English

• 0.   Introduction

  Reactive oxygen species (ROS) are integral to a variety of physiological and biochemical processes, including anti-inflammatory regulation, pathogen response, and mediation of biological signal transduction^[1-2]. Hypochlorous acid (HClO), a prototypical ROS^[3], is typically generated in vivo via myeloperoxidase-catalyzed oxidation of chloride ions (Cl^-) by hydrogen peroxide (H₂O₂)^[4-6]. HClO exhibits a dual nature in vivo ‒It effectively eliminates invading pathogens and bacteria owing to its potent oxidizing properties^{[4, 7-9]} and plays a crucial role in immune defense and inflammation^{[6, 10]}. However, excessive HClO levels can lead to various diseases, such as obesity, rheumatoid arthritis^{[8, 11]}, diabetes^[12], neurodegenerative disorders^{[4, 8, 13-14]}, atherosclerosis^{[6, 15]}, liver injury^{[5, 15]}, pulmonary fibrosis, and even cancer^{[7, 14-15]}. Therefore, developing a practical chemical tool for in situ HClO labeling that can provide valuable insights into the pathological functions of HClO is crucial.

  Scientists have used a variety of analytical techniques to detect HClO, including colorimetry, mass spectrometry, electrochemistry, polarography, and fluorescence imaging. Among these techniques, fluorescence imaging utilizing fluorescent probes has demonstrated significant advantages in terms of sensitivity, simplicity, response time, and spatiotemporal resolution^[3]. These inherent advantages make it suitable not only for in vitro analysis but also for in vivo imaging studies. A common strategy for designing HClO fluorescent probes involves linking an HClO reactive moiety to an organic fluorophore.

  Some of the major studies involving HClO include oxidation of o-(p-)quinone to o-(p-)benzoquinone by HClO, oxidation of diacylhydrazine by ClO^-, conversion of an aniline group to a nitro group, HClO-mediated intramolecular oxidation leading to ring formation or ring-opening, partial oxidation of thiourea to imidazoline by HClO, oxidative deprotection of thioheterocycles and sulfation of thioethers by HClO, oxidation of tolyl sulfides or selenides, oxidation of transition metal complexes, and oxidative cleavage of C=C, C=S, and C=N bonds by HClO^{[1-6, 8-10, 12-14, 16-30]}.

  In this study, we developed a dual-wavelength emitting fluorescent probe, N-(2-(7-(diethylamino)-2-oxo-2H-chromene-3-carboxamido)ethyl)-3, 7-bis(dimethylamino)-10H-phenothiazine-10-carboxamide (MBC), for detecting HClO. The molecular structure of MBC contains methylene blue (MB) and coumarin fluorophores. These fluorophores emit distinct fluorescence signals upon excitation at specific wavelengths of light, with non‑overlapping emission spectra. The two fluorophores are covalently linked via a carbonyl group, which effectively quenches their fluorescence. On exposing MBC to HClO, HClO induces oxidative cleavage of the carbonyl linkage owing to its strong oxidizing property. This leads to the release of MB and coumarin, generating red and green fluorescence, respectively. Through dual-fluorescence calibration and localization without mutual interference, the probe exhibited enhanced accuracy and anti-interference performance in biomarker detection and analytical applications.

  1.   Experimental

  1.1   Materials and measurements

  Sodium hypochlorite (NaClO), hydrogen peroxide (H₂O₂), potassium superoxide (KO₂), tert-butyl hydroperoxide (TBHP), hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium nitrate (NaNO₃), and ferrous sulfate heptahydrate (FeSO₄·7H₂O) were procured from Sinopharm Chemical Reagent Group. Other reagents (analytical grade), including dimethyl sulfoxide (DMSO), sodium perchlorate (NaClO₄), and sodium thiocyanate (NaSCN), were obtained from AladdinReagent (Shanghai) Co., Ltd. Organic solvents were obtained from Anhui Zesheng Technology Co., Ltd. Ultrapure water was produced using a Millipore Direct-Q system.

  Ultraviolet-visible (UV-Vis) absorption spectroscopy was performed with a UV-2600 spectrometer. Fluorescence spectra were collected on an FLSP920 spectrometer, and fluorescence quantum yield was measured with an RF-5301PC spectrometer. pH measurements were performed with a PHB-4 pH meter (Thunder Magnetic). NMR spectra were recorded on a Varian INOVA 400 MHz superconducting NMR spectrometer. High-resolution mass spectra were collected on a Bruker microTOF-Q Ⅱ quadrupole-time-of-flight mass spectrometer. Liquid chromatography‑tandem mass spectrometry (LC-MS/MS) was performed with an Agilent 1100 series LC/MS system. Confocal microscopy was performed with an Olympus FV1200 laser scanning confocal microscope designed for live cell imaging.

  1.2   Synthesis

  MBC comprises a MB component (which emits red fluorescence), a diethylaminocoumarin component (which emits green fluorescence), and an ester‑ethylenediamine linkage that can be cleaved by HClO. Details regarding the characterization of MBC are provided in the Supporting Information. The synthetic routes for MBC are illustrated in Scheme 1.

  Scheme 1

  

  Scheme 1.  Synthesis of MBC

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  1.2.1   Synthesis of tert-butyl (2-(7-(diethylamino)-2-oxo-2H-chromene-3-carboxamido)ethyl) carbamate (2)

  First, 7-(diethylamino)coumarin-3-carboxylic acid (1.31 g, 5.0 mmol) was dissolved in 15 mL of anhydrous dichloromethane. Subsequently, 1-(3-dimethylaminopropyl)‑3‑ethylcarbodiimide hydrochloride (EDC, 4.8 g, 21.0 mmol) and N-hydroxysuccinimide (NHS, 2.59 g, 22.5 mmol) were added to the solution, followed by stirring at room temperature for 6 h. Upon completion of the reaction, the solvent was removed under reduced pressure. The residue was re-dissolved in 50 mL of dichloromethane and washed with saturated sodium bicarbonate (20 mL×3) and saturated brine (20 mL×3). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a yellow powder, which was named compound 1 (1.13 g). The product was used directly without purification.

  Next, compound 1 (0.15 g, 0.419 mmol) was placed in a round-bottom flask and dissolved in 15 mL of dichloromethane. N-tert-butoxycarbonyl-1, 2-ethanediamine (0.30 g, 1.8 mmol) and N, N-diisopropylethylamine (0.20 g, 2 mmol) were added to the mixture, and the resulting solution was stirred at room temperature for 13 h. After the reaction was complete, the solvent was removed by vacuum distillation. The residue was re-dissolved in 15 mL of dichloromethane and washed successively with saturated ammonium chloride solution (20 mL×3), saturated sodium carbonate solution (20 mL×3), and saturated brine (20 mL×3). The organic phase was concentrated under reduced pressure, and the crude product was purified by column chromatography with CH₂Cl₂/MeOH (15∶1，V: V) solution as the eluent to yield 0.393 6 g of a light-yellow solid, which was named compound 2. The ¹H NMR analysis (400 MHz, chloroform-d) details were as follows: δ 8.97 (t, J=6.1 Hz, 1H), 8.69 (s, 1H), 7.43 (d, J=8.9 Hz, 1H), 6.65 (dd, J=9.0, 2.3 Hz, 1H), 6.50 (d, J=2.3 Hz, 1H), 5.11 (t, J=5.8 Hz, 1H), 3.56 (q, J=6.0 Hz, 2H), 3.46 (q, J=7.2 Hz, 4H), 3.36 (q, J=6.0 Hz, 2H), 1.44 (s, 9H), and 1.24 (t, J=7.1 Hz, 6H).

  1.2.2   Synthesis of N-(2-aminoethyl)-7-(diethylamino)-2-oxo-2H-chromene-3-carboxamide (3)

  Compound 2 (0.39 g, 0.976 mmol) was dissolved in 10 mL of a 4 mol·L^-1 hydrochloric acid solution prepared using ethyl acetate (EtOAc) as the solvent, and the resulting solution was stirred at room temperature until a white precipitate (compound 3) was obtained. Compound 3 was used directly without further purification.

  1.2.3   Synthesis of 3, 7-bis(dimethylamino)-10H-phenothiazine-10-carbonyl chloride (4)

  MB (1.69 g, 5.00 mmol) was dissolved in 25 mL of dichloromethane and transferred to a 100 mL round-bottom flask. An aqueous sodium hydrosulfide solution (1.04 g, 6.00 mmol) in 25 mL of water was then slowly added to the reaction mixture under continuous stirring. The temperature of the system was gradually increased to 40 ℃, and stirring was maintained for 1 h. The reaction mixture was then cooled by placing it in an ice-water bath. Subsequently, a solution of triphosgene (1.78 g, 6.00 mmol) in 25 mL of dichloromethane was added dropwise over 1 h. The resulting product was extracted with dichloromethane (20 mL×2) and washed with saturated brine (20 mL×3). The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The crude product was purified by column chromatography using CH₂Cl₂/petrol ether (5∶1，V/V) solution as the eluent to obtain a white solid, which was named compound 4. Owing to instability, compound 4 was utilized directly in subsequent reactions without additional isolation.

  1.2.4   Synthesis of MBC

  A 50 mL round-bottomed flask was charged with a solution of compound 4 (0.03 g, 0.09 mmol) and 5 (0.03 mg, 0.09 mmol) in anhydrous tetrahydrofuran (THF, 5 mL). Subsequently, sodium carbonate (0.02 mg, 0.179 mmol) was added, and the mixture was stirred at room temperature for 5 h. The reaction solvent was evaporated under reduced pressure, and the crude product was purified by column chromatography using dichloromethane/methanol (20∶1，V/V) as the eluent to yield 25 mg of MBC. The ¹H NMR (400 MHz, chloroform-d) analysis details were as follows: δ 8.93 (t, J=6.1 Hz, 1H), 8.65 (s, 1H), 7.40 (dd, J=14.1, 8.8 Hz, 3H), 6.69-6.63 (m, 3H), 6.61 (dd, J=8.8, 2.8 Hz, 2H), 6.51 (d, J=2.4 Hz, 1H), 5.36 (t, J=5.6 Hz, 1H), 3.58 (q, J=6.1 Hz, 2H), 3.45 (p, J=6.5, 5.7 Hz, 6H), 2.90 (s, 12H), and 1.24 (t, J=7.1 Hz, 6H). MS (electrospray ionization, ESI) spectra were also collected‒m/z calculated for C₃₃H₃₈N₆O₄S: 614.267 5; Found: 615.210 3 [M+H]⁺.

  1.3   Preparation of ROS

  ROS were prepared by methods adapted from established protocols. In this study, commercially available NaClO was used as a substitute for HClO. TBHP and H₂O₂ were diluted to their respective working concentrations using commercially available reagents. Sodium nitrite (NaNO₂) was dissolved in ultrapurewater to generate NO₂^- ions. Hydroxyl radicals (·OH) were generated via the Fenton reaction by reacting Fe²⁺ with 10 equivalents of H₂O₂. Superoxide anions (·O₂^-) were prepared by dissolving KO₂ in anhydrous DMSO, followed by ultrasonic treatment. Singlet oxygen (¹O₂) was produced by reacting H₂O₂ with 10 equivalents of HClO. Nitric oxide (NO) was generated by adding sodium nitroprusside to degassed ultrapure water under nitrogen, followed by vigorous stirring for 30 min. All ROS solutions were freshly prepared and used immediately. Other required ions were prepared with ultrapure water.

  1.4   Spectral measurements

  All spectral measurements were performed in 10.0 mmol·L^-1 phosphate-buffered saline (PBS, pH 7.4). Fluorescence responses of 5 μmol·L^-1 MBC to different species in PBS were collected in a 1 cm pathlength cuvette in two channels (blue: excitation wavelength of 405 nm, red: excitation wavelength of 620 nm). The resulting mixtures were incubated at 25 ℃ for 3 min before fluorescence spectroscopy analysis. Fluorescence spectra were recorded in two wavelength ranges: excitation at 405 nm with emission in the 430-620 nm range and excitation at 620 nm with emission in the 640-800 nm range.

  1.5   Cell culture and imaging

  1.5.1   Cell culture

  HepG2 cells were cultured in Dulbecco′s Modified Eagle Medium supplemented with a volume fraction of 10% fetal bovine serum, 100 U·mL^-1 penicillin, and 100 μg·mL^-1 streptomycin and maintained in a humidified incubator at 37 ℃ with a volume fraction of 5% CO₂. The cells were seeded in 96-well plates at a density of 8 000 cells per well and cultured for 24 h to facilitate cellular adherence. Subsequently, the cells were exposed to various concentrations of MBC (0, 2.0, 4.0, 6.0, 8.0, and 10.0 μmol·L^-1) for an additional 24 h. After treatment, the medium was carefully aspirated, and 110 μL of fresh medium containing 10 μL of cell counting kit-8 (CCK-8) reagent was added to each well under light-protected conditions. The plates were then incubated for 2 h, and absorbance was measured at 450 nm using a microplate reader to evaluate cytotoxicity.

  1.5.2   Fluorescence confocal imaging

  For the imaging experiment, HepG2 cells at a density of 30 000 cells per well in laser confocal culture dishes (diameter: 35 mm; glass bottom diameter: 10 mm) were incubated with 5.0 μmol·L^-1 MBC at 37 ℃ for 30 min before laser confocal imaging. To determine the effect of exogenous HClO, HepG2 cells pre‑ incubated with 5.0 μmol·L^-1 MBC were washed thoroughly and then incubated with 20.0 μmol·L^-1 HClO for 30 min. Subsequently, the cells were subjected to laser confocal imaging. All cells were washed three times with sterile PBS and imaged using an Olympus FV1200 laser confocal microscope (green channel: 488 nm of excitation wavelength, 490-540 nm of emission collection range; red channel: 633 nm of excitation wavelength, 650-700 nm of emission collection range).

  2.   Results and discussion

  2.1   Optical responses of MBC to HClO

  To determine the performance of MBC, first, its fluorescence response was investigated using 10.0 mmol·L^-1 PBS at pH 7.4 by adding three equivalents of HClO. As shown in Fig. 1, MBC (5.0 μmol·L^-1) exhibited minimal fluorescence in the absence of HClO. Upon the addition of three equivalents of HClO, MBC demonstrated distinct dual fluorescence. Specifically, under excitation at 405 nm, the fluorescence intensity in the 440-600 nm range was significantly enhanced, peaking at 480 nm. Moreover, when excited at 620 nm, MBC emitted visible fluorescence in the 640-740 nm range, peaking at 678 nm. Subsequently, a kinetic study was conducted to investigate the response of MBC to HClO by monitoring the changes in the fluorescence intensity at 480 nm (λ_ex=405 nm) with time. As illustrated in Fig.S4, following the introduction of HClO, the fluorescence signal intensity rapidly increased, reaching a maximum within 90 s. These findings confirmed the potential of MBC for rapid and sensitive detection of HClO.

  Figure 1

  

  Figure 1.  Intensity changes in the fluorescence spectra of MBC (5.0 μmol·L-1) upon addition of HClO (15.0 μmol·L-1): (a) excitation at 405 nm and (b) excitation at 620 nm

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  To investigate the selectivity of MBC, we examined the fluorescence spectral changes of 5.0 μmol·L^-1 MBC in the presence of various reactive species and ions (Fig. 2). Specifically, we used different ROS (15.0 μmol·L^-1): HClO, H₂O₂, NO, ¹O₂, and ·OH. In addition, we evaluated the effects of anions (0.1 μmol·L^-1) such as NO₂^-, ClO₄^-, SO₃^2-, Cl^-, SO₄^2-, NO₃^-, and PO₄^3-, along with 0.1 mmol·L^-1 cations and biological thiols, including K⁺, Na⁺, Fe²⁺, Fe³⁺, Mg²⁺, cysteine (Cys), and glutathione (GSH). The results indicated that uponexcitation at both 405 and 620 nm, only HClO caused a significant increase in the fluorescence signal of MBC. The other ROS, cations, anions, and biological thiols, commonly found in body fluids, did not induce any observable changes in the fluorescence of the probe. These findings demonstrated the high selectivity of MBC toward HClO.

  Figure 2

  

  Figure 2.  Fluorescence spectra of MBC (5.0 μmol·L-1) in the presence of various analytes upon excitation at wavelengths(a) 405 nm and (c) 620 nm, and the corresponding fluorescence intensities at emission wavelengths (b) 480 nmand (d) 678 nm

  1: Blank; 2: HClO; 3: H₂O₂; 4: NO; 5: ¹O₂; 6: ·OH; 7: NO₂^-; 8: ClO₄^-; 9: SO₃^2-; 10: Cl^-; 11: SO₄^2-; 12: NO₃^-; 13: PO₄^3-; 14: K⁺; 15: Na⁺; 16: Fe²+; 17: Fe³⁺; 18: Mg²⁺; 19: Cys; 20: GSH.

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  Moreover, we systematically evaluated the analytical performance of MBC using standard HClO solutions with varying concentrations in the aforementioned experiments. The experimental results are presented in Fig. 3. As shown, the fluorescence signal intensity gradually enhanced with increasing concentration of HClO. A strong linear relationship was observed between the fluorescence intensity at 480 nm (I_{480 nm}) and HClO concentration (c_HClO) in the 0-15.0 μmol·L^-1 range. The following linear equation was used: I_{480 nm}=122.45+58.49c_HClO; the correlation coefficient (R²) was determined to be 0.997 7. The standard deviation (σ) of the blank was 0.49. With this information, the HClO detection limit for MBC was calculated to be 25.13 nmol·L^-1. The fluorescence signal intensity at 678 nm (λ_ex=620 nm) also increased, and the relationship between the intensity (I_{678 nm}) and c_HClO was found to be linear. The following linear equation was used: I_{678 nm}=15.41+7.89c_HClO; R² was determined to be 0.997 6. Moreover, σ of the blank was 0.083, and the detection limit at this wavelength was determined to be 31.55 nmol·L^-1. Thus, the detection limits derived from the fluorescence signals at both wavelengths (480 and 678 nm) were consistent.

  Figure 3

  

  Figure 3.  (a) Fluorescence spectra of MBC upon excitation at 405 nm varying with c_HClO; (b) Linear relationship between I_{480 nm} and c_HClO; (c) Fluorescence spectra of MBC upon excitation at 620 nm varying with c_HClO; (d) Linear relationship between I_{678 nm} and c_HClO

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  2.2   Reaction mechanism

  The principle of sensing HClO by MBC for the optical changes is depicted in Scheme 2. With the addition of HClO, the free MBC undergoes a nucleophilic addition reaction through the process described in Scheme 2. The cleavage of the HClO-responsive site generates MB and coumarin ethylenediamine (CMEA) fluorophores, which, as two independent strong conjugated systems and induce fluorescence enhancement. To further investigate the sensing mechanism of MBC, the mass spectrometry analysis indicated corroborative evidence for the formation of MBC conjugate at m/z=326.518 5 (Obsd.) ([CMEA+Na]⁺, Calcd. 326.351 2 for C₁₆H₂₁N₃NaO₃⁺) and at m/z=285.102 4 (Obsd.) ([MB+H]⁺, Calcd. 285.121 6 for C₁₆H₁₈N₃S⁺), implying the addition product (Fig.S5).

  Scheme 2

  

  Scheme 2.  Schematic illustration of the reaction of MBC with HClO

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  2.3   Imaging of intracellular HClO using MBC

  The aforementioned experiments demonstrated the potential of MBC for rapid and effective in vitro HClO detection. To evaluate the practical applicability of the synthesized probe, we conducted cellular studies. Initially, the cytotoxicity of MBC was assessedusing the CCK-8 assay. After incubation for 24 h with varying concentrations of MBC (0, 2.0, 4.0, 6.0, 8.0, and 10.0 μmol·L^-1), HepG2 cells exhibited a survival rate exceeding 80% (Fig.S6), suggesting minimal cytotoxicity of MBC and its potential for practical applications.

  Subsequently, we evaluated the applicability of MBC in detecting exogenous HClO in cells. As shown in Fig. 4, untreated HepG2 cells did not exhibit any fluorescence in the green channel or red channel. Following incubation of HepG2 cells for 30 min with 5.0 μmol·L^-1 MBC at 37 ℃, a weak fluorescence signal was observed in both channels. This indicated that MBC possessed a certain degree of cell membrane permeability. To assess the capability of MBC in detecting intracellular HClO in living cells, we performed anexperiment wherein HepG2 cells were pre-incubated with fresh medium containing 15.0 μmol·L^-1 HClO for 30 min. Subsequently, the cells were treated with 5.0 μmol·L^-1 MBC for an additional 30 min before imaging. As shown in Fig. 4, significantly enhanced fluorescence was observed in both channels. These experimental results demonstrate that MBC can be effectively used for exogenous HClO detection in cells and biological applications.

  Figure 4

  

  Figure 4.  Fluorescence imaging of HepG2 cells: (a) untreatedcells; (b) cells treated with 5.0 μmol·L^-1 MBC for 30 min; (c) cells pre-incubated with 15.0 μmol·L^-1 HClO for 30 min followed by treatment with 5.0 μmol·L^-1 MBC for an additional 30 min

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  2.4   Rapid detection and imaging of HClO in aqueous samples

  To verify the rapid and convenient detection of HClO in water samples, we developed a colorimetric test strip based on MBC. Typically, a circular filter paper with a 1 cm diameter was immersed in 4 mL of 0.1 μmol·L^-1 MBC solution, followed by natural drying in a well-ventilated environment. To evaluate the performance, hypochlorite solutions with varying concentrations (25, 50, 75, and 100 μmol·L^-1) were applied to the prepared strips. Subsequently, fluorescence imaging analysis was performed under laser irradiation with an in vivo optical imaging system after a 1-minute incubation period. As shown in Fig. 5, with increasing concentrations of ClO^-, a distinct fluorescence intensity gradient was observed in both green and red channels, which confirmed the applicability of the developed colorimetric test strip in rapid semi-quantitative ClO^- analysis. Thus, this simple and effective method could be used for water quality monitoring.

  Figure 5

  

  Figure 5.  Fluorescence imaging of (a) green and (b) red fluorescence channels with the fluorescence imaging system underdifferent concentrations of hypochlorite solutions

  Green channel: excitation wavelength=420 nm, emission wavelength of green fluorescent protein (GFP) dye; Red channel: excitationwavelength=620 nm, emission wavelength of Cyanine5.5 (Cy5.5) dye.

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  3.   Conclusions

  In this study, we developed a near-infrared HClO fluorescence probe with dual-channel self-calibration capability. The probe featured a green-light coumarin fluorophore and a near-infrared methylene blue fluorophore integrated via a cleavable linkage that responded to HClO, allowing for dual-channel HClO monitoring in aqueous solutions. Utilizing this probe, we performed dual-channel detection of both endogenous and exogenous HClO in HepG2 cells. The probe exhibited consistent sensitivity and detection limits for HClO in both 480 and 620 nm channels. Our study demonstrates that the probe enables accurate detection of HClO in complex systems while effectively mitigating the interference from background fluorescence, inherent in single-channel detection.

  
  Conflicts of interest: The authors declare no competing financial interests.
  Supporting information is available at http://www.wjhxxb.cn
  
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  Scheme 1  Synthesis of MBC

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  Figure 1  Intensity changes in the fluorescence spectra of MBC (5.0 μmol·L-1) upon addition of HClO (15.0 μmol·L-1): (a) excitation at 405 nm and (b) excitation at 620 nm

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  Figure 2  Fluorescence spectra of MBC (5.0 μmol·L-1) in the presence of various analytes upon excitation at wavelengths(a) 405 nm and (c) 620 nm, and the corresponding fluorescence intensities at emission wavelengths (b) 480 nmand (d) 678 nm

  1: Blank; 2: HClO; 3: H₂O₂; 4: NO; 5: ¹O₂; 6: ·OH; 7: NO₂^-; 8: ClO₄^-; 9: SO₃^2-; 10: Cl^-; 11: SO₄^2-; 12: NO₃^-; 13: PO₄^3-; 14: K⁺; 15: Na⁺; 16: Fe²+; 17: Fe³⁺; 18: Mg²⁺; 19: Cys; 20: GSH.

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  Figure 3  (a) Fluorescence spectra of MBC upon excitation at 405 nm varying with c_HClO; (b) Linear relationship between I_{480 nm} and c_HClO; (c) Fluorescence spectra of MBC upon excitation at 620 nm varying with c_HClO; (d) Linear relationship between I_{678 nm} and c_HClO

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  Scheme 2  Schematic illustration of the reaction of MBC with HClO

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  Figure 4  Fluorescence imaging of HepG2 cells: (a) untreatedcells; (b) cells treated with 5.0 μmol·L^-1 MBC for 30 min; (c) cells pre-incubated with 15.0 μmol·L^-1 HClO for 30 min followed by treatment with 5.0 μmol·L^-1 MBC for an additional 30 min

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  Figure 5  Fluorescence imaging of (a) green and (b) red fluorescence channels with the fluorescence imaging system underdifferent concentrations of hypochlorite solutions

  Green channel: excitation wavelength=420 nm, emission wavelength of green fluorescent protein (GFP) dye; Red channel: excitationwavelength=620 nm, emission wavelength of Cyanine5.5 (Cy5.5) dye.

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