A Single Fluorescent Probe Separately and Continuously Visualize

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A Single Fluorescent Probe Separately and Continuously Visualize H2S and HClO in Lysosomes with Different Fluorescence Signals Mingguang Ren, Zihong Li, Beibei Deng, Li Wang, and Weiying Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05116 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019

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Analytical Chemistry

A Single Fluorescent Probe Separately and Continuously Visualize H2S and HClO in Lysosomes with Different Fluorescence Signals Mingguang Ren †, Zihong Li †, Beibei Deng †, Li Wang †, and Weiying Lin* † † Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials Science and Engineering, University of Jinan, Jinan, Shandong 250022, P.R. China. Fax: (+) 86-531-82769031, Email: [email protected]. ABSTRACT: A complicated relationship between the active small molecules exists in cells. On the organelle level, active small molecules also play an important role in the maintenance of organelle functions and roles. To investigate the relationship of biomolecules in subcellular, it is necessary and critical to develop molecular tools that can track two kinds of associated biomolecules within organelles with multiple fluorescence signals. However, this is still an unmet challenge up to date. Herein, we present the first single-fluorescent probe (Lyso-HA-HS) that can detect oxidative (HOCl) and reductive (H2S) substances within organelle (lysosomes) with multi-response signals. The reactions of the new probe with H2S and HOCl simultaneously result in the blue and red channels emission, respectively, providing different signal responses to the oxidative and reductive substances in the cellular lysosomes. Using a single fluorescent probe, we first achieved dual-channel imaging of the endogenous hypochlorous acid and hydrogen sulfide, respectively, in the lysosomes in the living cells. Moreover, the highly desirable attributes of the probe LysoHA-HS (such as high selectivity, good membrane-permeable and lysosomes enrichment ability) may enable it to be used in revealing the relationship of HOCl and H2S in lysosomes.

INTRODUCTION Various types of active small molecules (reactive nitrogen species, reactive oxygen species, reducing agents, etc.) are present in cells, which maintain the function of cells and play a very important role in a great deal of pathological and physiological processes. In addition, some active small molecules are in complex interactions. For example, HNO generation may associate with two gas transmitters NO and H2S. 1 In addition, H2S and Cys, 2 O2- and H2O2, 3 NO and ONOO, 4 H2O2 and HOCl, 5 etc. show a certain correlation between each other. In order to reveal the inter-relationship of active small molecules in different physiological processes, development of reporters that capable of displaying different signals for respective and continuous detection related active small molecules is highly valuable. Recently, there have been many outstanding achievements which could respectively or continuously detect two related active small molecules in cells with different signals models.614 However, all of these probes could only detect the related active small molecules in the whole cell instead of a specific organelle. More recently, well-designed fluorescent probes which could track the biomolecules of interest in the specific subcellular organelles have been constructed.15,16 However, there is scarce to describe a single fluorescent probe which could respectively or continuously detect two related active small molecules within organelles with different fluorescence signals. Lysosomes, spherical-shaped and catabolic organelles are the major digestive compartment within cells.17,18 The original knowledge of lysosomes was thought to be simple waste bags, but now they are involved in many cellular processes as advanced organelles and are considered key regulators of cell homeostasis.19 There was experimental evidence that

lysosomal dysfunction was associated with the pathogenesis of certain diseases such as Alzheimer’s disease (AD), cancer, neurodegenerative disorders, and cardiovascular diseases.19,20 Abnormal concentration of activity small molecule is regarded as an important factor leading to dysfunction of lysosome.21 Therefore, real-time detection and imaging of lysosomal biological species will help to understand the intracellular reaction kinetics and mechanisms, Furthermore, it further helped to formulate diagnostic and therapeutic strategies. Hypochorous acid (HOCl) is known to be one of the powerful microbicidal agent, which can be produced in immunological cells from hydrogen peroxide and chloride ions mediated by myeloperoxidase (MPO), which was also found in lysosome.22 In immune cells, HOCl plays important roles in defense against pathogens and microorganisms 23,24 Due to the important biological role of hypochlorite, some fluorescent probes for fluorescence imaging of intracellular HOCl have been designed and reported.25-30 At the organelle level, abnormal HOCl concentration leads to an imbalance of redox in the lysosome, further leading to lysosome dysfunction 22 In addition, high concentration of HOCl can induce apoptosis of cultured cells by lysosomal rupture.31 H2S was biosynthesis produced by enzymes catalyzed such as cystathionine β-synthase (CBS),32 cystathionine γ-lyase (CSE), 33 and 3-mercaptopyruvate sulphurtransferase (MST).34 At the cell organelles level, CBS is also found in the endosomal-lysosomal system.35 Furthermore, H2S also functions in lysosome organelles. H2S can induce cell death, which is related to the activation of calprotease protease, lysosomal instability and release of lysosomal protease.36 H2S and HOCl have interplaying roles in important physiological processes. H2S and HOCl are important

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mediators in brain function and disease. HOCl may contribute to the extensive oxidative stress and oxidative damage observed in human neurodegenerative diseases.37,38 H2S significantly inhibits plasmodium citrullinase inactivation and protein oxidation induced by HOCl, comparable to reduced glutathione.39 H2S also inhibits cytotoxicity induced by HOCl in cells, intracellular protein oxidation and lipid peroxidation.37,38 Hence, respective or continuous detection of H2S and HOCl would facilitate our understanding of the interplay and cross-talk of these two species in cells. In order to studies of the biological function of HOCl and H2S in lysosomes, much effort has been focused on the development of fluorescence probes to detect HOCl 40-44 and H2S 45-49 in lysosomes individually. For continuous detection of multiplex biomolecules in lysosomes, one way to solve the problem is to use multiple fluorescent probes in one system. However, the situation may be very complicated due to their spectral cross-talks, and the distinct localization and metabolisms. Therefore, to tackle the problem enumerated above, the development of an effective single molecule that possesses multiple recognition domains and distinct fluorescence signals to H2S and HOCl is in high demand and critical. Nevertheless, to our best knowledge, no single fluorescent molecular probes that could detect HOCl and H2S in lysosomes with multi-response signals have been revealed in the literature yet. To realize this goal, the major challenges include: 1) the probe should integrate multi-recognition domains, fluorescence reporters, and a lysosomal targeting group in a single molecule; 2) The fluorescence signals generated by responding to different analytes should have a sufficient signal interval to prevent interference between imaging channels; 3) The two reaction sites should have good selectivity and cannot interact with each other; 4) The probe should be able to respond to the two analytes separately or continuously with a reasonable signal response for different analytes. Herein, we reported the first lysosomes-targeted dualdetection fluorescent probe (Lyso-HA-HS) for sensing HOCl and H2S with different fluorescence signals. Lyso-HA-HS exhibited a dramatic fluorescence increase in the red channel in the presence of HOCl with excellent selectivity and high sensitivity. By contrast, with the addition of H2S, the probe displayed a huge fluorescence enhancement in the blue channel. In addition, the probe can be used for continuous detection of H2S and HOCl by fluorescence imaging in two channels. Significantly, Lyso-HA-HS is capable of respectively detecting endogenously produced H2S and HOCl by dual-color fluorescence imaging in lysosomes with a high localization coefficient between the probe and the lysosomal localization dye.

EXPERIMENTAL SECTION Materials and instruments. Reaction reagents used in the experiment and the stimulation and inhibitory reagents used in the biological experiments were purchased from the supplier and applied directly without further purification. The solvent used in the purification of the compound by column

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chromatography is analytically pure solvent without further purification. The water used in both the spectral and cell imaging experiments are twice-distilled water. The instruments used for absorption, emission and cell imaging are the same as those reported in previous literature. 50 Cells culture and Cytotoxicity assay. The protocols of the cells culture and cytotoxicity assay are similar to our previous report.51 Imaging of exogenous H2S and HOCl in HeLa cells. After incubation for 24 h in culture dishes, HeLa cells were washed with PBS and then treated with probe Lyso-HA-HS (5 μM) for further incubation for 20 min at 37 ˚C in the incubator. Probe-loaded HeLa cells were incubated with Na2S (50 μM) for 1 h or NaOCl (50 μM) 20 min at 37 ˚C after washing with PBS. Lastly, HeLa cells were washed with PBS three times, and prepared for imaging. Continuous imaging of exogenous H2S and HOCl in HeLa cells. After incubation for 24 h in culture dishes, HeLa cells were washed with PBS and then treated with probe Lyso-HAHS (5 μM) for further incubation for 20 min at 37 ˚C in the incubator. Probe-loaded HeLa cells were incubated with Na2S (50 μM) for 1 h and then washed with PBS three times, after that HeLa cells were further treated with the NaClO (50 μM) for 20 min. Lastly, HeLa cells were washed with PBS three times, and prepared for imaging. Determining the subcellular location of probe. For confirming the intracellular localization of the probe, HeLa cells or RAW 264.7 macrophage cells were used to co-localize of probe and Lysotracker. The cells were treated with LysoHA-HS (10 μM) and Lysotracker Green (0.5 μM) for 20 min and then treated with 100 μM Na2S or 50 μM NaOCl for another 20 min. Then the cells were washed by PBS prior to imaging. Test the co-localization coefficient of the blue channel (red channel) with the green channel separately. Imaging of endogenous H2S in HeLa cells. HeLa cells were divided into three groups and incubate for 24 h in culture dishes. The first control group of HeLa cells was incubated with 10 µM Lyso-HA-HS for 30 min. Then the cells were washed by PBS prior to imaging. The second group is the probe loaded HeLa cells incubated with Cysteine (100 µM/mL) for 2 h, then washed with PBS prior to imaging. For the third group, the probe loaded HeLa cells incubated with Cysteine (100 µM/mL) and PAG (200 µM/mL) were cultivation for 2 hours under the same conditions. HeLa cells were rinsed with PBS three times, and prepared for imaging. Imaging of endogenous HOCl in macrophage cells. RAW 264.7 macrophage cells were divided into three groups and incubate for 24 h in culture dishes. The first control group of RAW 264.7 cells was incubated with 10 µM Lyso-HA-HS for 30 min. Then the cells were washed by PBS prior to imaging. The second group is the probe loaded RAW 264.7 cells subsequently incubated with Lipopolysaccharide (LPS, 2 μg/mL) and Phorbol Myristate Acetate (PMA, 2 μg/mL) for 2 h prior to imaging. For the third group, the probe loaded RAW 264.7 cells subsequently incubated with Lipopolysaccharide (LPS, 2 μg/mL) / Phorbol Myristate Acetate (PMA, 2 μg/mL) and 4-aminobenzoic acid hydrazide (ABH, 200 μM) for 2 h prior to imaging. The cells were rinsed with PBS, and prepared for imaging.


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Analytical Chemistry Synthesis of compound Lyso-HA-HS. Compound PF-5 (50 mg，0.058 mmol, 1.0 eq) was dissolved in anhydrous DMF (5 mL), and then NaN3 (11.7 mg, 0.18 mmol, 3 eq) was added to the mixture and reacted at 100 oC overnight with inert atmosphere of nitrogen, the reaction solution was poured in to water 20 mL and extracted with EtOAc (3×30 mL). The organic layer was washed with water three times, brine one time and dried over Na2SO4. After filtration, the filtrate was removed under the reduced pressure and obtain crude product, and the crude product was further purified by column chromatography over silica gel eluting with methanol/dichloromethane (v/v 1:30). The final product LysoHA-HS is obtained of 47 mg (73 %) as a gray solid. 1H-NMR (400 MHz, DMSO-d6),  (ppm): δ 9.42 (s, 1H), 8.23 (s, 1H), 7.83 (dd, J = 18.3, 7.4 Hz, 2H), 7.67 – 7.51 (m, 2H), 7.25 (s, 1H), 7.19 (d, J = 7.9 Hz, 1H), 7.06 (d, J = 6.4 Hz, 1H), 6.65 (d, J = 7.2 Hz, 2H), 6.56 (d, J = 8.7 Hz, 1H), 6.46 (d, J = 8.7 Hz, 1H), 6.41 – 6.24 (m, 2H), 3.73 (s, 2H), 3.53 (s, 2H), 3.32 (dd, J = 23.9, 17.5 Hz, 10H), 3.19 (s, 2H), 2.84 (q, J = 14.3 Hz, 2H), 2.12 (s, 4H), 1.08 (t, J = 6.5 Hz, 6H); 13C-NMR (101 MHz, DMSO-d6)167.23 , 163.88, 163.35, 155.15, 153.46, 153.22, 151.82, 151.59, 148.93, 144.63, 142.72, 133.81, 131.02, 130.04, 129.84, 129.45, 129.15, 124.44, 123.49, 123.15, 116.66, 115.99, 111.76, 108.78, 108.40, 107.17, 104.27, 101.80, 97.29, 66.52, 65.40, 59.87, 52.86, 48.40, 47.77, 46.46, 44.11, 26.81, 12.86; HRMS (ESI) m/z calcd for C44H44N9O7 [M+1]+: 810.3364; found 810.3360.

RESULTS AND DISCUSSION Design and Synthesis of the Fluorescent probe LysoHA-HS.

Figure 1. Rational design of the probe Lyso-HA-HS reporting lysosomal HOCl, H2S, and HOCl/H2S with different fluorescence readouts.

In this work, we aimed to develop a single fluorescent probe which could response H2S, HOCl and H2S/HOCl in lysosomes using dual-channel imaging to present different fluorescence signal patterns. In order to image two analysts in dual-channel individually, two fluorophores linked with the corresponding recognition sites were needed in a single probe. We chose 7-azido-coumarin and dibenzoylhydrazinerhodamine as the platforms for imaging H2S and HOCl in blue channel and red channel, respectively. 7-aminocoumarin was

produced by 7-azidocoumarin reacted with H2S. Diformylhydrazine has been known to efficiently reaction site with HOCl by oxidizing diformylhydrazine into diformyldiimide.48 Morpholine, an directing group of lysosomes, is attached to rhodamine via a flexible chain, which can also improve the water solubility of the probe. Combined with the above elements, we designed and synthesized the first probe that could detect HOCl and H2S in lysosomes with multi-response signals. The synthetic route of the probe Lyso-HA-HS is shown in Scheme S-1, the related intermediates (shown in Scheme S-2) and the final product were fully characterized by standard 1H-NMR, 13C-NMR spectroscopy and MS.

Spectral Properties of Fluorescent probe Lyso-HAHS With Lyso-HA-HS in hand, the spectral properties of the probe in the absence or presence of the HOCl, H2S and HOCl/H2S were determined. As shown in Figure S1, LysoHA-HS has an obvious absorption peak at around 313 nm and almost no absorption above 400 nm. However, absorption peak around 380 nm significantly enhanced with the addition of Na2S (a commonly used H2S source). Compared with the control compounds C2 (Figure S2), this may be attributed to the changing of 7-azide to 7- amino group of the coumarin dye. This change in functional group also caused a significant change in the fluorescence quantum efficiency. The fluorescence quantum efficiencies of compounds C2 and C3 were 0.021 and 0.34, respectively, which was beneficial for the construction of fluorescent probes. Furthermore, the probe Lyso-HA-HS treated with NaOCl has apparent absorption at 550 nm, as their rhodamine dye trigger opened by HOCl (Figure S1). The absorption spectrum of C4 also has obvious absorption peak at 550 nm after treated with NaOCl, and the fluorescence intensity also increases with the addition of NaOCl with the fluorescence quantum efficiency increases from 0.017 to 0.28. (Figure S3). When excited at 380 nm or 550 nm, the probe shows no emission at 448 nm or 580 nm. However, when Na2S was added, 448 nm fluorescence will appear when excited at 380 nm and (Figure 2a), accompanied by obvious fluorescence color change from colorless to blue (Figure S4 and Figure S5). At room temperature, it only needs 3-4 min to reach plateau (Figure S6). While as the probe is treated with gradually increasing concentrations of HOCl and excited at 550 nm, the probe generates a red fluorescence centered at 580 nm, accompanied fluorescent color change from colorless to red (Fig 2b). The fluorescence intensity of the probe reaches its maximum within seconds (Figure S7). The emission intensity with the concentrations of Na2S (or NaOCl) shows a linear relationship (Figure S8 and Figure S9). This result indicated that Lyso-HA-HS was suitable for the quantitative determination of H2S. The detection limit was calculated to be 3.4×10-7 M and 7.3×10-8 M to H2S and HOCl respectively. These results indicate that the probe Lyso-HAHS selectively responded to H2S and HOCl respectively with different fluorescence signal pattern. Furthermore, the probe has a large signal resolution of 132 nm between the fluorescence peaks of H2S and HOCl responses, which reduces



mutual interference between the two fluorescent peaks and reduces the occurrence of cross-color during cell imaging.

Figure 2. (a) Changes of the emission spectra of Lyso-HA-HS (10 μM) with the addition of different equivalents of Na2S (0−10 equiv) excited at 380 nm (b) The emission spectra of Lyso-HAHS (10 μM) with the addition of different equivalents of NaOCl (0−5 equiv) excited at 550 nm. c) Emission spectra of Lyso-HAHS (10 μM) treated with 5 equiv NaOCl and then added with different equivalents of Na2S (0−20 equiv). d) Emission spectra of Lyso-HA-HS (10 μM) treated with 10 equiv Na2S and then added with different equivalents of NaOCl (0−15 equiv).

Moreover, we want to further explore the fluorescent signal response mode of the probe when successively treated with H2S and HOCl and different order of addition of them is there any effect on the fluorescence spectra. Whereas the fluorescence of the probe reaching the plateau at 580 nm after reacted with HOCl and then treated with H2S, the fluorescence at 448 nm also gradually enhanced with the increase of H2S concentration (Fig 2c). The same way, excited at 380 nm a dramatic fluorescence enhancement around 580 nm is shown with the addition of HOCl after treated the probe with H2S reaching the plateau (Fig 2d). At the same time, there is not much change in the fluorescence peak of 448 nm with the addition of HOCl. Due to a minimal spectral overlap between the emission spectral of 7- amino coumarin and the absorption spectral of rhodamine (Figure S10), the energy cannot be passed from coumarin moiety to rhodamine section by FRET. These results confirmed that although the FRET process does not exist in the molecule, the fluorescent molecules can be responded to H2S and HOCl with different fluorescence signal pattern. To confirm the sensing mechanism, the products of Lyso-HA-HS reacted with HOCl, H2S and H2S/HOCl was confirmed by HRMS respectively (Fig. S29-32). The product of the reaction Lyso-HA-HS reacted with HOCl, H2S and H2S/HOCl were determined to be compound P1-P3.

Effect of pH and sensing selectivity Due to the presence of several types of hydrolytic enzymes, the matrix in lysosomes maintains acidic environment (pH about 5.0).18 To monitor H2S and HOCl in lysosomes, the probe should remain stable and can respond to analytes under acidic environment. Under the pH of test solution around 5.0, the fluorescence spectra of the probe were measured with

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Na2S and NaOCl added separately and continuously (Figure S11). The experimental results show that the spectra changes were similar to the previously tested (Figure 2), except a weakened of the fluorescence intensity enhancement. As shown in Figure S12, the probe Lyso-HA-HS was stable over a wide pH range (2.8-11.3) and provides well spectral response to H2S and HOCl at pH 5-10, indicating that the probe Lyso-HA-HS can endure the acidity of the lysosome and has strong spectral response to the analyte. Compared with the previously reported literature, we found that the probe has a different sensitivity to acidity.47 We speculate that the difference between the sensitivity of Lyso-HA-HS probe and reported compound to acids was mainly due to the different types of atoms connected with spiral nitrogen, and the different test conditions. To study the selectivity of Lyso-HAHS towards H2S and HOCl, the probe was incubated with various cations, anions, reactive oxygen/nitrogen species and other relevant species. As shown in Figure S14, upon excited at 380 nm, the fluorescence enhancement of Lyso-HA-HS induced by H2S is much significant than that caused by other reducing substance (Cys, GHS and Hcy). However, other species have little interference with the probe emission spectra at 448 nm, indicating that the probe is a selective sensor for H2S within the emission wavelength of this segment. Similar to this, other species only induce minimum perturbation in fluorescence spectra of probe Lyso-HA-HS at 580 nm. While HOCl elicits a significant fluorescence increase at 580 nm (490-folds) While HOCl elicits the probe a large enhancement at the 580 nm emission and a 490-fold increase in fluorescence intensity (Figure S15), indicating that the probe has well selectivity to HOCl in this emission region. From what has been discussed above, the probe has highly selectivity to H2S and HOCl in two different fluorescent emission regions, and such good results allow the probe to perform fluorescence imaging in living cells at different fluorescent channels.

Fluorescence Imaging of H2S and HOCl in Live Cells

We have investigated the spectral response of the probe Lyso-HA-HS to H2S and HOCl, the effect of pH on response, and selectivity in vitro. The results show that the probe can detect H2S and HOCl separately or continuously at different emission wavelengths with well selectivity. We continue to evaluate the capabilities of the probe to image the intracellular H2S and HOCl. First, the cytotoxicity of the probe was tested by the MTT assay using HeLa cell lines, and the results show that the probe Lyso-HA-HS exhibited low cytotoxicity at low micromolar concentrations after a long period (24 h) (Figure S16). Thus, the low cytotoxicity properties of the probe may render the probe Lyso-HA-HS suitable for imaging H2S and HOCl in living cells. Through the spectrum test, it can be known that there is almost no overlap in the spectrum of the fluorescence response of the H2S and HOCl, about 130 nm between two emission peaks, which facilitates dual-channel imaging of the two analytes of the probe. So we used a dualchannel mode in the detections of H2S and HOCl in living cells. Based on the spectral data, blue channel (excitation at 405 nm and the emission collection at 425−475 nm) was set for the detection of H2S. Meanwhile, red channel (excitation at 561 nm and the emission collection at 570−620 nm) was used


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Analytical Chemistry for the detection of HOCl. HeLa cells only treated with LysoHA-HS almost no fluorescence is detected in both channels. However, the blue channel shows strong fluorescence emission when the probe-loaded HeLa cells further treated with Na2S (Figure 3b), but fluorescence signal still could not be detected in the red channel, meaning that the probe LysoHA-HS can be used to image H2S in the living cells in the blue channel. Further, we tested the ability of the probe to image HOCl in HeLa cell in the red channel. A strong fluorescent signal was detected in red channel when probeloaded HeLa cells were further treated with NaOCl (Figure 3f). At the same time, the enhancement of fluorescence signal in blue channel was not detected. These data indicate that the probe Lyso-HA-HS can be used to image intracellular HOCl in living cells. We further tested the continuous identification ability of Lyso-HA-HS to continuously respond to H2S and HOCl in living cells. The probe-loaded HeLa cells were incubated with Na2S for 1h and then washed with PBS. The living cells were further treated with the NaClO for 20 min. Significant fluorescence signals enhancement appear in both channels (Figure 3n and 3o). Adjusting the order of addition of the two analytes can get the same result (Figure 3r and 3s). Thus, the overall results demonstrate that Lyso-HA-HS can separately and continuously monitor intracellular H2S and HOCl in two channel imaging without interference between each other.

red channel(o) and merge image (p) of HeLa cells preincubated with Lyso-HA-HS (5 µM) for 30 min, then treated with Na2S (50 µM) for another 1h, washed with PBS three times and further loaded with NaOCl (50 µM) for another 20 min. (q-t) Bright field (q); blue channel(r); and red channel(s) and merge image (t) of HeLa cells preincubated with Lyso-HA-HS (5 µM) for 30 min, then treated with NaOCl (50 µM) for another 20 min, washed with PBS three times and further loaded with Na2S (50 µM) for another 1h.

Morpholine, as targeting group of lysosome, can significantly increase the probability of probe enrichment in lysosomes. 53,54 In order to verify whether the probe Lyso-HA-HS will be located in the lysosome, the probe was coincubated in HeLa cells with commercial lysosomal dye (LysoTracker Green), and then treated with Na2S or NaOCl respectively. As illustrated in Fig. 4, the fluorescence signal from the blue channel in the presence of H2S overlaid very well with the fluorescence of LysoTracker Green. In the presence of HOCl, there is also a well overlap between the fluorescence imaging of the red channel and the LysoTracker Green (Figure S17). Pearson's co-localization coefficients (describing the correlation of intensity distribution between the two channels) were calculated to be 0.89 and 0.86, respectively, confirming Lyso-HA-HS localization in lysosomes of living cells.

Figure 4 Probe Lyso-HA-HS and LysoTracker Green colocalization imaging in HeLa cells. a) brightfield image of HeLa cells; b) from blue channel (imaging of H2S); c) from the red channel; d) from the green channel (lysosomes staining); e) merge of green and blue channels; f) Intensity profile of linear region of (e) across the HeLa cell; g) Intensity scatter plot of blue and green channels.

Figure 3. Separately and continuously images of H2S and HOCl in HeLa cells using probe Lyso-HA-HS. (a-d) Bright field (a); blue channel(b); and red channel(c) and merge image (d) of HeLa cells only incubated with probe Lyso-HA-HS (5 µM) for 30 min. (e-h) Bright field (e); blue channel(f); and red channel(g) and merge image (h) of HeLa cells incubated with Lyso-HA-HS (5 µM) for 30 min and then further incubated with Na2S (50 µM) for another 1h. (i-l) Bright field (i); blue channel(j); and red channel(k) and merge image (l) of HeLa cells incubated with Lyso-HA-HS (5 µM) for 30 min and then further incubated with NaOCl (50 µM) for another 20 min. (m-p) Bright field (m); blue channel(n); and

Figure 5. (A) Imaging of endogenous H2S in HeLa cells using probe Lyso-HA-HS. (a) HeLa cells only treated with the probe (10 µM) for 30 min; (b) HeLa cells stimulated with cysteine (100 μM/mL) and then incubated with probe (10 µM) for 1h; (c) HeLa



cells stimulated with cysteine (100 μM/mL), PAG (200 μM/mL) and then incubated with probe (10 µM) for 1h. (B) Imaging of endogenous HOCl in Raw 264.7 macrophage cells using probe Lyso-HA-HS. (e) Raw 264.7 cells only treated with the probe (10 µM) for 30 min; (f) Cells were preincubated with probe (10 µM) for 30 min and then stimulated with LPS (2 μg/mL)/PMA (2 μg/mL) for 1 h; (g) Cells were preincubated with probe (10 µM) and treated with LPS (2 μg/mL)/PMA (2 μg/mL) and ABH (200 μM) for 1 h.

Imaging of endogenous H2S and HOCl in Live Cells

According to the above experiments, the probe Lyso-HA-HS can separately and continuously image exogenous H2S and HOCl in different channels. We were interested in further checking whether the probe can image endogenous H2S and HOCl in lysosomes with dual-color manner. Cysteine as a precursor can produce endogenous biosynthesis H2S under the catalysis of cystathionine β-synthase (CBS) inside the cell.2 HeLa cells were treated with 100 µM cysteine and incubated for 1 hour, and then treated with 10 µM Lyso-HA-HS exhibiting a dramatic fluorescence signal enhancement in the blue channel compared with the HeLa cells without cysteine stimulated. To further verify that the enhancement of this fluorescent signal observed above was due to endogenous H2S induced. Propargylglycine (PAG) acts as an inhibitor will limit the production of endogenous H2S.55 Comparison of the image data, when HeLa cells treated with cysteine (100 µM) and PAG (200 µM) at the same time, the fluorescence intensity from the blue channel is obviously reduced indicating that the enhancement of the fluorescent signal was indeed caused by endogenous production H2S. We further investigated whether the probe Lyso-HA-HS could fluorescence image endogenous HOCl in living macrophage cells in the red channel. According to the literature, endogenous HOCl will be produced when the RAW264.7 cells were stimulated by phorbol myristate acetate (PMA) and lipopolysaccharides (LPS) together.56 A marked fluorescent signal enhancement in red channel (Figure 5f) was observed in the RAW 264.7 cells, which was treated with LPS (2 µg/mL) and PMA (2 µg/mL). In contrast, the RAW264.7 macrophage cells which were incubated with Lyso-HA-HS almost no fluorescent signal was detected in the red channel (Figure 5e). In order to verify that the fluorescence enhancement is caused by the endogenous HOCl, we performed control experiment to inhibit the activity of MPO enzyme using ABH as an inhibitor, thereby reducing the concentration of endogenous HOCl.57 The intensity of the fluorescent signal in red channel does have suppressed when the stimulated RAW 264.7 cells were treated with 200 µM ABH, meaning that the change of fluorescence signal in the red channel was caused induced by endogenous HOCl. The data above suggested that the probe Lyso-HA-HS was capable of fluorescence imaging of endogenous H2S and HOCl in living cells in the blue and red channel respectively. Furthermore, we tried to simultaneously fluorescence image endogenous H2S and HOCl by means of dual stimulation of RAW 264.7 cells, as treated cells with cysteine (100 µM) and LPS/PMA (2/2 µg/mL) together. However, we only detected the endogenous H2S in blue channel, while endogenous HOCl was not detected in the red channel. We speculate that it may be due to interference caused by two stimulation pathways. In the future work, we will further study and try to solve this problem.

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CONCLUSION In summary, we have engineered the first fluorescent probe, Lyso-HA-HS, which can simultaneously detect of HOCl and H2S in lysosomes with multi-response signals. In addition, the probe displays highly favorable properties; high selectivity, well membrane-permeability and high colocalization coefficient. These critical attributes enable tracking of endogenous H2S and HOCl at lysosomes in living cells using a single fluorescence probe for the first time. Thus, we expect that the probe will be a powerful molecular tool for studying the relationship between the redox balance and the function of the lysosomal in living cells. Moreover, the rational design strategy may be extended to construct powerful fluorescent probes for tracking related active small molecules in other organelles.

ASSOCIATED CONTENT Supporting Information

Experimental procedures and some spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author [email protected]

Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT This work was financially supported by NSFC (21472067, 21502067, 21672083, 21877048), the Natural Science Foundation of Shandong Province, China (ZR2014BP001), Taishan Scholar Foundation (TS 201511041), and the startup fund of University of Jinan (309-10004, 160082101).

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Analytical Chemistry

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A Single Fluorescent Probe Separately and Continuously Visualize

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