in MeCN/H₂O (8 : 2, v/v) solution at pH 7.2 (Fig. 3). The free probe was weakly fluorescent and the fluorescence intensity was strengthened at 683 nm with increasing amounts of NaSH and displayed a linear response to the amount of NaSH up to 100 μM (Fig. 3 inset). The detection limit for NaSH was estimated to be 0.5 μM (3σ/k, wherein σ is the standard deviation of blank measurement, k is the slope between the fluorescence intensity versus NaSH concentration). Thus, the sensitive and linear response of BDP-680 to NaSH may allow quantitative measurement of NaSH concentration at its biological level.

Fig. 3 Fluorescence responses of BDP-680 (10 μM in MeCN/H₂O = 8/2, v/v; pH = 7.2, λ_ex = 610 nm) upon reacting with NaSH in 1, 3, 5, 10, 20, 40, 60, 80, 100, 200, 300, 400, 500 μM measured at 30 min after addition at 25 °C. The inner panel displays the linear fluorescence enhancement of BDP-680 toward NaSH of 1, 3, 5, 10, 20, 40, 60, 80, 100 μM.

For comparison purposes, a comprehensive summary of the recently developed fluorescent probes for NaSH is collected in Table S1.† BDP-680 showed appropriate linear response range and detection limit.

Selectivity/competition assays

The selectivity profile of BDP-680 towards various analytes was provided in Fig. 4 in the presence of 50 equiv. of neutral or anionic analyte such as HS⁻, GSH, Cys, Hcy, HSO₃⁻, SO₄²⁻, S₂O₃²⁻, S₂O₅²⁻, Cl⁻, Br⁻, I⁻, NO₂⁻, N₃⁻, H₂O₂, HCO₃⁻, CO₃²⁻, CH₃COO⁻. All of the analytes, except NaSH, caused little change from that of the probe alone, which demonstrates the excellent selectivity of BDP-680 to NaSH.

Fig. 4 Relative responses of BDP-680 toward various analytes. Relative fluorescence intensity of BDP-680 (10 μM) in MeCN/H₂O (8/2, v/v; pH = 7.2) was measured at 683 nm (λ_ex = 610 nm) after incubation at 25 °C for 30 min in the presence of 500 μM (final concentrations) of analytes. (0) free; (1) HS⁻; (2) GSH; (3) Cys; (4) Hcy; (5) HSO₃⁻; (6) SO₄²⁻; (7) S₂O₃²⁻; (8) S₂O₅²⁻; (9) Cl⁻; (10) Br⁻; (11) I⁻; (12) NO₂⁻; (13) N₃⁻; (14) H₂O₂; (15) HCO₃⁻; (16) CO₃²⁻; (17) CH₃COO⁻.

The excellent selectivity of BDP-680 to H₂S was further demonstrated in the competition experiments shown in Fig. 5. As shown in Fig. 5, strong fluorescence was observed for BDP-680 in the presence of 50 equiv. of analytes e.g. GSH, Cys, Hcy, HSO₃⁻, SO₄²⁻, S₂O₃²⁻, S₂O₅²⁻, Cl⁻, Br⁻, I⁻, NO₂⁻, N₃⁻, H₂O₂, HCO₃⁻, CO₃²⁻, CH₃COO⁻ after addition of 20 equiv. of NaSH.

Fig. 5 Relative responses of BDP-680 toward various analytes. Relative fluorescence intensity of BDP-680 (10 μM) in MeCN/H₂O (8/2, v/v; pH = 7.2) was measured at 683 nm (λ_ex = 610 nm) at 25 °C 30 min later after addition of analytes. The black bars represent the fluorescence intensity of BDP-680 in the presence of individual analyte (500 μM) (1) free; (2) GSH; (3) Cys; (4) Hcy; (5) HSO₃⁻; (6) SO₄²⁻; (7) S₂O₃²⁻; (8) S₂O₅²⁻; (9) Cl⁻; (10) Br⁻; (11) I⁻; (12) NO₂⁻; (13) N₃⁻; (14) H₂O₂; (15) HCO₃⁻; (16) CO₃²⁻; (17) CH₃COO⁻. The red bars represent the fluorescence intensity after addition of NaSH (200 μM) to the mixture of BDP-680 and corresponding analyte.

pH dependence

The pH-dependence experiments on the fluorescence behaviour of BDP-680 (10 μM) towards H₂S (500 μM) were carried out as well (Fig. 6). Experimental results indicated that the response of BDP-680 to H₂S works nicely in the pH range of 5.0–11.0. Therefore, the probe could function over a broad range of pH values and react selectively toward H₂S. These results strongly suggest that BDP-680 could have potential value for H₂S analysis in biological environments.

Fig. 6 The effect of pH on the relative fluorescence intensity of BDP-680 (10 μM) upon addition of 500 μM NaSH in MeCN/H₂O (8/2, v/v). All the data was measured at 683 nm (λ_ex = 610 nm) at 25 °C 30 min after addition of NaSH (black square: BDP-680; red circle: BDP-680 + NaSH).

Biological application

We next acquired the detection of H₂S in diluted (10%) deproteinized fetal bovine serum (FBS). The concentration-dependent fluorescence responses of BDP-680 to NaSH in 10% deproteinized FBS sample were measured. As shown in Fig. 7, the free probe was weakly fluorescent in 10% deproteinized FBS sample, with the increase of NaSH concentration the fluorescence intensity of the serum sample with BDP-680 increased accordingly, the emission maximum of the probe (λ_em = 672 nm) was slight red-shifted to 683 nm and a linear calibration curve with the added NaSH concentration from 0 to 20 μM was obtained. The detection limit of BDP-680 for NaSH in 10% deproteinized FBS sample was calculated to be about 1 μM (3σ/k, wherein σ is the standard deviation of blank measurement, k is the slope between the fluorescence intensity versus NaSH concentration) which is well in consistent with the aforementioned value obtained from serum free solutions. These results indicate that BDP-680 can be used to detect NaSH in complex biological serum samples quantitatively.

Fig. 7 Fluorescence spectra changes of BDP-680 (10 μM, λ_ex = 610 nm) upon addition of various concentrations of NaSH in MeCN/H₂O (8/2, v/v; pH = 7.2) containing 10% deproteinized fetal bovine serum (FBS, v/v) at 37 °C. Concentrations of NaSH are used: 0, 1, 3, 4, 5, 10, 15, 20 μM. Inset: linear response of BDP-680 at 683 nm to a function of NaSH concentrations. Each spectrum was collected after 30 min NaSH addition.

To further explore the biological application of BDP-680, the cytotoxic and fluorescence imaging experiments were carried out. BDP-680 exhibited very low cytotoxicity evaluated by classic MTT assays towards Human Hepatoma SMMC-7721 incubated for 24 h in the presence of 80 μM BDP-680 (Fig. S3, ESI†).

For cell imaging experiments, when Human Hepatoma SMMC-7721 cells were incubated with 10 μM of BDP-680 only in culture medium at 37 °C for 1 h, no obvious fluorescence was observed (Fig. 8). In contrast, addition of NaSH to the cells pre-incubated with BDP-680 resulted in significant red fluorescence. With this bioimaging experiment we have demonstrated the potential of BDP-680 for the imaging of NaSH in living cells.

Fig. 8 Confocal laser scanning microscopic images of BDP-680 (λ_ex = 633 nm) to NaSH in SMMC-7721 cells. (a–c) Cells incubated with BDP-680 (10 μM) for 60 min: (a) bright-field images, (b) red channel, (c) merged image from (a) and (b); (d–f) cells incubated with BDP-680 (10 μM) for 60 min, further incubated with NaSH (100 μM) for another 60 min: (d) bright-field images, (e) red channel, (f) merged image from (d) and (e).

Conclusions

In conclusion, we have developed a novel fluorescent turn-on NIR BDP-680 with high selectivity and sensitivity for H₂S. BDP-680 is weakly fluorescent in MeCN/H₂O (8 : 2, v/v) (Φ_f = 0.03). Upon addition of NaSH to BDP-680, dramatic increase in fluorescence (Φ_f = 0.95) was observed. Moreover, this new probe is stable and works in a wide pH range (5.0–11.0). Such a NIR probe has been applied successfully to the detect H₂S in 10% deproteinized FBS. The detection limit of BDP-680 for NaSH in 10% deproteinized FBS sample is calculated to be about 1 μM which is well consistent with the value obtained from serum free solutions (0.5 μM). The probe is low toxic and has been successfully used to detect H₂S in living cells.

Experimental section

General

¹H NMR spectra were recorded on a VARIAN Mercury 400 MHz spectrometer. Mass spectrometric measurements were performed by AB SCIEX TripleTOF™ 5600⁺ mass spectrometer. The refractive index of the medium was measured by 2 W Abbe's refractometer at 20 °C. Fluorescence spectra were recorded on FluoroSENS spectrophotometer. UV/vis spectra were recorded on Perkin-Elmer Lambda 35 UV/vis spectrophotometer.

The fluorescent quantum yields (Φ_f) of the BODIPY systems were calculated using the following relationship (eqn (1)):

Φ_f = Φ_refF_samplA_refn_sampl²/F_refA_sampln_ref² (1)

here, F denotes the integral of the corrected fluorescence spectrum, A is the absorbance at the excitation wavelength, and n is the refractive index of the medium (n = 1.4448 in CHCl₃; n = 1.3461 in MeCN/H₂O (v/v = 80/20)), ref and sampl denote parameters from the reference and unknown experimental samples, respectively. The reference systems used was boronazadipyrromethene compound aza-BODIPY (Φ_f = 0.36 in chloroform).⁵⁵

The detection limit was calculated based on the fluorescence titration. In the absence of NaSH, the fluorescence emission of BDP-680 was measured with five parallel repeat and the standard deviation of blank measurement was achieved. Then the responses to the concentration of NaSH in the range from 0–100 μM after incubation of 30 min were recorded. To gain the slop, the fluorescence intensity at 683 nm was plotted to the concentration of NaSH. The detection limit was calculated with the following equation:

Detection limit = 3σ/k

where σ is the standard deviation of blank measurement, k is the slop between the fluorescence intensity versus NaSH concentration.

Cell culture and confocal microscopy study

Human Hepatoma SMMC-7721 cells were incubated in DMEM with 10% (v/v) fetal bovine serum, 1% penicillin/streptomycin at 37 °C in 5% CO₂. Human Hepatoma SMMC-7721 cells were seeded at a density of 5 × 10⁴ cells per well (200 μL) in a 6-well plate.

The cells were imaged by using a confocal laser scanning microscope (λ_ex = 633 nm). BDP-680 (20 μM, 1 mL) in DMSO/PBS buffer (1 : 1, v/v, 10 mM, pH 7.4) was added to Human Hepatoma SMMC-7721 cells in a six-compartment cell culture plate that contained 1.0 mL culture medium, and was incubated at 37 °C for 60 min. After removing the culture medium and washing with PBS twice, the fluorescence images of cells were taken. For the control experiment, the cells in a six-compartment cell culture plate that contained 2.0 mL culture medium were treated with 100 μM NaSH in culture media for 60 min at 37 °C in a humidified incubator. After washing with PBS for twice, the cells were further incubated with 10 μM of BDP-680 for 60 min. After washing with PBS for twice, the fluorescence images of cells were taken.

Synthesis of BDP-680

Under N₂, BODIPY 2 (48.4 mg, 0.1 mmol) was dissolved in dry dichloromethane. To the suspension was added triethylamine (0.05 mL, 0.36 mmol) and ethyl cyanoacetate (0.05 mL, 0.47 mmol) stirred for 2 h at 80 °C. The reaction mixture was extracted with saturated aqueous NaHCO₃ and brine. The combined organic solutions were dried over MgSO₄. The solvent was evaporated, and the resulting crude mixture was purified by chromatography on silica gel followed by recrystallization from CH₂Cl₂/n-hexane to afford a coppery solid of BDP-680 (51.5 mg, 89%). Mp > 250 °C. ¹H NMR (CDCl₃, 400 MHz): δ (ppm): 8.70 (d, J = 9.2 Hz, 2H), 8.41 (s, 1H), 6.97 (dd, J = 9.2, 2.4 Hz, 2H), 6.81 (d, J = 2.8 Hz, 2H), 6.78 (s, 2H), 4.44 (q, J = 7.2 Hz, 2H), 3.89 (s, 6H), 2.90 (t, J = 7.2 Hz, 4H), 2.74 (t, J = 7.2 Hz, 4H), 1.44 (t, J = 7.2 Hz, 3H) ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 161.3, 161.0, 157.6, 150.2, 143.4, 133.5, 133.2, 130.7, 122.2, 121.2, 114.4, 113.3, 112.7, 104.2, 100.0, 63.4, 55.3, 30.8, 29.7, 14.1. HRMS-ESI: calcd for C₃₃H₂₈BF₂N₃NaO₄ [M + Na]⁺ 602.2039, found 602.2038.

Acknowledgements

This research was supported by NSFC (21372063) and Program for Changjiang Scholars and Innovative Research Team in University, No. PCS IRT1126. We thank Ms Jiamin Wang for X-ray crystal structure analysis of BDP-680.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Experimental details, MTT assays and ¹H, ¹³C NMR spectra. CCDC 1023076. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra06952e

‡ These authors contributed equally to this work.

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