A Phenylselenium-Substituted BODIPY ... - ACS Publications

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A Phenylselenium-Substituted BODIPY Fluorescent Turn-off Probe for Fluorescence Imaging of Hydrogen Sulfide in Living Cells Deyan Gong,† Xiangtao Zhu,‡ Yuejun Tian,§ Shi-Chong Han,‡ Min Deng,† Anam Iqbal,† Weisheng Liu,† Wenwu Qin,*,† and Huichen Guo*,‡ †

Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, P. R. China ‡ State Key Laboratory of Veterinary Etiological Biology and Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou, Gansu Province 730046, P. R. China § Institute of Urology, The Second Hospital of Lanzhou University, Lanzhou, Gansu Province, P. R. China S Supporting Information *

ABSTRACT: Herein a phenylselenium-substituted BODIPY (1) fluorescent turn-off sensor was developed for the purpose to achieve excellent selectivity and sensitivity for H2S detection based on the substitution reaction of the phenylselenide group at the 3-position with H2S. The excess addition of hydrogen sulfide promoted further substitution of the phenylselenide group at the 5-position of the probe and was accompanied by a further decrease in fluorescence emission intensity. Sensor 1 demonstrated remarkable performance with 49-fold red color fluorescence intensity decrease at longer excitation wavelength, a low detection limit (0.0025 μM), and specific fluorescent response toward H2S over anions, biothiols, and other amino acids in neutral media. It showed no obvious cell toxicity and good membrane permeability, which was well exploited for intracellular H2S detection and imaging through fluorescence microscopy imaging.

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raphy,10 and sulfide precipitation.11 However, these techniques do not allow the temporal and spatial monitoring of reactive and transient H2S, and complicated sample preparation and tissue or cell destruction are required.12,13 Fluorescent imaging methods are highly desirable and offer high sensitivity as well as real-time imaging. A variety of innovative H2S fluorescent probes based on different reaction mechanisms have been reported, which rely mainly on the reduction of azide and/or nitroso to amine mediated by H2S,14−16 nucleophilic addition of H2S,17−19 copper precipitation,20,21 or thiolysis of leaving groups.12,22−24 Several fluorescent sensors containing selenium as leaving groups for detection of the sulfhydryl groupcontaining biomolecules have been explored.25−27 Seungyoon et al. have synthesized a phenylselenium-substituted coumarin sensor to detect glutathione (GSH) over cysteine (Cys)/ homocysteine (Hcy) with excellent selectivity and fast reaction

ydrogen sulfide (H2S) is a weak acid in aqueous solution, equilibrating mainly with HS− and notorious for its unique odor of rotten eggs. It has emerged as a member of the endogenous gaseous transmitters of signaling molecules existing in the human body, which regulates cardiovascular, neuronal, and immune systems.1−3 Research has also indicated that an abnormal level of H2S is related to diseases such as Down syndrome,4 diabetes, arterial and pulmonary hypertension, and Alzheimer’s disease.5,6 The biological concentration of sulfide levels range from 10 to 100 μM in blood plasma.7 Despite the fact that H2S has been linked to diverse physiological and pathological processes, a large part of its underlying molecular events are still indistinct and could be explored in depth.5 Obviously, it is highly valuable to develop superior techniques or prospective chemicals with remarkable selectivity and sensitivity for H2S detection in complicated biological systems. Coincident with the increased biological importance of H2S, new approaches for H2S quantification are rapidly emerging. Current strategies that are available for the detection of H2S include colorimetry,8 electrochemical assay,9 gas chromatog© 2017 American Chemical Society

Received: October 19, 2016 Accepted: January 13, 2017 Published: January 13, 2017 1801

DOI: 10.1021/acs.analchem.6b04114 Anal. Chem. 2017, 89, 1801−1807

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Analytical Chemistry

Scheme 1. Chemical Structure of Fluorescent Probe BOD-PhSe (1) and a Plausible Mechanism of Its Reaction with H2S

Hz, H-a), 6.83 (d, 2H, J = 4.0 Hz, H-b), 7.51 (m, 4H (Ph)), 7.58 (t, 1H, J = 7.6 Hz, H-c); mass spectrum (ESI), m/z 336.9 (M); 316.9 (M − HF, 100%) (C15H9BCl2F2N2 requires m/z 336.9592). Synthesis of BOD-PhSe (1): 100 mg (0.297 mmol) of compound 2 was dissolved in 25 mL of acetonitrile, and then 150 mg of anhydrous potassium carbonate (1.085 mmol) and 88 μL of benzeneselenol (90%, 0.742 mmol) were added successively; then the reaction mixture was heated under reflux. The reaction was completed after about 10 h (monitored by silica gel TLC using 5:1 petroleum ether−ethyl acetate). The organic layer was concentrated under diminished pressure and then fractionated by silica gel flash chromatography (petroleum ether/ethyl acetate =10:1) to give compound 1 as a deep purple solid (106 mg) in 62% yield, mp 107−109 °C. Rf 0.34 (petroleum ether/ethyl acetate = 5:1). 1H NMR (CDCl3): δ 5.892 (d, 2H, J = 4.4 Hz, H-a), 6.561 (d, 2H, J = 4.4 Hz, H-b), 7.430 (m, 11H (Ph)), 7.753 (d, 4H, J = 6.4 Hz, H-d); 13C NMR (CDCl3): δ 120.73, 126.71, 127.81, 128.38, 129.10, 129.27, 129.57, 129.81, 129.98, 130.29, 131.59, 133.77, 136.55, 137.29, 138.37, 153.41. HRMS: calcd for 1 (C27H19BF2N2Se2) 579.9940, found 579.9940. Measurements of Photophysical Properties and Time-Resolved Emission Spectra Experiments. Surfactant Triton X-100 was used to improve the water-solubility of the probe. 1 was diluted in Triton X-100/DMSO/HEPES buffer (0.01:1:9, v/v/v, 10 mM, pH 7.4) and then incubated at 37 °C in a thermostatic water bath for the titration and selectivity experiment. Time-resolved emission spectra experiments (TRES) were recorded using an FLS920 fluorescence spectrometer. The recorded column diagrams were fitted by Gaussian-weighted nonlinear least-squares in the software package, and accurate results were obtained after changing parameters to get the minimum χ2. Global analyses were calculated through FAST software. Cell Culture and Confocal Microscopy Imaging. Baby hamster kidney (BHK) cells were incubated in culture medium overnight at 37 °C under a 5% CO2 and 95% relative humidity atmosphere. Cells were plated on glass coverslips, allowed to adhere for 24 h, and incubated at 37 °C. Cells lines were treated with 1 (10 μM) in 1.0 mL of culture medium subjoined with

rate, which is based on the substitution reaction of the phenylselenide group with the sulfhydryl group of GSH.27 To our knowledge, reports on fluorescent probes containing the phenylselenide group as the leaving group to detect H2S are scarce. Various BODIPY derivatives as fluorescent probes demonstrating excellent performance have been synthesized for the detection of a variety of sensing targets, such as pH,28 cations,29 biothiols, and so on.30−32 3,5-Diphenylselanyl-8-phenyl-4,4difluoro-4-bora-3a,4a-diaza-s-indacene (1) as a fluorescence turn-off H2S sensor has been synthesized in this study according to the method of Seungyoon et al.27 Scheme 1 illustrates a feasible mechanism for its excellent sensitivity to H2S, which is based on the substitution reaction of the phenylselenide group at the 3-position with H2S. Excess addition of hydrogen sulfide promoted further substitution at the 5-position of the probe, achieving excellent sensitive and selective H2S detection for H2S-triggered fluorescence turn-off nature.22,31,33 The excellent H2S sensing performance of 1 as well as its biological fluorescent labeling has been well visualized.

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EXPERIMENTAL SECTION Instruments and Reagents. Benzeneselenol (90%) was procured from Heowns. Sodium sulfide nonahydrate was purchased from Guangfu and was used as the hydrogen sulfide source in all the experiments. (Caution! Benzeneselenol and H2S have a very unpleasant smell and are poisonous at higher concentrations. They should be handled with great care and in small amounts.) All experiments were carried out with commercially available reagents and solvents and used without further purification. Deionized water was used. NMR spectra were acquired on a Bruker spectrometer, and mass spectra were taken on Bruker DRX-400 and Bruker microTOF-Q II mass spectrometers. Fluorescence spectra were recorded on an FLS920 fluorescence spectrometer and Hitachi F-7000 spectrofluorimeter. Cresyl violet in methanol (λex = 582 nm, ϕf = 0.54) was used as fluorescence standard for the quantum yield test.34 Synthesis of Probe BOD-PhSe (1). As shown in Scheme 1, 3,5-dichloro-BODIPY (2) was prepared according to the known procedure.35 1H NMR (CDCl3): δ 6.42 (d, 2H, J = 4.0 1802


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Analytical Chemistry 5% DMSO for 2 h; then Na2S solution was added to the cells, incubated for another 30 min, and washed three times with prewarmed phosphate-buffered saline before imaging with an Olympus FV1000-IX81 laser confocal microscope at an excitation wavelength of 559 nm. The other group was used as the control group, which was incubated merely with 1 (10 μM) under identical conditions for 2.5 h. Cytotoxicity Tests. The in vitro cytotoxicity was tested using a standard 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide (MTT) assay in cell lines. After BHK cells were incubated in culture media in 96-well plates overnight, different concentrations of probe 1 were added to the wells of the treatment group and incubated for another 24 h. A German Berthold Mithras2LB943 reader was used to measure the absorbance value at 450 nm.

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RESULTS AND DISCUSSION Spectroscopic Studies of BOD-PhSe as a Na2S Fluorescent Probe. The absorption and emission spectra of 1 were recorded and are shown in Figure S1. In UV−vis absorption spectra, 1 exhibited a narrow band centered at 587 nm that belongs to the 0-0 vibrational band of S0-S1 transition and a less pronounced shoulder (0-1 vibrational band of S0-S1 transition) at shorter wavelengths, which resemble those from previously reported BODIPY dyes. Moreover, the weaker, broader absorption band centered at ∼390 nm belongs to the S0-S2 transition. 1 showed a fluorescence emission band centered at ∼610 nm, and the ϕ f value was 0.299. Spectroscopic/photophysical data of 1 are summarized in Table S1. In comparison with the phenoxy (phenyl-O-, λabs = ∼520 nm and λem = ∼530 nm)36 and sulfur (-S-, λabs = ∼570 nm and λem = ∼580 nm)37 disubstituted BODIPY analogues, not only the absorption but also the emission maxima of the phenylselenium-substituted BODIPY became deeper red. The properties of the BODIPY dye show interesting optical changes when substitution groups change from oxygen to sulfur to selenium.38 Incubation of 100 μM Na2S with 1 (10 μM) in Triton X100/DMSO/HEPES buffer (0.01:1:9, v/v/v, 10 mM, pH 7.4) at 37 °C after 20 min resulted in a significant (∼71 nm; from 587 to 516 nm) blue shift in the absorption spectrum and was accompanied by a significant fluorescence intensity decrease (Figure S1, Table S1). The UV−vis absorption and fluorescence spectral changes of 1 in the reaction with Na2S are shown in Figure 1. With the increasing Na2S concentration in the range of 0−20 μM, the intensity of the maximum absorption wavelength band centered at 587 nm of 1 increased with a slightly hypsochromic shift, while new signals appeared at ∼390 nm and ∼516 nm with increasing intensity. The related peak changes at 587/390 nm with increasing Na2S concentration are attributed to the substitution of the phenylselenide group with H2S at the 3-position of 1. In the Na2S concentration range of 20−85 μM, excess addition of hydrogen sulfide promoted further substitution of the phenylselenide group at the 5-position of the probe and was accompanied by a decrease in the intensity of the absorption band at around 516 and 552 nm. The absorption band at 587 nm disappeared (Figure S1). A ratiometric absorption change with an isosbetic point at 540 nm was readily detected in the Na2S concentration range of 20−85 μM, indicating the presence of different species. The course of the reaction demonstrated color variation from purple to pink in the sunlight (Figure 1a, inset). Probe 1 exhibited a strong and

Figure 1. (a) Absorption and (b) fluorescence changes of 1 (10 μM) with Na2S. Inset: (a) visible and (b) fluorescence change, under a 365 nm UV lamp, of 1 with Na2S. (c) The calibration curve of 1 quenched by Na2S excited at 582 nm. Relative standard deviation (RSD) is 7.5%, n = 4. All spectra were obtained after 20 min of incubation of 1 with100 μM Na2S.

broad emission band centered at 610 nm when excited at 582 nm. An increasing concentration of Na2S quenched the fluorescence emission intensity of 1 with a slightly hypsochromic shift from 610 to 602 nm. The quantum yield ϕf of 1 decreased from 0.299 in the absence of Na2S to 0.008 with addition of 10 equiv of Na2S. Upon reacting with H2S, the phenylselenide group in 1 was substituted by the sulfhydryl group of H2S, causing a 37-fold decrease in quantum yield and demonstrating a H2S-triggered fluorescence turn-off nature. As 1803


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Figure 2. Partial 1H NMR spectra in DMSO-d6 at 25 °C: (a) Probe 1; (b) probe 1 with Na2S·9H2O (1 equiv), 30 min; (c) probe 1 with Na2S·9H2O (7 equiv), 30 min.

shown in Figure S1c, when excited at 500 nm, probe 1 exhibited two weak broad emission bands centered at 558 and 609 nm. The weaker emission centered at 558 nm is assigned to the 0-1 vibrational band of the S0-S1 transition. Upon addition of different concentrations of Na2S within 20 min, the fluorescence intensity of 1 at 609 nm (red) underwent a significant decrease while the broad emission at 558 nm (green) increased gradually. A distinct ratiometric fluorescence change with an isoemissive point at 588 nm was readily detected as the concentration of Na2S increased. The changes in fluorescence wavelengths also illustrate that the chemical transformation and the increasing emission at 558 nm are attributed to the formation of sulfhydryl derivatives. The quantum yield of the emission band at 558 nm was very low, and the emission increases slightly at 558 nm after the addition of Na2S; as shown in Figure 1b, inset, there was almost no green fluorescence under a 365 nm UV lamp. It is not novel enough to apply 1 as a ratiometric fluorescence probe in consideration of the low sensitive detection of H2S; thus, further work was not carried out. The time-dependent fluorescence response of 1 (10 μM) in the presence of Na2S was investigated. Upon addition of 10 equiv of Na2S, the emission band centered at 610 nm quickly decreased within 10 min and then slowly decreased and reached a relative minimum after about 20 min (Figure S2). Therefore, the detection of Na2S was achieved through the detection of probe 1 within 20 min. Figure 1c shows a plot of 1 quenched by Na2S and the correction curve of fluorescent strength (F) versus Na2S amount.

The emission band centered at 610 nm quickly decreased in correspondence with the amount of Na2S in the range of 0−15 μM and slowly decreased upon increasing the Na 2 S concentration in the range of 15−100 μM. The relative contributions of this trend are attributed in the same way, as absorption signals change in the two different ranges of Na2S concentration. The detection limit (DL) of 1 for Na2S was calculated to be 0.0025 μM (0−15 μM) from the calibration curve with the following equation: DL = 3.3σ/k (σ: the standard deviation of fluorescence spectrometer, k: the slope of the correction curve). As shown in Table S2, the DL acquired by our strategy was well below the reported ranges. Comparative tests have been conducted to examine the effect of the usage of Triton X-100 and DMSO on the fluorescent detection experiments. When no Triton was added, the usage of DMSO should be increased to dissolve the probe 1 well; DMSO/HEPES buffer was used at a suitable proportion (1:1, v/v) for our test. Figure S3 shows time-dependent fluorescence spectra, titration, and selective fluorescence spectra of probe 1 for Na2S. The reaction of Na2S in DMSO/HEPES buffer (1:1, v/v) showed a similar fluorescence response upon addition of Na2S and biothiols as in Triton X-100/DMSO/HEPES buffer (0.01:1:9, v/v/v) (Figure S3b,c). On the one hand, DMSO of up to 50% volume ratio had to be used together with the probe. A high percentage of organic solvent is probably harmful for the cells and limits its range of use. On the other hand, as shown in the time course in Figure S3a, upon addition of 10 equiv of Na2S, the emission band centered at 610 nm decreased slowly and reached a relative minimum in about 50 min. Therefore, the reaction rate of Na2S in DMSO/HEPES buffer (1:1, v/v) was slower than in Triton X-100/DMSO/HEPES buffer (0.01:1:9, v/v/v). Thus, Triton X-100/DMSO/HEPES buffer (0.01:1:9, v/v/v) was a better choice for the detection of H2S. Mechanism Exploration. According to the reported literature, upon reacting with H2S, the fluorophore group

F = 3472.09 − 223.14 × [Na 2S] (r = 0.9895, [Na 2S] = 0−15 μM)

(1) 1804


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Analytical Chemistry substituted by the H2S sulfhydryl group quenches the fluorescence for the H2S-triggered fluorescence turn-off nature. Scheme 1 illustrates a feasible mechanism for its excellent sensitivity to H2S. To identify the substitution reaction with H2S, the 1H NMR spectra of 1 in a solution of DMSO-d6 with Na2S·9H2O (1 equiv or 7 equiv) at 30 min reaction time were monitored at 25 °C (Figure 2). Because of the symmetrical structure of 1, the signals of the pyrrole rings’ four protons of 1 appear at 5.86 and 6.70 ppm, respectively. After 30 min of incubation with 1 equiv of Na2S·9H2O, the 1H NMR spectral peaks belonging to the phenylselenide group at 7.75 ppm moved to the multiple peaks range of 7.16−7.30 ppm for the phenylselenide leaving group. The 1H NMR peaks belonging to the pyrrole rings’ protons at 5.86 ppm moved to 5.98 ppm, and two new peaks at 6.03 and 6.10 ppm assigned to the pyrrole rings emerged concomitantly. It is confirmed that the symmetrical structure of 1 was destroyed by the substitution reaction of the phenylselenide group by H2S at the 3-position. The MS spectrum of 1 (Figure S4) in EtOH with Na2S (1 equiv) at 2 h reaction time at 25 °C was measured to characterize this reaction further. The predicted product is BOD-SH (the molecular weight of [BOD-SH] (C21H15BF2N2SSe) is 456.0). In Figure S4, m/z 495.1 (calcd = 495.0) belonging to [BOD-SH + K] was clearly observed. The 1H NMR peaks belonging to the pyrrole rings all moved to the range of 6.52−6.60 ppm and formed multiple peaks with 7 equiv of excess Na2S (Figure 2). The multiple peaks range of 6.52−6.60 ppm was assigned to the symmetrical pyrrole rings’ protons of BOD-2SH because of the further substitution of the phenylselenide group at the 5-position of 1. After treatment with Na2S, the two phenylselenide groups of 1 were substituted by the sulfhydryl group in a two-step substitution reaction. Selective Fluorescence Response of 1 Turn-off Fluorescence toward Na2S. To investigate the selectivity in aqueous solution, various representative intracellular species were added to a solution of chemodosimeter 1 (10 μM) in Triton X-100/DMSO/HEPES buffer (0.01:1:9, v/v/v, 10 mM, pH 7.4). As shown in Figure 3, only Na2S decreases the fluorescence intensity dramatically at about 49-fold, while other analytes were silent to 1 under the same condition. Figure S5a shows the detailed emission response spectra of 1 upon addition of 10 equiv of biothiols and Na2S, and Figure S5b shows the competition graph of 1 in the presence of 10 equiv of biothiols with Na2S. This clearly shows that the turn-off fluorescence response of 1 with Na2S was also demonstrated significantly and was scarcely disturbed by concomitant biothiols. In addition, 1 showed pH-dependence in the detection of H2S. As shown in Figure S6, the fluorescence intensity of 1 was slightly changed in the pH value scope of 5−9. Moreover, in the incubation of Na2S, the turn-off fluorescent response was also demonstrated significantly and is relatively steady in a wide pH scope of 6−9; however, an exception displayed at pH 5 implied that the thiolysis reaction of the phenylselenide group could not proceed to a great extent with Na2S in the acidic surrounding. 1 can be used for the detection of H2S at a wider pH range (pH 6−9), within which the majority of biological samples (5.25−8.93) can be detected. pH value alterations show no strong disturbance to H2S detection of chemodosimeter 1 in solution. To examine the reversibility of the processes, an amount of benzeneselenol was added to the 1−Na2S solution mixture (1 (10 μM) reacted with 40 μM Na2S in Triton X-100/DMSO/

Figure 3. Fluorescence responses at λem = 610 nm of 1 (10 μM) reacted with 0.1 mM different kinds of species. All spectra were obtained after 50 min of incubation with different analytes, RSD is about 6.4%, n = 4, λex =582 nm. (1) Probe 1, (2) Na2S, (3) HSO3−, (4) F−, (5) Cl−, (6) Br−, (7) I−, (8) CH3COO−, (9) SCN−, (10) N3−, (11) NO3−, (12) SO42−, (13) CO3−, (14) H2PO4−, (15) ClO−, (16) H2O2, (17) Pro, (18) Ile, (19) Ala, (20) His, (21) Glu, (22) Tyr, (23) Lys, (24) Met, (25) Asp, (26) Phe, (27) Arg, (28) Gly, (29) Ser, (30) Cys, (31) Hcy, (32) GSH, (33) ascorbic acid.

HEPES buffer (0.01:1:9, v/v/v, 10 mM, pH 7.4) at 37 °C for 20 min was prepared as the 1−Na2S solution mixture). The fluorescence spectra of 1−Na2S as a function of benzeneselenol concentration are shown in Figure S7. Upon increasing benzeneselenol concentration, the emission intensity with a maximum at 610 nm increased slightly in the range of 0−180 μM and then remained constant. It is not novel enough to apply 1 as a fluorescence probe for the detection of benzeneselenol in consideration of the low substitution reaction rate, probability, and sensitivity with benzeneselenol; thus, further work was not carried out. Fluorescence Decay Traces of 1 toward Different Concentrations of Na2S. Fluorescence decay traces of 1 or 1 at different concentrations of Na2S in Triton X-100/DMSO/ HEPES buffer (0.01:1:9, v/v/v, 10 mM, pH 7.4) at 37 °C were tracked at several emission wavelengths through a singlephoton timing method.39 For the 1−Na2S solution mixture, global analyses in terms of decay time τi and αi were performed, incorporating in a single decay surface curves recorded 20 min after Na2S addition at the same excitation wavelength (560 nm) but at different emission wavelengths (590, 610, 630, and 650 nm) (Table S3, Figure S8, Figure S9). τi were linked for decay traces measured at different λem by using FAST software. It can be expected that global analysis with four decay traces would estimate the {τi, αi} values with higher accuracy than singlecurve analysis. The fluorescence decay spectra of probe were recorded and globally analyzed as biexponential profiles. With the increasing detection wavelength λem in the range of 590−650 nm, an enhancement was displayed in the related amplitude of the slow constituent τ1 (α1 = 1% at 590 nm, α1 = 4% at 610 nm, α1 = 5% at 630 nm, α1 = 5% at 650 nm) (Table S3). τ2 = ∼1.04 ns is the major decay constituent belonging to the fluorescence lifetime of the BODIPY chromophore, while τ1 = ∼2.73 ns can be attributed to the lifetime of formation of aggregates (formed because of the π−π-stacking effect) of 1 in Triton X-100/ DMSO/HEPES buffer. 1805


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cells were treated with 1 to assess the capability and its potential in bioimaging of H2S. Prior to the bioimaging studies, a good level of cell viability, low cytotoxicity, and good biocompatibility in the presence of 1 was confirmed by MTT assay (Figure S10). As a control, fluorescent confocal images of BHK cells treated with and without Na2S were both taken and compared. They clearly indicated that the 1-stained cells not only kept good shape and appeared viable but also displayed remarkable red emission attributed to the fluorescence of 1 under monoemission mode imaging (Figure 5a). Overlays of

Upon addition of different concentrations of Na2S to 1, the fluorescence decay obviously changed in the presence of Na2S at a concentration of 0, 10, 25, and 100 μM after 20 min (Figure 4). Triexponential analyses were performed, incorpo-

Figure 4. Representative time-resolved emission spectra of 1 (10 μM) in the absence and presence of different concentrations of Na2S in Triton X-100/DMSO/HEPES buffer (0.01:1:9, v/v/v, 10 mM, pH 7.4) and 1. All spectra were obtained after 20 min of incubation with Na2S. λex = 560 nm, λem = 610 nm. Figure 5. BHK cells confocal fluorescence microscopy imaging: (a) Fluorescence images of BHK cells incubated with 1 (10 μM). (b) Images of BHK cells incubated with 100 μM Na2S and 1 (10 μM). Scale bar = 7.5 μm.

rating decay spectra; detailed photophysical properties of sensor 1 regarding decay times τ1, τ2, and τ3 are listed in Table S3. At a detection emission wavelength of 610 nm, τ1 and τ2 of the solution mixture were almost constant (∼2.73 and ∼1.04 ns), but the longer lifetime increased with the amplitude (4% to 40%) and the shorter lifetime decreased with the amplitude (96% to 43%). A new decay time τ3 (∼0.49 ns) appeared, and then the amplitude (20% to 17%) remained almost constant upon increasing the concentration of Na2S. A new fast decay can be attributed to the presence of Na2S. The intramolecular charge transfer (ICT) from the negative charge of S− to the electron-withdrawing BODIPY moiety was enhanced. In general, the decay profiles of 1 for the addition of of Na2S are complicated due to the involvement of at least two or three different species and the likely presence of multiple states. A possible explanation for the absorption, fluorescence emission, and lifetime changes of 1 with Na2S involves a monoor disubstituted process of -SH, indicating the presence of three different species. One is disubstituted phenylselenium 1 (λabs = 587 nm and λem = 610 nm) with strong fluorescence, and the other is monosubstituted phenylselenium BOD-SH (λabs = 558 nm) with low fluorescence. The last one is BOD-2SH (λabs = 500 nm) with low fluorescence. It is proposed that the acid− base equilibrium of BOD-SH ⇋ BOD-S− occurs at pH = 7.4. The ϕf values of mono- or disubstitution of the 3,5-dichloroBODIPY chromophore with the S-nucleophile group are quite high.37 Because of the excited-state ICT effect from the negatively charged -S− (BOD-SH ⇋ BOD-S−) to the BODIPY acceptor which is electron deficient, the ϕf values of the sulfhydryl derivatives product were low. Visualizing Na2S in Living Cells. As mentioned above, 1 showed little fluorescence response to other anions and biological thiols, which makes 1 promising for specific recognition of aqueous sulfide in live cells. Initially, BHK

confocal fluorescence and bright-field images demonstrated that the fluorescence was evident in the cytoplasm at levels higher than in the nucleus and membrane. The other group treated with Na2S displayed negligible fluorescence when 1-treated cells were then exposed to 100 μM Na2S for 30 min at 37 °C (Figure 5b). Clearly, these data demonstrate the potential of 1 as a sensitive fluorescent probe for specific recognition of aqueous sulfides and fluorescent image analysis, which can be successfully used to monitor the presence of Na2S.

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CONCLUSIONS A novel fluorescent turn-off sensor 1 for H2S based on a twostep substitution of the phenylselenide group of 1 by H2S was identified, which is very helpful for the further design of fluorescent probes for H2S detection. Sensor 1 displayed an excellent selective sensitivity fluorescence response toward H2S, which afforded a DL of 0.0025 μM (0−15 μM). More notably, the chemodosimeter 1 could be demonstrated to label cells and monitor H2S in living cells by confocal fluorescence imaging successfully. The design strategy and H2S-triggered fluorescence turn-off nature reported here should be applicable to develop a wide range of practically H2S fluorescent sensors with high selectivity and sensitivity.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b04114. 1806


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Spectroscopic/photophysical data and figures (Figure S1−S10). Supplementary data and compound characterization data (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Fax: +86-931-8912582. E-mail: [email protected]. *E-mail: [email protected]. ORCID

Wenwu Qin: 0000-0002-9782-6647 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the National Natural Science Foundation of China (no. 21271094), the Ministry of Science and Technology of China (2014DFA31890), and the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (grant no. J1103307).

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