Two ratiometric fluorescent probes for hypochlorous acid detection and imaging in living cells

Author’s Accepted Manuscript Two Ratiometric Fluorescent Probes for Hypochlorous Acid Detection and Imaging in Living Cells Xiang Han, Chang Tian, Jingjing Jiang, Mao-Sen Yuan, Shu-Wei Chen, Juan Xu, Tianbao Li, Jinyi Wang www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(18)30360-6 https://doi.org/10.1016/j.talanta.2018.04.015 TAL18554

To appear in: Talanta Received date: 13 January 2018 Revised date: 27 March 2018 Accepted date: 7 April 2018 Cite this article as: Xiang Han, Chang Tian, Jingjing Jiang, Mao-Sen Yuan, ShuWei Chen, Juan Xu, Tianbao Li and Jinyi Wang, Two Ratiometric Fluorescent Probes for Hypochlorous Acid Detection and Imaging in Living Cells, Talanta, https://doi.org/10.1016/j.talanta.2018.04.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Two Ratiometric Fluorescent Probes for Hypochlorous Acid Detection and Imaging in Living Cells Xiang Han,a Chang Tian,b Jingjing Jiang,a Mao-Sen Yuan,a Shu-Wei Chen,a Juan Xu,a Tianbao Lia and Jinyi Wang *ab a

College of Chemistry & Pharmacy, Northwest A&F University, Yangling, Shaanxi 712100, P. R. China b

College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, P. R. China

* Corresponding author. Tel.: +86 29 87082520; fax: +86 29 87082520. E-mail address: [email protected] (J. Wang).

ABSTRACT. Hypochlorous acid plays a very important role in living cells, because it can resist microorganism attack and has a lethal effect on pathogens. The unnormal generation of ClO- can cause tissue damage and corresponding diseases. Detection of ClO- is very necessary in biological systems. In this article, we respectively hybridized the two different water-soluble coumarin (7-hydroxycoumarin and 7-diethylaminocoumarin) fluorophores with a longer-wavelength emissive rhodamine fluorophore to construct an intergrant, and then isothiocyanate was modified with the intergrant to recognized ClO-. The two ratiometric fluorescent probes RHClO-1 and RHClO-2 were developed for ClO- detecting with high selectivity and sensitivity. Especially, the probe RHClO-2 has lower detection limit (42 nM) for ClO- in 5 seconds. What's more, the probe RHClO-2 was successfully used in monitoring endogenous ClO- in living cells.

Graphical Abstract:

Ratiometric fluorescent probes RHClO-1 and RHClO-2 were developed for detecting hypochlorite in solutions and living cells.

Keywords: Ratiometric; Fluorescent probe; Hypochlorite; Imaging; Living cells.

1. Introduction Hypochlorous acid (HClO), one of the highly reactive oxygen species (ROS), is weakly acidic (pKa = 7.53) and partially exists in the form of hypochlorite ion (ClO-) in physiological conditions [1,2]. In immunological cells, ClO- can be produced from hydrogen dioxide (H2O2) and chloride ions (Cl-) assisted by myeloperoxidase (MPO, an enzyme presented in phagocytes). It is relevant to host defense in organism and exhibits a special capacity in killing multiple pathogens [3]. The unregulated generation of ClO- is thought to be related to some diseases including Parkinson’s disease, Alzheimer’s disease and cancer. [4-8]. However, the roles of ClO- in these diseases are still not explained clearly [3]. Seeking an effective method for ClOdetection, especially in situ detection, still keeps the indispensability. To data, there are various methods for ClO- detecting such as microelectrodes [9], nuclear magnetic resonance [10] and optical imaging (fluorescence, chemi-luminescence and bioluminescence imaging) [11]. Among them, fluorescence probes exhibited the unique advantages as an effective tool for monitoring bio-molecules within the bio-systems because of their high selectivity, good sensitivity, readily operation and potentially of real time monitoring [12-21]. Based on coumarin [22-24], BODIPY [25,26], cyanine [27] or rhodamine [28-34], many turn-on fluorescence probes have been reported for the detection/imaging of ClO- in recent years. Generally, these probes have high sensitivity owing to their high fluorescence quantum yield. However, their applications were limited because of single-wavelength emission, short stokes shifts, high background signal and so on [35-40]. The ratio-fluorescence probes can effectively solve these problems, because they have longer pseudo-stokes shifts, and provide two different emission wavelength with built-in correction [41-47]. As far as we know, ratiometric fluorescent probes for ClO- remain exceedingly rare [39,42]. The develop of new

ratiometric fluorescent probes for ClO- remain in urgent demand [11,23]. Generally, the desired fluorescent probes for ClO- should meet the following conditions: (a) there are two different excitation wavelengths to distinguish the background bioluminescence, (b) high sensitivity and good selectivity for precisely monitoring of ClO- formation, (c) relatively long emission wavelength to avoid self-fluorescence interference, and (d) excellent biocompatibility and hypotoxicity to reduce the biological damage. On the basis of the previous works, we are attempted to obtain ratiometric fluorescent probes for ClO- with two enough-far changing wavelength to achieve remarkable fluorescent colour change. Previous studies demonstrate that connecting electron donor and acceptor through the reaction site of ClO- is an effective designing strategy to realize this purpose [48,49]. However, such structural probes usually exhibited a low sensitivity for ClO-. Inspired by the ratiometric design [37,39-42], in this study we synthesised two rhodamine-coumarin-based ratiometric fluorescent probes RHClO-1 and RHClO-2 for ClO- detection (Scheme 1). We hybridized the coumarin fluorophore with a longer-wavelength emissive rhodamine fluorophore to construct an intergrant as the principal part of the probe. In addition to the excellent photophysical properties such as high fluorescent quantum yields and long emission wavelength, the framework of rhodamine derivatives behave the change in structure between spirocyclic (non-fluorescent) and ring-opening (fluorescent) forms in response to external stimulation. Simultaneously, the coumarin was chosen as the constructed fragment because of its good light stability, water solubility, and short-wavelength absorption, especially. We anticipated that the intergrant of rhodamine and coumarin can possess the merit of the two fluorophore, moreover, a large stokes shifts and the enough obvious fluorescent change. Thiosemicarbazide moiety was chosen as the

ClO- reaction site. To our delight, the probe RHClO-2 exhibited a perfect ability to monitor endogenous ClO- in living cells stimulated by lipopolysaccharide (LPS) and phorbol myristate acetate (PMA), and also showed high selectivity, good sensitivity, quick response and low cytotoxicity.

2. Experimental section 2.1. Materials and apparatus 3-(Diethylamino)phenol,

3-(1-piperazinyl)

phenol,

4-diethylaminosalicylaldehyde

and

2,4-dihydroxybenzaldehyde were purchased from Aladdin (Shanghai, China). Phenyl isothiocyanate, diethylmalonate and methanesulfonic acid were obtained from Alfa Aesar (Lancaster, England). All the other reagents and solvents were of analytical grade and supplied by local commercial suppliers. Prior to use, the dichloromethane (DCM) and ethyl alcohol (EtOH) were purified using the standard procedures. The liver hepatocellular (HepG2) cells, human breast adenocarcinoma (MCF-7) cells and mouse macrophage (RAW 264.7) cells were purchased from the Chinese Academy of Sciences (Shanghai, China). Ultrapure water was supplied by a Milli-Q system (Millipore) and was used in all the experiments. High resolution mass spectrometry (HR-MS) was undertaken using an AB SCIEX Triple TOF 5600+ spectrometer (AB Sciex, Boston, MA, USA). The other apparatus used in the experiments were the same as those in our previous studies [50]. 2.2. Synthesis and structural characterization The synthesis and characterization of the probes can be found in the supplementary material. 2.3. Spectroscopic characterization

The stock solutions (0.1 M) of small biological molecules and zwitterion in water were prepared from cysteine (Cys), glutathione (GSH), sodium citrate (Vc), adenosine-triphosphate (ATP), K2SO4, Na2SO3, MgCl2•6H2O, Zn(NO3)2•6H2O, CaCl2•2H2O and FeCl2•4H2O. RHClO-1 was exactly weighted and dissolved in ethanol and RHClO-2 was dissolved in dimethyl sulfoxide (DMSO) provided to the stock solution of probe (1 mM). To detect ClO- by colorimetry and fluorescence method, the stock solution of probe RHClO-1 was diluted in PBS (pH 7.4)-ethanol (7꞉ 3, v/v) and RHClO-2 was diluted in PBS (pH 7.4)-DMSO (99꞉ 1, v/v). Both probes were prepared into 20-µM working solution. Then, different concentration solutions of the analytes were added to 3 mL of the working solution in a 1-cm cuvette. Unless otherwise specifically stated, all the spectral studies were performed in PBS (pH 7.4) at room temperature. The fluorescence excitation of RHClO-1 was at 413 nm, and emission was collected in the range of 420-800 nm. The fluorescence excitations of RHClO-2 were respectively at 325 nm (coumarin fluorophore) and 565 nm (rhodamine derivatives), and the emission was collected in the range of 350-800 nm. 2.4. Preparation of ROS and RNS Hydroxyl radical (•OH), stable peroxynitrite anion (ONOO-) and singlet oxygen (1O2) were prepared through the method reported [51-53]. Sodium hypochlorite (NaOCl), H2O2 and tert-butyl hudroperoxide (t-BuOOH) directly configured to be 0.1 M stock solutions in water. ONOO- was prepared by diluting 0.3 mL of 30% hydrogen peroxide solution and 0.08 mL of 96% sulfuric acid solution to 5 mL solution with water. The solution quickly pour into 5 mL NaNO2 (0.04 g/mL) solution, and then add to 10 mL of NaOH (0.05 g/mL) solution to obtain the stock solution after the addition of 0.08 g MnO2 [51]. •OH was prepared by mixing FeSO4•7H2O and

H2O2 with equal quality, and the concentration was evaluated through Fe2+ [52]. Singlet oxygen 1

O2 was prepared by mixing 1:2 molar-ratio of NaOCl with H2O2 as its application previously

[53]. 2.5. Cell culture and cytotoxicity assay The liver hepatocellular cells (HepG2), human breast adenocarcinoma cells (MCF-7) and mouse macrophage cells (RAW 264.7) were cultured following the method reported previously [54]. HepG2 and MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Grand Island, NY) and RAW 264.7 cells were cultured in Roswell Park Memorial Institute medium 1640 (RPMI-1640 medium). The cytotoxicity assay was performed by using colorimetric methyl thiazolyl tetrazolium (MTT) assay. HepG2 and MCF-7 cells were first seeded in 96-well tissue culture plates with fresh DMEM and incubated for 24 h, respectively. RHClO-2 (0, 5, 10 15, 20 and 25 μM) was then added to the cell suspension and continue to cultivated. After 24 h, 10 μL MTT (5 mg/mL) was added to each well and cultivated for 4 h. DMEM were removed and added 100 μL DMSO. The absorbance was measured at 490 nm using a microplate reader (Bioread, model 680). The cytotoxic effect of the probe was determined by quantified the ratio of the absorbance (the experimental cell cells / the control cells) [54]. 2.6. Fluorescence image Prior to exogenous ClO- imaging experiments, HepG2 and MCF-7 cells (1 × 105 cells/mL) were first seeded into plate. After 24-h incubation, medium was removed and the cells were washed with PBS, incubated with 5 μM RHClO-2 for 0.5 h at 37 oC, and then washed again with PBS. Afterward, the cells were further incubated with 5 μM ClO- for 0.5 h at 37 oC and

then were used to perform fluorescent imaging, Blue channel λem: 330-380 nm and red channel λem: 510-550 nm. The similar procedures were used to image endogenous ClO- in living cells, RAW 264.7 cells (1 × 105 cells/ml) were first imaged after incubated with 1 μM lipopolysaccharide (LPS) for 12 h at 37 oC, and then the cells were incubated with RHClO-2 (5 μM) and phorbol myristate acetate (PMA) (1 μM) for 30 min at oC, during which fluorescent images were obtained every 5 min. Blue channel λem: 330-380 nm and red channel λem: 510-550 nm.

3. Results and discussion 3.1. Structure and synthesis of the probes The probe RHClO-1 and RHClO-2 were synthesized according to the synthetic route (Scheme S1). 3-Diethylaminophenol was first reacted with benzoic anhydride to give compound 1. Compound 2 was prepared via an acid-promoted Friedel–Crafts acylation between compound 1 and 3-(1-piperazinyl) phenol. Then, the amidation of compound 2 with hydrazine hydrate in anhydrous

ethanol

afforded

compound

3

with

66.8%

yield.

Meanwhile,

4-diethylaminosalicylaldehyde was reacted with diethylmalonate to give compound 4 with 81.5% yield, and then give compound 5 by acylation. Compound 6 was synthesized by the reaction of compound 3 with 5 in dry CH2Cl2 at room temperature. In addition, 2,4-dihydroxybenzaldehyde was reacted with diethylmalonate to give compound 7 with 72.8% yield, and then give compound 8 by acylation. Compound 9 was synthesized by the reaction of compound 3 with 8 in dry CH2Cl2 at room temperature. The probe RHClO-1 and RHClO-2 were synthesized by the reaction of compound 6 with phenyl isothiocyanate and compound 9 with phenyl isothiocyanate in DMF at 50 oC, respectively. The chemical structures of RHClO-1

and RHClO-2 were carefully characterized by 1H NMR, 13C NMR and HR-MS. The detailed synthetic procedures and relevant spectral data can be found in the supplementary material. It is worth noting that the solubility of RHClO-2 with the group of 7-hydroxycoumarin was better than RHClO-1 with the group of 7-diethylaminocoumarin, which lead to RHClO-1 can dissolved in EtOH-PBS (EtOH꞉ PBS = 3/7, v/v, pH = 7.4, 20 μM) and RHClO-2 can dissolved in DMSO-PBS (DMSO꞉ PBS = 1/99, v/v, pH = 7.4, 20 μM). 3.2. Spectral properties of the probes The optical responses of RHClO-1 and RHClO-2 towards ClO- were first explored. As shown in Fig. 1, the normalized absorption and emission spectra of RHClO-1 and RHClO-2, as well as the fluorescent titration of the probes to ClO- were collected. RHClO-1 shows a maximum absorbance and emission at 413 nm and 473 nm, respectively. And the probe RHClO-2 respectively shows a maximum absorbance and emission at 325 nm and 460 nm in the absence of ClO-. The results showed that the spirolactone structure of rhodamine is closed at the moment. Upon the addition of ClO-, a new absorption band (RHClO-1) at 565 nm appeared in the visible range, along with a color change from yellow to pink (Fig. S1a). Stimulation of RHClO-1 with ClO- produced a strong fluorescence emission at 575 nm. The probe RHClO-2 appeared a new absorption band at 565 nm and a new emission band at 585 nm, accompanied by a color change from colourless to red (Fig. S1b), indicated that the rhodamine ring-open amide form was produced in the presence of ClO-. These results demonstrated that the two probes can detect ClO- by spectroscopic method, and enabled us to further study the practicability for ClOdetection in real environment.

Subsequently, the ability of the two probes for quantitative analysis of ClO- was thoroughly evaluated. The absorbance and emission spectra of RHClO-1 and RHClO-2 (20 μM) were measured after adding ClO- at different concentrations. As shown in Fig. S2, the free RHClO-1 (20 μM) showed a maximal absorption at 413 nm and no absorption appeared within the scope of 450 nm to 650 nm. The absorption at 413 nm was likely assigned to the coumarin fluorophore framework. There is no absorption between 450 and 650 nm because the spirolactam form of rhodamine is closed. However, with the addition of ClO- to the RHClO-1 solution, the absorption of RHClO-1 at around 413 nm slightly reduced. Synchronously, a new absorption band at around 565 nm appeared. Similar to the absorption spectra, the free RHClO-1 only showed the characteristic emission band of the coumarin fluorophore at 473 nm and displayed no emission band for the rhodamine derivative moiety. By comparison with increasing the concentration of ClO-, a significant increase was observed in the fluorescence intensity at around 575 nm. At the same time, the emission band at around 473 nm gradually decreased (Fig. S3). All the results indicate that the probe RHClO-1 has an excellent signal resolution performance with the addition of ClO-, which bring about a longish red shift (∆λ=102 nm) in the emission spectra. The ratio of the fluorescence intensities at 575 and 473 nm (F575/F473) showed a significant change from 0.063 (in the absence of ClO-) to 0.15 [in the presence of ClO- (20 μM)], showing a 2.4-fold increase in the emission ratios (the fluorescence quantum yields (∅)=0.0210, Table S1). Fig. S4 showed that there was a linear dependence of the fluorescence intensity on the ClO- concentration in the range of 0-20 μM, with a regression equation as R = 0.9957. The results indicate that the probe is potentially available for the quantitative detection of ClO-. The detection limit (LOD, 3s/k) for ClO- was estimated to be 0.64 μM.

As shown in Fig. S5, the free RHClO-2 (20 μM) showed a maximal absorption band at 325 nm and no absorption in the range of 500 to 650 nm. However, upon adding ClO- to the RHClO-2 solution, the absorption of RHClO-2 at around 325 nm slightly reduced. At the same time, a new absorption band at 575 nm appeared corresponding to the open spirolactam form of rhodamine. Different from the absorption spectra, the free RHClO-2 only showed the characteristic emission peak of the coumarin fluorophore at 460 nm and displayed no emission for the rhodamine moiety. With increasing the concentration of ClO- (Fig. 2a), a furious fluorescent increase was observed at 585 nm. At the same time, the emission band at around 460 nm gradually reduced. In comparison, the probe RHClO-2 exhibited a better signal resolution performance with the addition of ClO- than RHClO-1 to obtain a large red shift (∆λ=125 nm) in the emission. The ratios of the fluorescence intensity at 585 and 460 nm (F586/F460) showed a drastic change from 0.051 (in the absence of HOCl) to 7.4 [in the presence of ClO- (20 μM)], a 145-fold variation in the emission ratios (Φ =0.2290, Table S1,). Fig. 2b showed the linear dependence of the fluorescence intensity on the ClO- concentration in the range of 0-20 μM, with a regression equation as R = 0.9931. The results indicate that the probe RHClO-2 can be used to the quantitative determination of ClO-. The LOD for ClO- was estimated to be 0.042 μM, which was sensitive enough for determining ClO- in cells [55]. The response time of the probe RHClO-1 and RHClO-2 toward ClO- was also examined under the condition of pH 7.4. As shown in Fig. S6, over 90% of the fluorescence reaction of RHClO-1 with ClO- was completed within 20 seconds. Compared to RHClO-1, RHClO-2 showed a faster response, reaching the highest fluorescence intensity ratio in less than 5 seconds (Fig. 2c). The possible reason may be that the solubility of RHClO-2 was better than RHClO-1,

leading to the reactivity of probe RHClO-2 was enhanced. The rapid response of RHClO-2 towards ClO- indicated that it could serve as a pointer to real-time monitor ClO- in organisms [39]. 3.3. Selectivity of the probes To evaluate the selectivity of the probe RHClO-1 and RHClO-2 towards ClO-, various biologically relevant analytes, different reactive oxygen species/reactive nitrogen species (ROS/RNS) and ions enriched in living cells, including Cys, GSH, Vc and ATP, K+, Na+, Mg2+, Zn2+, Fe2+, Ca2+, NO2-, SO32-, SO42-, were added independently into the probe solutions, and then measuring their emission spectra. Under the condition of pH 7.4, other biologically relevant analytes, ROS/RNS or ions almost could not cause the fluorescence changes of RHClO-1 and RHClO-2 (Fig. S7-9 and Fig. 2d). These results indicated that the two probes had excellent selectivity toward ClO- over other tested species. Ultimately, the spectral data show that our designed probes, especially RHClO-2, have a better sensitivity, detection limit and water solubility than most of the reported ClO- probes (Table S2). 3.4. pH and time dependence of the probes The ratiometric responses of RHClO-1 and RHClO-2 toward ClO- at different pH were investigated. The results showed that the emission spectra of RHClO-1 and RHClO-2 were essentially pH-insensitive at pH 6-8 (Fig. S10). After the addition of 20 μM ClO-, the fluorescence intensity ratio (F575/F473) of the probe RHClO-1 increased significantly at pH 6-12. Same phenomenon was also observed for the fluorescence intensity ratio (F585/F460) of RHClO-2 at pH 6-8. The results indicated that the two probes could detect ClO- under

physiological condition (pH = 7.4), suggesting that the assay is compatible with most biological applications [56]. 3.5. Reaction mechanism of the probes A possible reaction mechanism of the probe RHClO-1 and RHClO-2 with ClO- was further investigated by using high-resolution mass spectrometry (HR-MS) spectra. Upon the addition of ClO-, the thiosemicarbazide of the probes can be converted to 1,3,4-oxadiazoles, which will consecutively facilitate the spirolactam ring-opening reaction. Consequently, the rhodamine moiety produced a strong red fluorescence. The generated 1,3,4-oxadiazoles have been confirmed by HR-MS. Their M+ base peaks were at 814.3628 for RHClO-1 and 759.2959 for RHClO-2 (Fig. S11). 3.6. Fluorescence imaging of exogenous ClO- in living cells Encouraged by the prominent results above, we proceeded to investigate the feasibility of the probe RHClO-2 to image ClO- in living cells due to its better water solubility and biocompatibility, compared with RHClO-1. We first studied its cytotoxicity, because cytotoxicity is a crucial parameter used to assess whether a probe can be applied to intracellular detection. In this study, the standard MTT assay was used to evaluate RHClO-1 cytotoxicity. The result (Fig. S12) indicated that RHClO-2 had no cytotoxicity to HepG2 and MCF-7 cells at low concentrations (5-25 μM), meaning that RHClO-2 can be used for cell imaging. To evaluate the imaging ability of RHClO-2 in living cells, HepG2 and MCF-7 cells were incubated with RHClO-2 (5 μM) for 30 min. The cells showed a strong fluorescence in the blue channel (Fig. 3b and 3f), but there is hardly no fluorescence in the red channel (Fig. 3c and 3g). However, after treated with 5 μM ClO- for 10 min, the blue fluorescence of the cells is slightly

weaker in the blue channel (Fig. 3d’ and 3f’). At the same time, the cells exhibited strong red fluorescence in the red channel (Fig. 3c’ and 3g’). According to the fluorescence intensity of the images, the ratio of the red fluorescence intensity to blue fluorescence intensity showed significant increase, indicating that ClO- can cause the ratio reaction of RHClO-2 in cells. (Fig. 3i and 3j). The imaging test was consistent with the spectroscopy data of RHClO-2. All the results indicated that the probe RHClO-2 could be used as ratiometric fluorescence imaging of ClO- in living cells. 3.7. Fluorescence imaging of endogenous ClO- in living cells The detection of endogenous ClO- was examined in the murine live macrophage cell line (RAW 264.7), because lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) can stimulate RAW 264.7 cells to produce ClO- [57]. As shown in Fig. 4, after incubated with RHClO-2, RAW 264.7 cells displayed almost no fluorescence in the red channel with excitation at 561 nm (Fig. 4a’, Fig. 4b’, and 4b’’). However, after incubated with PMA and RHClO-2, the red fluorescent label intensity increased in the red channel (Fig. 4c’’) because of the reaction between the probe and endogenous ClO- [29]. These results indicated that the probe RHClO-2 was able to detect endogenous ClO- in living cells. Based on the study above, ClO- in RAW 264.7 cells can be real-time detect using RHClO-2 (Fig. 5a). After in turn treated with LPS, PMA, and RHClO-2, we observed blue fluorescence in RAW 264.7 cells. As time goes on, it slowly diminished. At the same time, the red fluorescence grew rapidly (Fig. 5b) and the corresponding ratio (red to blue) of fluorescence intensity increased (Fig. 5c). These data indicated that the probe RHClO-2 could be used to image endogenous ClO- produced processes in living macrophage cells.

4. Conclusions In summary, two rhodamine-coumarin-based ratiometric fluorescent probes RHClO-1 and RHClO-2 for ClO- detection were synthesized, which showed excellent response to ClO- under physiological condition. The probe RHClO-1 can detect ClO- about 20 s and the detection limit is 0.64 μM. The ratiometric probe RHClO-2 displays a 136-nm red-shift in emission and remarkable fluorescent color change from blue to red upon the addition of ClO-. The response time of RHClO-2 to ClO- is 5s and the detection limit is 42 nM. In addition, the two probes exhibited outstanding biocompatibility and low biotoxicity. The results of fluorescence imaging confirmed that the RHClO-2 was suitable for ratiometric visualization of exogenous and endogenous ClO- in living cells, which may be also used as an alternative to assess deleterious effect of ClO-.

Acknowledgements This work was supported by the National Natural Science Foundation of China (21675126, 21375106 and 21405121). The Fund of Youth Science and Technology Stars by Shaanxi Province (2015KJXX-15).

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of

β-galactosidase

stimulated

activity

in

hepatocellular

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Captions to Figures Scheme 1. The structures of RHClO-1 and RHClO-2 and ClO- sensing mechanism.

Fig 1. Normalized absorbance spectra (a) and fluorescence spectra (b) of RHClO-1 and RHClO-2.

Fig 2. (a) Fluorescence titration profiles of 20-μM RHClO-2 (for RHClO-2, Ex@420 and 575 nm for coumarin and the rhodamine part) towards different concentrations (0–20 μM) of NaClO. (b) Linear correction between the fluorescence ratio (F585/F460) towards the concentrations of 0-20 μM NaClO. (c) Temporal profile of fluorescence intensity of RHClO-2 before and after NaOCl (20 μM) was added. (d) Fluorescence responses of 20 µM RHClO-2 upon the addition of various different ROS/RNS and ions enriched in living cells (50 µM, Cys, GSH, Vc and ATP, 100 µM; Mx is Na+, K+, Mg2+, Zn2+, Fe2+, Ca2+, NO2-, SO32- and SO42-).

Fig 3. Fluorescence images of living HepG2 and MCF-7 cells (a-c). HepG2 cells were treated with RHClO-2 (5 μM) incubation 30 min. (d) b and c overlay chart. (a’-c’) HepG2 cells were pre-incubated with 5 μM RHClO-2 for 30 min, and then treated with NaClO (5 μM) for 10 min. (d’) b’ and c’ overlay chart. (e-g) MCF-7 cells were treated with RHClO-2 (5 μM) for 30 min. (h) f and g overlay chart (e’-g’). MCF-7 cells were pre-incubated with 5 μM RHClO-2 for 30 min, and then treated with NaClO (5 μM) for 10 min. (h’) f’ and g’ overlay. (i) The fluorescence ratio (Fred/Fblue) of HepG2 cells (i) and MCF-7 cells (j). Scale bar = 50 μm. The results are presented as means ± standard error (SE) with replicates n = 3 (**p < 0.01, ***p < 0.001).

Fig 4. Fluorescence images of living RAW 264.7 cells (a-a’’). RAW 264.7 cells were treated with RHClO-2 (5 μM) for 30 min. (b-b’’) RAW 264.7 cells were treated with LPS (1 μM) for 12 h. (c-c’’) RAW 264.7 cells were pre-incubated with 1 μM LPS for 12 h, and then treated with PMA (1 μM) and RHClO-2 (5 μM) for 20 min. Scale bar = 50 μm.

Fig 5. (a) Fluorescence images of living RAW 264.7 cells (RAW 264.7 cells were preincubated with 1 μM LPS for 12 h, and then reated with PMA and RHClO-2 (5 μM) for 5, 10, 15, and 20 min, respectively). (b) A variation of fluorescent colors (black line: blue fluorescent, red line: red fluorescent.). (c) The fluorescence ratio (Fred/Fblue) of RAW 264.7 for different times. Scale bar = 50 μm. The results are presented as means ± SE with replicates n = 3 (*p < 0.05, ***p < 0.001).

Highlights:

● Two ratiometric fluorescent probes RHClO-1 and RHClO-2 were developed for ClO- detection. ● RHClO-1 can detect ClO- in 20 s with the detection limit of 0.64 μM. ● RHClO-2 can detect ClO- in 5 s with the detection limit of 42 nM. ● RHClO-2 can in situ monitor ClO- in living RAW 264.7 cells.

Revised Fig. 1

Revised Fig. 2

Fig. 3

Fig. 4

Fig. 5

Revised Scheme 1