Two-Photon Fluorescent Probe for Detection of Exogenous and

Feb 6, 2015 - Endogenous Hydrogen Persulfide and Polysulfide in Living. Organisms. Lingyu Zeng,. †. Shiyu Chen,. †. Tian Xia,. ‡. Wei Hu,. §. C...
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Two-Photon Fluorescent Probe for Detection of Exogenous and Endogenous Hydrogen Persulfide and Polysulfide in Living Organisms Lingyu Zeng,† Shiyu Chen,† Tian Xia,‡ Wei Hu,§ Chunya Li,§ and Zhihong Liu*,† †

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, and ‡College of Life Science, Wuhan University, Wuhan 430072, China § College of Chemistry and Material Science, South-central University of Nationalities, Wuhan 430074, China S Supporting Information *

ABSTRACT: Hydrogen persulfide and polysulfide (H2Sn) are newly discovered intracellular reactive species considered to have high protein S-sulfhydration efficiency. The detection of H2Sn in living systems is essential for studying their functions but is quite challenging. In this work, we report a two-photon excited fluorescent probe, QSn, capable of tracking H2Sn in living organisms. QSn exhibited turn-on two-photon fluorescence response upon reaction with H2Sn. With a favorable photophysical property, high specificity, and low cytotoxicity, QSn was able to recognize exogenous H2Sn in living cells. More importantly, it realized for the first time the visualization of endogenous H2Sn generated in cells overexpressing cystathionine β-synthase and cystathionine γ-lyase, the enzymes responsible for producing endogenous H2Sn. Taking advantage of two-photon microscopy, the probe was also applied to achieve H2Sn detection in zebrafish embryos and to observe H2Sn distribution in living organisms.

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H2Sn have rarely been reported. By using mass spectrometry (MS), H2Sn can be monitored directly in aqueous solution or after being derived by monobromobimane, but the MS-based methods are unsuitable for detecting H2Sn at near neutral pH and the derivatives are unstable during measurement.7,11 There are also spectroscopic methods for H2Sn assay, in which the absorption peaks at 300 and 372 nm were either directly used for H2Sn determination8 or through a H2Sn oxidation/ regeneration process at a GaAs electrode with in situ UV−vis spectroelectrochemistry.12 The detection by UV−vis absorption is straightforward but is limited by rather low sensitivity. More importantly, all the above methods are unsuitable for in vivo monitoring, which highlights the demand for fluorescent probes. Molecular probe-based fluorescence microscopy is wellknown for its high specificity and sensitivity, which can be applied in living systems with nondestructive visibility and high temporal/spatial resolution.13 There has been only one H2Sn fluorescent probe reported so far, by Xian and Ma’s group, which was the pioneering work in this field. However, the fluorescein-based probe was not sensitive enough for endogenous H2 Sn and it realized only exogenous H2 Sn detection in cultured cells.14 Unlike the exogenous species

s a gaseous signaling molecule, the diverse functions of hydrogen sulfide (H2S) in physiological and pathological processes have been well studied and widely accepted.1−4 However, recent years have witnessed a controversy regarding whether some reported biological effects of H2S are actually executed by hydrogen per- and polysulfide (H2Sn, n ≥ 2). In vivo, H2Sn can be generated from H2S through rapid oxidation by oxygen or enzyme and possesses reaction potency (e.g., nucleophilicity and reducibility) superior to that of H2S.5,6 It is found that H2Sn possesses high protein S-sulfhydration efficiency, which enables its promising regulatory function in altering enzyme activity and potentially important role in H2S signaling. Several very recent studies reveal that H2Sn shows nearly 300 times higher potential than that of parental H2S in inducing Ca2+ influx in astrocytes by activating TRAP1 channels7 and acts significantly on lipid phosphatase PTEN8 and Keap 1 which regulate Nrf2 signaling.9 Additionally, some studies on the regulatory functions of persulfide and polysulfide also suggest that the H2Sn-participated protein S-sulfhydration may represent an alternative mechanism.6,10 In all, there are accumulating assumptions and evidence showing that H2Sderived H2Sn may compensate for some physiological functions that H2S cannot fulfill, and the research on H2Sn is just in the initial stage with many issues yet to be resolved. To shed light on the signaling or regulatory function of H2Sn, it is urgent to develop a tool for the recognition and tracking of the species in living systems. However, detecting methods for © XXXX American Chemical Society

Received: December 11, 2014 Accepted: February 6, 2015

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DOI: 10.1021/acs.analchem.5b00172 Anal. Chem. XXXX, XXX, XXX−XXX

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mixed in 10 mL of anhydrous DMF. When the mixture was heated to 80 °C, benzyl chloride (0.556 mL, 4.83 mmol) was added and reacted for 24 h. The reaction solution was quenched by water and extracted by ethyl acetate. The organic layer was dried with anhydrous Na2SO4. Then the solvent was removed and subjected to silica gel column chromatography using petroleum ether:ethyl acetate (3:1) as the eluent to afford 2 as white solid (515 mg, 51% yield). 1H NMR (300 MHz, DMSO-d6), δ 2.80 (s, 3H), 5.21 (s, 2H), 7.19 (d, J = 2.4 Hz, 1H), 7.31 (d, J = 8.5 Hz, 1H), 7.45 (ddt, J = 31.6, 24.1, 7.3 Hz, 6H), 8.04 (d, J = 8.4 Hz, 1H), 8.11 (d, J = 9.0 Hz, 1H). HRMS (MALDI): calcd for C17H16ON [M + H]+ 250.1226, found 250.1222. Synthesis of Compound 3. A solution of 2 (461 mg, 1.85 mmol) in 5 mL of 1,4-dioxane was heated to 60 °C. Next, SeO2 (246.4 mg, 2.22 mmol) was added to the solution, and then the temperature was increased to 80 °C for 4 h. The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with petroleum ether:ethyl acetate (5:1) to give a light yellow solid (292 mg, 60% yield). 1H NMR (300 MHz, DMSO-d6), δ 5.26 (s, 2H), 7.25 (d, J = 2.7 Hz, 1H), 7.38−7.48 (m, 3H), 7.53 (d, J = 7.2 Hz, 2H), 7.58 (dd, J = 9.3, 2.8 Hz, 1H), 8.03 (d, J = 8.5 Hz, 1H), 8.18 (d, J = 4.2 Hz, 1H), 8.21 (d, J = 3.5 Hz, 1H), 10.22 (s, J = 0.7 Hz, 1H). HRMS (MALDI): calcd for C17H14O2N [M + H]+ 264.1019, found 264.1015. Synthesis of Compound 4. A solution of 3 (390 mg, 1.48 mmol) and 2-aminobenzenethiol (190 μL, 1.78 mmol) in 5 mL of DMSO was stirred under argon atmosphere at 130 °C for 6 h. Then the reaction solution was quenched by water and extracted by ethyl acetate. The organic layer was dried with anhydrous Na2SO4. Then the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography using petroleum ether:ethyl acetate (8:1) as the eluent to afford 4 as a light yellow solid (476 mg, 87% yield). 1H NMR (300 MHz, DMSO-d6), δ 5.25 (s, 2H), 7.24 (s, 1H), 7.38−7.50 (m, 4H), 7.55 (dd, J = 12.2, 7.7 Hz, 4H), 8.02 (d, J = 7.8 Hz, 1H), 8.19 (dd, J = 15.7, 8.7 Hz, 3H), 8.51 (d, J = 8.4 Hz, 1H). HRMS (MALDI): calcd for C23H17ON2S [M + H]+ 369.1056, found 369.1046. Synthesis of Compound 5. 4 (470 mg, 1.28 mmol) was fully dispersed in 10 mL of anhydrous CH2Cl2 under 0 °C, and then BBr3−CH2Cl2 solution (5.12 mL, 1 mol/L) was added to the mixture dropwise. After being stirred for 3 h at room temperature, the reaction was quenched with water. Then the mixture was neutralized with saturated NaHCO3 solution and extracted by anhydrous CH2Cl2. The organic layer was dried with anhydrous Na2SO4. The solvent was removed under reduced pressure, and crude product was purified by silica gel column chromatography using petroleum ether:ethyl acetate (4:1) as the eluent. The product was obtained as a yellow solid (250 mg, 70% yield). 1H NMR (300 MHz, DMSO-d6), δ 7.28 (d, J = 2.6 Hz, 1H), 7.43 (dd, J = 9.1, 2.7 Hz, 1H), 7.52 (s, 1H), 7.59 (d, J = 1.0 Hz, 1H), 8.01 (d, J = 9.1 Hz, 1H), 8.13 (d, J = 7.7 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 8.34 (d, J = 8.6 Hz, 1H), 8.37 (s, 1H), 10.42 (s, 1H). 13C NMR (100 MHz, d6-DMSO), δ 119.30, 119.55, 120.77, 120.89, 127.97, 128.01, 130.27, 130.39, 144.07, 144.10, 144.27, 163.51, 163.67, 163.71. HRMS (MALDI): calcd for C16H11ON2S [M + H]+ 279.0587, found 279.0583. Synthesis of the Probe QSn. 5 (167 mg, 0.6 mmol), 2fluoro-5-nitrobenzoic acid (167 mg, 0.9 mmol), and 4-

with controllable addition amounts in experiments, the probing of endogenous H2Sn generated in real cellular events is more challenging and should present more values. However, the detection of endogenous H2Sn in living systems has not yet been explored. An attractive approach for living systems imaging is two-photon microscopy (TPM), which processes deep z-axis depth-of-field, weak autofluorescence and selfabsorption, reduced photodamage to biological samples, and lowered photobleaching.15,16 Herein, we developed a twophoton fluorescent probe which, for the first time, realized the detection of both exogenous and endogenous H2Sn in living cells and was applicable in living zebrafish embryo imaging as well.

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EXPERIMENTAL SECTION Materials and Apparatus. Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. High resolution mass spectrometry was performed on an LTQ FT Ultra (Thermo Fisher Scientific, America) with MALDI-DHB mode. LC-MS analysis was performed on a high performance liquid chromatograph (Agilent 1200, America) connected to a quadrupole time-offlight mass spectrometer (Q-TOF MS, Agilent 6520, America). Absorption spectra were recorded on a UV−vis spectrophotometer (Shimadzu UV-2550, Japan), and one-photon fluorescence spectra were obtained with a fluorometer (Shimadzu RF-5301 PC, Japan). Two-photon fluorescence spectra were excited by a mode-locked Ti:sapphire femtosecond pulsed laser (Chameleon Ultra I, Coherent, America) and recorded with a DCS200PC photon counting with Omnoλ5008 monochromator (Zolix, China). Two-photon microscopy images were collected from a spectral confocal and multiphoton microscope (Carl Zeiss, LSM 780 NLO, Germany) with a mode-locked titanium-sapphire laser source (Mai Tai HP, Spectra Physics, America). The synthesis route of QSn is depicted in Scheme 1, and synthesis details are described below. Compound 1 was prepared by the literature method.17 Synthesis of Compound 2. 2 was synthesized by modifying a literature procedure.18 Under argon atmosphere, 1 (0.56 g, 4.02 mmol) and K2CO3 (0.72 g, 5.22 mmol) were Scheme 1. Synthesis Route for 5 and QSna

Reagents and conditions: (a) 2-Butenal, HCl, H2O, 100 °C, 12 h; (b) benzyl chloride, K2CO3, DMF, Ar, 80 °C, 24 h; (c) SeO2, 1,4-dioxane, 80 °C, 4 h; (d) 2-aminobenzenethiol, DMSO, 130 °C, 6 h; (e) BBr3, anhydrous CH2Cl2, rt, 3 h; (f) 2-fluoro-5-nitrobenzoic acid, EDC, DMAP, anhydrous CH2Cl2, rt, 10 h. a

B

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(NEST). For labeling, the cells were washed with serum-free DMEM and then incubated with 5 μM QSn (containing 1% DMSO in serum-free DMEM) for 30 min at 37 °C. For exogenous polysulfide imaging, the cells were washed two times with serum-free DMEM and incubated with 0.5 mM Na2S2 or Na2S4 for another 30 min at 37 °C. For the thiol-consumed group, the cells were washed with PBS and incubated with 2 mM N-methylmaleimide (NMM) for 1 h and then treated with the same labeling procedure. For endogenous polysulfide imaging, the cells transfected with pRK-Flag or CBS/CSE plasmids were washed and incubated with QSn under the same conditions. Two-Photon Fluorescence Imaging of H2Sn in Zebrafish. The fertilized AB genotype zebrafish eggs were purchased from China Zebrafish Resource Center (CZRC). The embryos were transferred to 90 mm Petri dishes (NEST) filled with E3 embryo media (E3M: 15 mM NaCl, 0.5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 0.15 mM KH2PO4, 0.05 mM Na2HPO4, 0.7 mM NaHCO3, 10−5% methylene blue; pH 7.5).22 The embryos were kept in the incubator at 28 °C for 3 days, replenishing fresh E3M everyday. Before imaging, the 3-day-old zebrafish were transferred to 24-well microplates and incubated with 5 μM QSn (containing 0.2% DMSO in E3M) for 30 min at 28 °C. After the zebrafish were washed with E3M to remove the remaining QSn, they were further incubated with 0.5 mM Na2S4 in E3M for another 30 min at 28 °C. Before being subjected to TPM, the embryos were anesthetized with tricane and embedded onto 0.5% agarose gel according to the literature protocol.23 All the pictures collected were of living embryos with an observed heartbeat.

dimethylaminopyridine (6 mg, 0.05 mmol) were dispersed in anhydrous CH2Cl2, and then 1-(3-(dimethylamino)propyl)-3ethylcarbodiimide hydrochloride (138 mg, 0.72 mmol) was added. The reaction was stirred at room temperature for 10 h. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography using petroleum ether:ethyl acetate (10:1) as the eluent. 1 H NMR (300 MHz, DMSO-d6), δ 7.26 (d, J = 2.6 Hz, 1H), 7.41−7.48 (m, 5H), 7.68−7.75 (m, 5H), 7.91 (d, J = 8.4 Hz, 1H). HRMS (MALDI): calcd for C23H13O4N3FS [M + H]+ 446.0605, found 446.0605. Measurement of Two-Photon Cross-Section. The twophoton cross-section (δ) was determined by using the femtosecond (fs) fluorescence measurement technique as described.19 5 and QSn were dissolved in PBS buffer (50 mM, pH 7.4, containing 0.9% NaCl and 25 μM CTAB), and the two-photon induced fluorescence intensity was measured at 710−800 nm by using rhodamine B as the reference, whose two-photon property has been well characterized in the literature.20 The intensities of the two-photon-induced fluorescence spectra of the reference and sample emitted at the same excitation wavelength were determined. The twophoton cross-section was calculated by using δ = δr(SsΦrϕrcr)/ (SrΦsϕscs), where the subscripts s and r stand for the sample and reference molecules, respectively. The intensity of the signal collected by a CCD detector was denoted as S. Φ is the fluorescence quantum yield. ϕ is the overall fluorescence collection efficiency of the experimental apparatus which can be approximated by the refractive index of the solvent. The number density of the molecules in solution is denoted as c. δr is the two-photon absorption cross-section of the reference molecule. The fluorescence quantum yield was determined by using quinine sulfate in 0.1 M sulfuric acid as the reference according to the literature method.21 Cell Culture. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Scientific) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) and incubated in an atmosphere of 5/95 (v/v) of CO2/air at 37 °C. Construction of CBS/CSE Overexpressing Cells. The cDNA clones for human CBS and CSE were purchased form Origene (lot no.: RC201755 and RC202195, respectively), and the Flag-tagged CBS and CSE mammalian expression plasmids were prepared by standard molecular biology techniques. After 1 day, the constructed plasmids were transfected by FuGENE (Roche) into HeLa Cells (∼1 × 105) seeded in 24-well plates. pRK-Flag was transfected as negative control. Western Blot Assay. HeLa cells were lysed with NP-40 lysis buffer (1.0% NP-40, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0). The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membrane. To block nonspecific antibody binding, the membrane was treated with 5% nonfat milk (dissolved in TBS) for 30 min. Next, the membrane was incubated with antibodies for Flag (1:1000, Sigma-Aldrich) or β-actin (1:1000, Santa Cruz Biotechnology). Antibodies were washed with TTBS three times (10 min/time), incubated with HRP-conjugated secondary antibodies (1:1000), washed, and finally visualized using a chemiluminescence (ECL) system. Two-Photon Fluorescence Imaging of Exogenous and Endogenous H2Sn in HeLa Cells. Two days before imaging, the cells were passed and plated into glass-bottomed dishes

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RESULTS AND DISCUSSION Preparation and Photophysical Properties of the Probe. We synthesized the fluorescent probe QSn (synthesis route shown in Scheme 1) using 2-fluoro-5-nitrobenzoate as the H2Sn recognition domain and 2-benzothiazol-2-yl-quinoline-6-ol (5) as the fluorophore. The quinoline core has been proved to possess good two-photon absorption ability in our previous studies.18,24 The principle of sensing H2Sn by QSn is depicted in Scheme 2, with the reaction product 5 as the Scheme 2. QSn−H2Sn Reaction Mechanisma

a

Numbers in brackets are the extracted mass ([M + H]+) of the related species.

signaling molecule. The reaction of the probe with H2Sn was monitored by liquid chromatograph−mass spectrometry (LCMS) analysis, which confirmed the formation of 5 (Figure S-1, Supporting Information). In aqueous buffer, QSn and 5 exhibit absorption maxima 4 −1 −1 (λabs max) at 368 nm (ε = 2.22 × 10 M ·cm ) and 362 nm (ε = 1.57 × 104 M−1·cm−1) and fluorescence maxima (λflmax) at 536 C

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Information) and Figure 1b,c, respectively. The target concentration-dependent turn-on fluorescence signals were observed under both excitation modes, with a maximal fluorescence enhancement factor of ca. 24-fold. The detection limit was calculated as 0.5 μM according to the 3sb/m criterion, where m is the slope for the range of the linearity and sb is the standard deviation of the blank (n = 11). We also conducted the titration of QSn by Na2S2 under one-photon mode, which exhibited similar fluorescence enhancement along with the increase of Na2S2 amount (Figure S-5, Supporting Information). Furthermore, we tested the specificity of QSn toward H2Sn by measuring the fluorescence responses to other reactive species including reactive nitrogen species (RNS: NO2−, NO3−, NO, ONOO−), reactive oxygen species (ROS: H2O2, ClO−, O2−, · OH, 1O2), and other reactive sulfur species (RSS: GSH, Cys, Hcy, S2−, S2O32−, SO32−, SO42−, S8). Except for the targets Na2S2 and Na2S4, all the other tested species caused very weak or negligible fluorescence enhancement of QSn (Figure 1d). Two-Photon Fluorescence Imaging of Exogenous and Endogenous H2Sn in HeLa Cells. Before utilizing QSn in bioimaging, the cytotoxicity and photostability of QSn were examined. The cytotoxicity test by tetrazolium-based colorimetric assay (MTT assay) demonstrates its low toxicity in that 95% of cells were variable after incubation with 5 μM QSn for 24 h (Figure S-6, Supporting Information). The photostability of the probe was tested by continuously illuminating QSnlabeled cells under TPM imaging conditions (which were adopted in the following bioimaging experiments) for 30 min. A negligible decrease of fluorescence intensity was observed (Figure S-7, Supporting Information), which guarantees the long-time observation of living organisms under two-photon excitation. As the first step of bioapplication, we used QSn to detect exogenous H2Sn in living cells. One group of HeLa cells were incubated with 5 μM QSn for 30 min, using another group without QSn loading as the negative control. The two-photon microscopy images show negligible background fluorescence in the negative control group (Figure 2a) and weak fluorescence in the QSn-labeled group (Figure 2b), which suggests the existence of physiological H2Sn in cells. To confirm the source of the weak fluorescence, we pretreated another group of cells with 2 mM NMM, a thiol-depleting agent, to remove physiological H2Sn. (The depletion of H2Sn by NMM was first verified in a solution assay, as shown in Figure S-8, Supporting Information). After 1 h of NMM-treatment, 5 μM QSn was added and allowed for 30 min incubation. Compared with Figure 2b, the fluorescence of this NMM-treated group (Figure 2c) is remarkably weakened, which demonstrates that QSn is sensitive enough to illuminate basal-level H2Sn in living cells. Thereafter, to examine the ability of QSn to recognize exogenous H2Sn, two other QSn-labeled groups were added with 0.5 mM Na2S2 and 0.5 mM Na2S4, respectively, and were incubated for another 30 min. It is clearly seen that the QSnlabeled cells added with Na2S2 or Na2S4 show much brighter fluorescence than that treated only with QSn (Figure 2d−f), which confirms the ability of QSn to recognize exogenous H2Sn in cells. Further, we made an effort to examine the capability of QSn for endogenous H2Sn detection. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are commonly regarded as the major enzymes for H2S production, which often jointly present and primarily locate in cytosol.29,30 Increasing studies in

nm (Φ = 0.01) and 534 nm (Φ= 0.57), respectively (Table S-1, Supporting Information). We also obtained the photophysics of QSn in varying solvents (Table S-2, Supporting Information). It is seen that the probe QSn is very weakly fluorescent, while the reaction product 5 shows 57-fold enhancement in quantum yield in the aqueous buffer, enabling highly sensitive target recognition. We contribute the fluorescence “off-on” switch to the hydroxyl bound−release process, which has been well documented and worked in many fluorescent probes.13,14,25−27 The maximal two-photon action cross-section (δmaxΦ, in which δ is the two-photon absorption cross-section and Φ is the quantum yield) value of 5 in PBS buffer was detected to be 50 GM at 730 nm (Figure 1a), whereas the δΦ value of QSn at this

Figure 1. (a) Two-photon active cross-section (δΦ) of 5 under 710− 800 nm excitation wavelengths. (b) Two-photon fluorescence spectra of QSn (10 μM) in the presence of increasing amounts of Na2S4 (0, 1, 2.5, 3.75, 5, 10, 15, 20, 30, 50, 100, 250, 500, 750, 1000 μM) and (c) the titration curve (inset: linear response at lower Na 2 S 4 concentrations). (d) Responses of 10 μM QSn to 100 μM RNS, ROS, RSS, and targets (1: QSn alone; 2−5: NaNO2, NaNO3, NO, ONOO−; 6−10: H2O2, ClO−, O2−, ·OH, 1O2; 11−18: GSH, Cys, Hcy, Na2S, Na2S2O3, Na2SO3, Na2SO4, S8; 19: Na2S2; 20: Na2S4, respectively). The above experiments were performed in PBS buffer (50 mM, pH 7.4, containing 0.9% NaCl and 25 μM CTAB).

wavelength was nearly undetectable, which implies that QSn could be an efficient two-photon probe for the target. The solubility of QSn, as determined with a fluorescence method (Figure S-2, Supporting Information), is approximately 5 μM in water, which is sufficient for cell staining. To ensure the biological application, we also tested the effect of pH on the fluorescence of both QSn and 5. As shown in Figure S-3, Supporting Information, the fluorescence emissions of the molecules are insensitive to pH within the physiological range 6.0−8.0. H2Sn Sensing Performance of QSn in Aqueous Buffer. Next, we investigated the response of QSn to H2Sn in phosphate-buffered saline (PBS, 50 mM, pH 7.4, 0.9% NaCl), containing 25 μM cetrimonium bromide (CTAB). As Scheme 2 demonstrates, H2S2 is the species that reacts with the probe. Considering the ready transformation from polysulfides to H2S2 under physiological conditions,6,7,9,28 we chose the sodium salts of polysulfides as the equivalents in the present study. The fluorescence titration of QSn by Na2S4 was performed under both one-photon (368 nm) and two-photon (730 nm) excitation, with the results shown in Figure S-4 (Supporting D

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Figure 3. (a−c) TPM images of HeLa cells incubated with 5 μM QSn for 60 min at 37 °C. The cells were transfected with (a) pRK-Flag vector, (b) CBS, and (c) CSE recombinant plasmids. Images were obtained with 730 nm excitation, and the emission was collected in the range of 410−650 nm. Scale bar: 20 μm. Cells shown are representative images from replicate experiments (n = 5). (d) Protein expression levels of HeLa cells analyzed by Western blot assay. (e) Normalized mean fluorescence intensity of images a−c.

Figure 2. TPM images of HeLa cells (a) untreated or (b) incubated with 5 μM QSn for 30 min at 37 °C. (c) Cells were pretreated with 2 mM NMM for 1 h and then incubated with QSn under the same condition. (d) Cells incubated with 5 μM QSn for 30 min at 37 °C and then further incubated with 0.5 mM Na2S2 or (e) 0.5 mM Na2S4 under the same condition. Images were obtained with 730 nm excitation, and the emission was collected in the range of 410−650 nm. Scale bar: 20 μm. Cells shown are representative images from replicate experiments (n = 5). (f) Normalized mean fluorescence intensity of images a−e.

exogenous and endogenous H2Sn verified, we last evaluated the usage of the probe in living zebrafish embryo, in view of both the satisfying sensing performance of QSn and the advantages of TPM. To this end, we incubated 3-day-old zebrafish embryo with 5 μM QSn in zebrafish embryo medium (E3M) at 28 °C for 30 min. The probe-loaded zebrafish embryo were then washed with E3M and incubated with 0.5 mM Na2S4 for another 30 min, followed by TPM imaging after being anesthetized. The probe-loaded sample exhibits a moderate fluorescence signal (Figure 4a), indicating the existence of

recent years propose that H2S produced by those enzymes can be stored as bound sulfane sulfur including polysulfides and persulfides (RSnR, R = alkyl or H, n ≥ 2) in the presence of oxygen within organisms.7,9,10,28,31 We thus inferred that the overexpression of CBS/CSE could result in the elevation of endogenous H2Sn level. Hence, we constructed the overexpression systems by transfecting CBS/CSE recombinant plasmids into HeLa cells (positive groups). At the same time, HeLa cells transfected with the empty vector, pRK-Flag, were set as negative control. Consistent with the condition of exogenous H2Sn cell imaging, the plasmid-transfected cells were incubated with 5 μM QSn for a total of 60 min. Images were also taken with two-photon excitation at 730 nm. As shown in Figure 3a−c, the positive groups exhibit fluorescence much stronger than that from cells of the negative group, thus suggesting the increased endogenous level of H2Sn in these CBS/CSE overexpressing cells. To provide experimental evidence for the protein overexpression, the cells were digested and subjected to Western blot assay. The results unambiguously proved the overexpression of CBS and CSE in the positive groups (Figure 3d). Furthermore, as illustrated by cell images and calculated average fluorescence intensities (Figure 3e), the CSE overexpressing cells show luminescence significantly brighter than that of the CBS overexpressing cells, with the same transfection amount. Our results demonstrate the higher H2Sn production in CSE overexpressing system, which agrees with the differentiated roles of CBS/CSE elucidated by a recently reported LC-MS/MS study32 as well as the enzymes and biochemistry involved in persulfide and H2S cross-talk.33 The above results have revealed the capability of QSn to discriminate endogenous H2Sn levels in living cells, which may lead to versatile applications of the probe in H2Snassociated biological/physiological processes. Two-Photon Fluorescence Imaging of H2Sn in Zebrafish. With the probing ability of QSn in cells for both

Figure 4. TPM images of zebrafish pretreated with 5 μM QSn for 30 min and then (a) washed with E3M and incubated in fresh E3M for 30 min or (b) washed and further incubated with 0.5 mM Na2S4 for 30 min. Scale bar in full view merge image: 500 μm. Scale bar in magnified images: 200 μm. (Magnified regions are marked with yellow squares in full view images.) Images were obtained with 730 nm excitation, and the emission was collected in the range of 410−650 nm. Zebrafish shown are representative images from replicate experiments (n = 3). E

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endogenous H2Sn in the zebrafish embryo. The sample treated with external Na2S4 shows enhanced fluorescence (Figure 4b), which represents the sum of both endogenous and exogenous H2Sn. We also manipulated z-scan on the Na2S4-treated embryo, from which the uneven staining at varying penetration depths can be observed (Figure 5a−c). To present clearer

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 86-27-8721-7886. Fax: 86-27-6875-4067. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support from the National Natural Science Foundation of China (no. 21375098). We also acknowledge the technical support from China Zebrafish Resource Center (CZRC) and Wuhan National Laboratory for Optoelectronics.

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Figure 5. TPM images of 5 μM QSn-labeled zebrafish after 0.5 mM Na2S4 treatment, incubated for 30 min at 28 °C. (a) Merged image of bright field and fluorescent images of the whole fish. (b) Accumulated fluorescent image of the whole fish. (c) Separate fluorescent images of the whole fish at different z-axis depth. Scale bar: 500 μm. (d−f) Accumulated fluorescence images from the head and body (0, 122.9, 245.8 μm), and tail (0, 61.5, 122.9 μm). Scale bar: 200 μm.

observations, we collected fluorescence images at different parts of the fish. The accumulated fluorescence images of selected regions in the embryo clearly demonstrate the distribution of H2Sn in its head, body, and tail (Figure 5d−f). Note that we used only 5 μM QSn for staining, and the embryos we used in the experiment were still alive after laser exposure, thus further suggesting the application prospect of QSn for in vivo imaging.

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CONCLUSIONS We have developed a two-photon excited fluorescent probe QSn for H2Sn. The probe is able to specifically detect H2Sn with a distinct fluorescence turn-on response. Combining its favorable two-photon fluorescence property, low cytotoxicity, and high photostability, we have used QSn to achieve the visualization of endogenous H2Sn in living cells for the first time. Moreover, the probe is also applicable for H2Sn detection in living zebrafish embryos. The results of this work suggest that the developed probe may find applications for in vivo H2Sn detection, which will help to clarify the roles and functions of H2Sn, the newly discovered reactive species, in various physiological and pathological processes. Furthermore, by using other fluorophores with a larger two-photon-active cross-section, it is reasonably expected that more H2Sn probes can be developed for better imaging performance in the future.

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ASSOCIATED CONTENT

S Supporting Information *

Solution preparation, water solubility assay, cytotoxicity assay, LC-MS trace, photophysical properties, and additional figures. This material is available free of charge via the Internet at http://pubs.acs.org. F

DOI: 10.1021/acs.analchem.5b00172 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry (31) Ishigami, M.; Hiraki, K.; Umemura, K.; Ogasawara, Y.; Ishii, K.; Kimura, H. Antioxid. Redox Signaling 2009, 11, 205−214. (32) Ida, T.; Sawa, T.; Ihara, H.; Tsuchiya, Y.; Watanabe, Y.; Kumagai, Y.; Suematsu, M.; Motohashi, H.; Fujii, S.; Matsunaga, T.; Yamamoto, M.; Ono, K.; Devarie-Baez, N. O.; Xian, M.; Fukuto, J. M.; Akaike, T. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 7606−7611. (33) Miranda, K. M.; Wink, D. A. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 7505−7506.

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DOI: 10.1021/acs.analchem.5b00172 Anal. Chem. XXXX, XXX, XXX−XXX