Visualization of in Vivo Hydrogen Sulfide Production by a

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Visualization of In Vivo Hydrogen Sulfide Production by a Novel Bioluminescence Probe in Cancer Cells and Nude Mice Xiaodong Tian, Zhiyan Li, Choiwan Lau, and Jianzhong Lu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03712 • Publication Date (Web): 20 Oct 2015 Downloaded from http://pubs.acs.org on October 25, 2015

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Analytical Chemistry

Visualization of In Vivo Hydrogen Sulfide Production by a Novel Bioluminescence Probe in Cancer Cells and Nude Mice Xiaodong Tian‡, Zhiyan Li‡, Choiwan Lau, and Jianzhong Lu* School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China Email: [email protected]; Tel: 0086-21-51980058

ABSTRACT: Hydrogen sulfide (H2S) has emerged as an exciting endogenous gasotransmitter in addition to nitric oxide and carbon monoxide. However, its precise measurement in living cells and animals remains a challenge. In this study, a novel bioluminescence H2S probe was designed and synthesized by modifying the 6’-amino group of D-aminoluciferin into a 6’-azido group, which was highly selective against other reactive sulfur, nitrogen, and oxygen species. Our H2S probe azidoluciferin sensitively reacted with H2S to release D-aminoluciferin with a strong bioluminescence signal. Based on its high selectivity and sensitivity, the H2S probe was used to detect H2S production in live cancer cells and nude mice. The bioluminescence signal decreased in mice treated with propargylglycine, an inhibitor of H2S, suggesting that our H2S probe can detect endogenous H2S in real time, in vivo. Overall, the excellent sensing properties of the probe combined with its bioimaging capability make it a useful tool to study H2S biological roles.

INTRODUCTION Hydrogen sulfide (H2S) is known as the third endogenous gasotransmitter1 after carbon monoxide (CO) and nitric oxide (NO),2 although H2S is long known for its toxicity and unpleasant smell of rotten eggs.3,4 The significance of H2S has been validated in a variety of physiological processes such as cardiovascular protection,5 vasodilation,6 cell growth,5 and neuromodulation.2 Abnormal H2S regulation is related with diabetes,7 hypertension,6 Alzheimer's disease,8 and Down syndrome.3 In addition, H2S plays roles in some specific cellular H2S targets such as nitric oxide,9 heme proteins,10 cysteine residues on KATP channels,11 and many other emerging targets. In these biosystems, endogenous H2S is derived primarily from the catalytic action of several enzymes: cystathionine-βsynthase (CBS), cystathionine-γ-lyase (CSE), 3mercaptopyruvate sulfurtransferase (3-MST), and cysteine lyase (CL) on the cysteine-related substrates.10,12 Therefore, a highly selective and sensitive analytical tool is urgently needed to detect H2S in these complicated biosystems. Traditional analytical methods such as gas chromatography,13 electrochemical method,14 colorimetric assay,15 and metal-induced sulfide precipitation,16 which are highly sensitive and could satisfy the needs for their application, have been used for the detection of H2S. However, they are often limited by poor compatibility with live cells, because of post-mortem destruction of tissues or cell lysates.2 Fortunately, reaction-based methods of H2S detection have been rapidly developed, which can offer a higher spatial and temporal resolution and more favorable compatibility in biological samples than these traditional analytical methods. The mechanisms behind these new methods involve H2S as a nucleophile to attack activated

electrophiles17-19 or precipitated metal salts20 or as a reductant to reduce azide21-26 or nitro group27 on masked fluorophores. In particular, the azide-H2S reaction is well established chemistry for the design of H2S probes, and several fluorescent probes for the specific detection of H2S have been reported recently23-26. However, there are few examples of in vivo imaging of H2S, and imaging deep mammalian tissue remains a frontier field for H2S detection. At the heart of the matter is a lack of sensitivity and depth penetration due to background autofluorescence, light scattering, photoactivation/photobleaching, etc. Bioluminescence imaging obviates many of the intrinsic limitations of fluorescence imaging. The technique is based on the sensitive detection of visible light produced during luciferase-mediated oxidation of a small molecule substrate (luciferin or aminoluciferin) with ATP, Mg2+, and O2.28 Bioluminescence imaging is gaining appreciation for its use in real-time animal imaging, as the total absence of tissue luminescence bestows negligible background, enabling exquisite sensitivity.29 Many types of luciferase transgenic mice as well as disease specific models are now commercially available. Therefore, designing a selective and sensitive firefly luciferase-based probe to detect H2S in cancer cells and nude mice is a thoughtprovoking topic. Based on previous reports using H2S to reduce azide, we hypothesized that a bioluminescent H2S probe could be developed by combining H2S-mediated azide reduction with a luciferin-derived sensing platform (Scheme 1). This highlights that the 6’-amino (or 6’-hydroxyl) group is a key part for enzyme combinations. Therefore, caging 6’amino (or 6’-hydroxyl) of aminoluciferin (or luciferin) can

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hide its interaction with firefly luciferase, which consequently quenches the bioluminescence.30

Scheme 1. Reduction of azidoluciferin with H2S generates the parent aminoluciferin. Subsequent reaction with luciferase generates bioluminescence.

EXPERIMENTAL SECTION Materials and Apparatus: Millipore water was used to prepare all aqueous solutions. The bioluminescent images of the cells and model mice were determined with a Xenogen IVIS Spectrum imaging system (Caliper Life Sciences, USA). Silica gel plates were used, with UV light (254 nm) for visualization. All NMR data was collected using a Bruker 600 MHz. Mass spectroscopy data was collected on a Triple TOFTM 5600 instrument. Infrared spectroscopy data was collected on a Thermo Nicolet instrument. Luciferase was purchased from Promega. DL-propargylglycine (PAG), ATP and NaHS were purchased from Sigma-aldrich. Synthesis of D-Aminoluciferin: D-Aminoluciferin was synthesized by modifying a literature procedure.31 Dcysteine hydrochloride monohydrate (2830 mg, 13.00 mmol) was dissolved in PBS (10 mM, pH 7.4). The appropriate 2-cyano-6-aminobenzothiazole (910 mg, 5.20 mmol) was dissolved in methanol. Then, the solution of 2-cyano6-aminobenzothiazole was slowly injected into the solution of D-cysteine hydrochloride monohydrate. After the reaction mixture was stirred for 24 h at room temperature. Equal volume of water was added to dilute the reaction mixture. Following that, the pH of the mixture was adjusted to 8.5. Volatile components of the mixture were then removed under vacuum. The aqueous layer was extracted with ethyl acetate for three times. Next, the aqueous layer was acidified (pH 3.5) back. Ultimately, the aqueous layer was extracted with ethyl acetate for three times, organics dried over MgSO4, filtered and subjected to rotary evaporation to yield D-aminoluciferin as an orange solid (943 mg, 65 %). 1H NMR (600 MHz, DMSO-d6) δ = 13.02 (s, 1H)，7.78 (d, J=8.8 Hz, 1H), 7.08 (d, J=2.1 Hz, 1H), 6.86 (dd, J=8.8, 2.2 Hz, 1H), 5.85 (s, 2H), 5.37 (dd, J=9.7, 8.2 Hz, 1H), 3.73 (dd, J=11.1, 9.8 Hz, 1H), 3.64 (dd, J=11.1, 8.2 Hz, 1H); 13C NMR (151 MHz, DMSO-d6) δ = 171.2, 164.2, 153.0, 149.1, 144.0, 137.7, 124.4, 115.8, 102.9, 77.8, 34.4;

HRMS C11H9N3O2S2 278.0064.

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calcd.278.0063,

found

Synthesis of Azidoluciferin: Our H2S probe was synthesized as follows.3,32 The appropriate D-aminoluciferin (180 mg, 0.65 mmol) was dissolved in 18 mL dry CH3CN. After cooling the solution to 0℃, 0.12 mL (0.91 mol) of tert-butyl nitrite (tert-BuONO) was added drop-wise. The reaction mixture was stirred for about 1 h and then 0.12 mL (1.04 mmol) of azidotrimethylsilane (TMS-N3) was added. The reaction mixture was transferred to room temperature and stirred overnight. After the completion of reaction by TLC monitoring (ethyl acetate/methanol 3:1), the solvent was evaporated, the residue was further purified by the silica gel chromatography to afford azidoluciferin as a yellow solid (184 mg, 93 %). 1H NMR (600 MHz, DMSO-d6) δ =13.25 (s, 1H), 8.16 (d, J=8.8 Hz, 1H), 8.06 (d, J=2.2 Hz, 1H), 7.32 (dd, J=8.8, 2.3 Hz, 1H), 5.45 (dd, J=9.7, 8.4 Hz, 1H), 3.80 (dd, J=11.0, 10.1 Hz, 1H), 3.71 (dd, J=11.2, 8.3 Hz, 1H); 13C NMR (151 MHz, DMSO-d6) δ = 170.9, 164.2, 160.1, 149.9, 138.9, 136.8, 125.0, 119.3, 112.6, 78.0, 34.7; IR (cm-1): 2123 [ν(N3)], 1741 (νCO), 1588, 1489, 1441, 1296, 1220, 1128, 1032, 911, 867, 812; HRMS C11H7N5O2S2 [M-H]calcd.303.9968, found 303.9958. Optimization of Azidoluciferin Concentration in vitro: Because of its poor solubility in water, 32 mM of azidoluciferin was prepared in buffered (10 mM pH 7.4 PBS) aqueous DMSO solution (H2O/DMSO = 1:9, V/V). Then the probe was diluted into varied concentrations using PBS (pH 7.4). Accordingly, its concentrationsdependent luminescence changes (6.25, 12.5, 25, 50, 100, 200, 400, 800, 1600 and 3200 μM) with NaHS (250 μM) were tested in PBS (pH 7.4). As corresponding blank controls, PBS (pH 7.4) buffer were added instead of NaHS under the same experimental conditions. After 60 min incubation at room temperature in the dark, the solution of each tube (100 μL) were added 1 μL ATP (100 mM) and 3 μL (100 μg/mL) luciferase, and then the total flux was immediately measured. Selectivity Measurement: Azidoluciferin (100 μM) were mixed with the solutions of 0 μM NaHS (PBS), 160 μM NaHS, 1 mM glutathione (GSH), 15 μM cysteine (Cys), 1 mM Na2S2O3.5H2O, 1 mM (NH4)2SO4, 1 mM Na2SO3, 1 mM NaNO3, 1 mM NaNO2, 1 mM NaCl or 1 mM NaHCO3 in PBS (pH 7.4), respectively. The reactions were performed at room temperature in the dark for 60 min, 1 μL of ATP (100 mM) and 3 μL (100 μg/mL) of luciferase were added into each tube, and then the total flux was immediately measured. Sensitivity Measurement: Azidoluciferin (100 μM) was respectively mixed with various concentrations of NaHS in PBS buffer (pH 7.4). As a blank control, the same volume of PBS (pH 7.4) was mixed with the probe (100 μM) instead of NaHS. Samples and blank control were incu-


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bated for 60 min at room temperature in the dark prior to analysis, 1 μL of ATP (100 mM) and 3 μL (100 μg/mL) of luciferase were added, and then the total flux was immediately measured. Azidoluciferin Kinetics in Bulk PBS: The reactions of azidoluciferin (100 μM) with three concentrations of NaHS (0.25, 0.50 and 1.00 mM) were carried out in PBS (pH 7.4). As corresponding blank controls, PBS (pH 7.4) were added instead of NaHS solution under the same experimental conditions. After each pair of reaction and blank solutions (100 μL) were incubated at room temperature in the dark for 0, 5, 10, 15, 30, 60, 120, 180, 240 and 300 min, 1 μL (100 mM) of ATP and 3 μL (100 μg/mL) of luciferase were added into each tube, and then the total flux was immediately measured. Toxicity Analysis: Cell viability was assessed by CCK8 in HEK 293T cell line. Firstly, cells were seeded onto 96-well plates (5000 cells per well) in the growth medium and incubated until 80 % density. Then, azidoluciferin (0, 15.63, 62.50, 125, 250, 500, 1000 and 2000 μM with 0.5 % DMSO) were added into each well, respectively. After an overnight incubation at 37 ℃ in a humidified atmosphere in a 5 % CO2 incubator, 10 μL of CCK8 solution was added into each well. After 3 h incubation, absorbance at 450 nm was measured. Cell Bioluminescence Imaging of Various Concentrations Exogenous H2S: Huh7 cells were grown in cell culture flask. After a 24-h incubation, the cells were collected and suspended with appropriate volume PBS (pH 7.4). Then, the cells (5 x 105 cells per well), azidoluciferin (200 μM) were mixed with various concentrations of NaHS (0, 50, 100, 250, 500, 750, 1000 and 1500 μM) in black 96-well plates. Luminescent signal (photons per second) for each well was immediately measured by an imaging system. Cell Bioluminescence Imaging of Endogenous H2S: Huh7 cells were grown in cell culture flask. After a 24-h incubation, the cells were collected and suspended with appropriate volume PBS (pH 7.4). Equal cells were added into three wells of black-96-well plates (5 x 105 cells per well). After PAG (50 μM), an external inhibitor of endogenous H2S, was incubated with the cells for 30 min in the first well, azidoluciferin (200 μM) was added into the first, second and third well, respectively. Following that, NaHS (250 μM) were immediately mixed into the second well. Meanwhile, the third well was added equal volume PBS (pH 7.4) as a control. Bioluminescence response was quickly measured at 1, 2, 4, 6, 8 and 15 min by an imaging system.

trol in the absence of exogenous H2S on the first day. Light production was measured at 4, 9, and 18 min. The next day, 200 μL of azidoluciferin (5 mM with 10 % DMSO) was injected as described above into the same mouse, followed by an intratumoral injection of 100 μL NaHS (30 mM). Light production was also measured at 4, 9, and 18 min. Bioluminescence Imaging of Endogenous H2S in vivo: Briefly, 200 μL of azidoluciferin (5 mM with 10 % DMSO) was injected into another mouse as a control on the first day. Light production was then measured at 3, 9, and 13 min. On the second day, the same mouse was intratumorally injected with 50 μL of PAG (20 mM). After incubation for 10 min, 200 μL of azidoluciferin (5 mM with 10 % DMSO) was injected into the mouse and the light production was measured at 3, 9, and 13 min. RESULTS AND DISCUSSION Synthesis: Consequently, as depicted in Figure 1, our bioluminescent H2S probe, i.e., azidoluciferin, was synthesized in two steps from commercially available starting material (2cyano-6-aminobenzothiazole) with a good yield.

Figure 1. Synthesis of azidoluciferin. a) H-D-CysOH.HCl.H2O (2.5 equiv), room temperature, 24 h, 65%. b) tert-BuONO (1.4 equiv), in dry CH3CN, 0°C, 1h, then TMS-N3 (1.6 equiv), room temperature, overnight, 93%.

Optimization of Azidoluciferin Concentration in vitro: Azidoluciferin concentration was evaluated by comparing the F-F0, where F represents the total flux of samples and F0 represent the blank total flux. In the probe concentration optimization experiments, the probe concentration ranged from 6.25 to 3200 μM was investigated. As expected, both the background and target signal increased with the probe concentration at the beginning whereas target signal gradually decreased after peaking (data not shown). For the F-F0, it first increased as the probe concentration was elevated, and reached a maximum at 100 μM and then decreased. Figure 2 showed that 100 μM of azidoluciferin was a suitable concentration for monitoring H2S in vitro.

Bioluminescence Imaging of Exogenous H2S in vivo: Briefly, 200 μL of azidoluciferin (5 mM with 10% DMSO) was injected through the tail vein into a mouse as a con-



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Sensitivity Measurement: Sensitivity is another significant parameter to evaluate the performance of a new probe. Based on the 3σ method, our limit of detection was estimated to be 0.1 μM, which was much lower than that of most of the existing small-molecule probes for H2S.3,19,37Additionally, azidoluciferin also showed a good log-linearity (R2=0.9959) between the F-F0 and NaHS concentrations in the range of 10 to 1000 μM (Figure 4). The high sensitivity of our H2S probe may allow the detection of intracellular H2S, which is a problem with most known probes.5,38

Figure 2. Concentration-dependent F-F0 changes of azidoluciferin with NaHS at 250 μM. Samples and blanks were incubated for 60 min in pH 7.4 PBS buffer at room temperature prior to analysis.

Selectivity Measurement: To our delight, azidoluciferin is a poor substrate for firefly luciferase, which leads to an extremely weak luminescence. In such a case, we first tested the selectivity of azidoluciferin in vitro by comparing the F-F0 of the reaction products between our probe and biologically relevant reactive sulfur, nitrogen, and oxygen species. Note that the concentrations of Cys were reported to be about 13 and 10 μM in rat and human physiological conditions, respectively.33,34 In mammals, the concentration of H2S was around 160 μM in tissues.35,36 Therefore, we chose Cys (15 μM) and H2S (160 μM) as our tested concentrations. As shown in Figure 3, 15 μM of Cys indeed triggered weak bioluminescence, however, it is almost negligible in comparison with that induced by 160 μM NaHS (about 8 fold luminescence enhancement than that induced by 15 μM Cys). Therefore, our H2S probe azidoluciferin indeed showed an excellent selectivity toward H2S over other testing species, indicating that azidoluciferin has a reasonable activity to capture H2S.

Figure 4. The log-linear relationship between F-F0 and the concentration of NaHS. Azidoluciferin (100 μM) was respectively mixed with various concentrations of NaHS (10-1000 μM) in PBS buffer (pH 7.4). As a blank, the same volume of PBS (pH 7.4) was mixed with azidoluciferin (100 μM) instead of NaHS. Samples and blank were incubated for 60 min at room temperature prior to analysis.

Azidoluciferin Kinetics in Bulk PBS: Time-dependent luminescence responses of our H2S probe to different concentrations of NaHS (0.25, 0.50 and 1.00 mM) were evaluated in PBS. As shown in Figure 5, NaHS concentrations were varied, and the luminescence response was NaHS-dependent with higher NaHS concentrations increasing the reaction rate, where a maximum was reached much faster at a higher NaHS concentration and the reaction dynamics behavior of the probe with NaHS was thus mainly controlled by the concentration-related collision probability. Data showed that a 60-min incubation was suitable for the investigation of the reaction between azidoluciferin and NaHS in bulk PBS.

Figure 3. F-F0 changes of azidoluciferin (100 μM) with the tested species in PBS (pH 7.4): 1, 160 μM NaHS; 2, 15 μM Cys; 2− 2− 2− 3, 1 mM GSH; 4, 1 mM S2O3 ; 5, 1 mM SO4 ; 6, 1 mM SO3 ; 7, − − − − 1 mM NO3 ; 8, 1 mM NO2 ; 9, 1 mM HCO3 ; 10, 1 mM Cl .


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Analytical Chemistry conventional measurements in bulk PBS, the velocity of the reaction was increased for the small-volume reactions. These differences can be attributed to differences in the reaction format. The bulk PBS is a closed system, and consequently reaction products are contained and can result in NaHS depletion and product inhibition, this can give the appearance of a decreased reaction rate. The cell system allows continuous exchange of NaHS and product inside or outside the cells. This exchange, coupled with the small volume of the reaction, helps to maintain NaHS levels, reduce product inhibition, and prevent mass diffusion limitations. Thus, a short incubation time was suitable for the investigation of the reaction between azidoluciferin and NaHS in the cells.

Figure 5. Time-dependent F-F0 changes of azidoluciferin with NaHS (azidoluciferin, 100 μM; NaHS, 0.25, 0.50 and 1.00 mM) in PBS (pH 7.4) at room temperature.

Toxicity Analysis: To prove that azidoluciferin can be used to monitor H2S in living cells, a key question is whether the cells maintain viable and healthy during the test. Therefore, the influence of azidoluciferin on cell viability and proliferation was examined by the CCK8 assay. Figure 6 showed that cell viability was slightly affected by azidoluciferin at the concentration used.

Figure 6. Cell viability of azidoluciferin in HEK 293T cell line shown by CCK8 assay.

Cell Bioluminescence Imaging of Various Concentrations Exogenous H2S: Based on our result in vitro, azidoluciferin was further used for detecting exogenous H2S in living Huh7 cells. It was found the bioluminescence intensity was dependent on the concentration of NaHS (0 - 1500 μM) (Figure 7a, b) and as low as 50 μM of H2S could be detected in the living cells. Since the cells belong to the small-volume reaction containers, the reaction kinetic characteristics of azidoluciferin were further evaluated using three concentrations of NaHS (0.25, 0.50 and 1.00 mM). As depicted in Figure 7c, the probe kinetics in cells was sharply different from that in PBS, a much faster kinetics was observed in the cells. When compared to the

Figure 7. Imaging of exogenous H2S in Huh7 cells. a) Bioluminescence imaging of Huh7 cells incubated with azidoluciferin and various concentrations of NaHS. b) Linear relationship between the total flux and NaHS concentration. c) Time-dependent total flux changes of azidoluciferin with



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NaHS (azidoluciferin, 200 μM; NaHS, 0.25, 0.50 and 1.00 mM) in cells.

Cell Bioluminescence Imaging of Endogenous H2S: Incubation of Huh7 cells with our probe azidoluciferin yielded obvious bioluminescence, which was likely dependent on the H2S produced endogenously by the living cells. To date, a few probes detecting intracellular H2S have been reported.4,17,21,39-46 Therefore, our major concern was whether our probe could detect endogenously produced H2S. Previous reports have shown that GSH is reduced to Cys by γ-glutamyl transpeptidase47 and Cys is converted to H2S by CSE and CBS.48 Therefore, we considered PAG to be an inhibitor of CSE, which can decrease endogenous H2S level in living Huh7 cells. Our results (Figure 8), as especially at 6 min point, clearly showed that endogenous H2S was gradually decreased with the addition of PAG, since the intensity of bioluminescence of PAG group was weaker than that of control group. Data clearly demonstrated that our probe was possible for detection of endogenous H2S in biological samples.

Figure 9. Bioluminescence imaging of exogenous H2S in Huh7 xenografts in nude mice. a) Bioluminescent images acquired at 4, 9, and 18 min from one mouse treated with exogenous H2S or the control. b) Total flux measured at 4, 9, and 18 min from one mouse treated with exogenous H2S or the control. Figure 8. Bioluminescence images of PAG-treated cells, exogenous H2S and control were acquired at 1, 2, 4, 6, 8 and 15 min in PBS (pH 7.4).

Bioluminescence Imaging of Exogenous H2S in vivo: To further investigate whether azidoluciferin could detect H2S in vivo, a live animal imaging test should be conducted. Hence, the subcutaneous tumor xenografts of a Huh7 cell line that constitutively expressed luciferase in nude mice were used as the animal model (the details see the supporting information). Due to individual difference, tumor size is often different sharply for different mouse, the same mouse acts as its own control. As shown in Figure 9, compared with the control group, higher bioluminescence signals were observed at different time points after the injection of NaHS. The results further confirmed that our probe could be used to image exogenous H2S activity in xenografted liver cancer tumors in mice.

Bioluminescence Imaging of Endogenous H2S in vivo: Interestingly, mice, which only received the probe, also displayed a modest, but measurable bioluminescence in the xenografted liver cancer tumors, which possibly results from endogenous H2S in these living animals. Therefore, we further determined whether this emission signal was partially due to the detection of endogenous H2S. Since the inhibition effect of PAG was rather weak at 18 min, we thus chose 13 min instead of 18 min as our observation point. Data showed (Figure 10) that the PAGtreated animal exhibited an obvious decrease in the bioluminescence signal compared with the control group at different time points. Therefore, the results well suggested that azidoluciferin can even detect endogenous H2S in real time in vivo, which may help us to further study the biological roles of H2S in the future.


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Analytical Chemistry Supporting Information

Supplementary experimental methods, probes characterization are available free of charge via the Internet at http://pubs.acs.org.”

AUTHOR INFORMATION Corresponding Author *[email protected].

Author Contributions ‡

These authors contributed equally

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the Natural Science Foundation of China (No. 21375025, 21175027).

REFERENCES

Figure 10. Bioluminescence imaging of endogenous H2S in Huh7 xenografts in nude mice. a) Bioluminescence images acquired at 3, 9, and 13 min from one mouse treated with PAG or the control. b) Total flux measured at 3, 9, and 13 min from one mouse treated with PAG or the control.

CONCLUSION In summary, a novel bioluminescence H2S probe azidoluciferin was successfully designed, synthesized, and identified as a new and promising tool for H2S detection. So far, all experimental results suggested that our probe exhibited high sensitivity, selectivity, and fast response for real-time detection of H2S in vitro, in cells, and in vivo. Furthermore, our probe demonstrated excellent imaging characteristics in vivo. Therefore, the influence of H2S levels on health and diseases might be uncovered by applying a bioluminescence method. Considering the increasing interest for H2S biological research, we believe that the novel bioluminescent H2S probe can serve as a functional and elucidative tool to further explore the biological roles of H2S in physiological and pathological processes.

ASSOCIATED CONTENT

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for TOC only


9

Visualization of in Vivo Hydrogen Sulfide Production by a

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