Rational Design and Bioimaging Applications of Highly Specific “Turn

Publication Date (Web): July 30, 2018 ... Here we propose an easy-to-fabricate coumarin-based fluorescent probe incorporated with an aryl dihydrazide ...
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Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Rational Design and Bioimaging Applications of Highly Specific “Turn-On” Fluorescent Probe for Hypochlorite Teng Li, Leikun Wang, Shiqi Lin, Xiao Xu, Meng Liu, Shiyang Shen, Zhengyu Yan,* and Ran Mo* State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Center of Advanced Pharmaceuticals and Biomaterials, China Pharmaceutical University, Nanjing 210009, China

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ABSTRACT: Hypochlorite (OCl−), an essential part of reactive oxygen species (ROS), plays a crucial role in cellular redox balance. OCl− has been shown to be implicated in many physiological and pathological processes, including liver injury and cancer. Exploitation of fluorescent probes for the OCl− helps to reveal its function in the genesis and progression of the aforementioned diseases. Here we propose an easy-to-fabricate coumarin-based fluorescent probe incorporated with an aryl dihydrazide linker for highly specific detection and biological imaging of OCl− in living cells and tissues. The p-nitrophenylmodified dihydrazide-linked coumarin derivative (Cou-dhz-PhNO2) was screened from a series of candidate molecules and served as a “turn-on” probe with a low background fluorescence interference due to the nitro-group-based quenching on the coumarin fluorescence. The Cou-dhz-Ph-NO2 probe showed high selectivity and fast response with excellent linear relationship for detection of OCl−. A specific OCl−-responsive mechanism that the dihydrazide linker could be oxidatively cleaved by OCl− was deduced. The exogenous OCl− and endogenous OCl− were successfully visualized using the Cou-dhz-Ph-NO2 probe in living cells, such as MDA-MB-231 cells, RAW 264.7 cells and neutrophils, and the pathologic tissues, including the tumor tissue and the acutely injured liver tissue. This study paves the way for utilizing the aryl dihydrazide linker as OCl−-responsive module, which can aid the evolution of increasingly specific probes for detection of OCl− and diagnosis of OCl−-coupled diseases.

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INTRODUCTION Reactive oxygen species (ROS) are highly active metabolites of oxygen, such as hydrogen peroxide (H2O2), hypochlorous acid/hypochlorite (HOCl/OCl−), singlet oxygen (1O2), peroxynitrite (ONOO−), hydroxyl radical (OH·) and ozone (O3).1 Redox homeostasis is maintained in normal cells and tissues, while oxidative stress is involved in a variety of diseases and disorders.2 As the oxidation product of chloridion (Cl−) by myeloperoxidase (MPO) and H 2 O 2 , OCl − is an indispensable ROS with potent oxidation potential.3 OCl− can interact with DNA, proteins, and lipids and is implicated in multiple biological processes, typically in eliminating invading microorganisms, which is an important part of passive immunity.4−7 Nonetheless, OCl− also plays an essential role in a number of pathological processes, including kidney disease,8 diabetes,9 carcinogenesis,10 liver injury,11 atherosclerosis.12 For instance, OCl− promotes progression of atherosclerosis by oxidizing low-density lipoprotein (LDL) to oxidized LDL (oxLDL),13 induces severe cell damages through chlorination in the patients with type 2 diabetes mellitus,14 and causes tissue injuries in many neurodegenerative diseases.15 In particular, the MPO G-463A polymorphism has been demonstrated to be related to carcinogenesis in many types of cancers, such as lung cancer,16 gastric cancer,17 colorectal cancer,18 and hepatocellular carcinoma.19 OCl− and other © XXXX American Chemical Society

oxygen radicals generated by MPO can induce DNA damage and block DNA repair, which is regarded as a mechanism for tumorigenesis. The physiological concentration of OCl− in normal tissues is reported to be 5−25 μM,20 while the OCl− concentration was much higher than 100 μM in the pathological conditions.21−23 The variation of endogenous OCl− has been considered as an indicator of the development of ROS-coupled diseases and disorders. To this end, precise detection of OCl− change in the body would be a potential strategy for timely diagnosis of the OCl−-related diseases. Numerous fluorescent probes have been increasingly designed for the analyte determination due to favorable sensitivity, visualization ability, fast response, simplicity of preparation, and high spatial−temporal resolution. The past a few years have witnessed the development of fluorescent probe for detection of OCl−. Typical probes with distinctive characteristics of ratiometric signal,24,25 fluorescence “off− on”,26−28 fluorescence “on−off”,29 two-photon microscopy,30,31 upconversional fluorescence32 have been extensively reported. Besides, the exploitation of the fluorescent nanoprobes32−35 is also moving into high gear boosted by the rapid Received: June 18, 2018 Revised: July 13, 2018

A

DOI: 10.1021/acs.bioconjchem.8b00430 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

a model fluorescent molecule. A series of substituted benzene groups are conjugated to the coumarin molecule via the aryl dihydrazide linker. The impact of these substituent groups on the fluorescence property of coumarin is exploited. The pnitrophenyl-modified aryl dihydrazide-linked coumarin derivative (designated as Cou-dhz-Ph-NO2) is screened with a low background fluorescence interference by the quenching effect of nitro group on the fluorescence of coumarin, which serves as an “off−on” probe for highly selective detection of OCl−. The aryl dihydrazide linker was validated to degrade in the presence of OCl− rather than other ROS, which showed a preferable OCl−-selectivity. Exposure to OCl− triggers the cleavage of the dihydrazide linkage and the removal of the nitro group, which results in the fluorescence “turn on” of the probe. The obtained Cou-dhz-Ph-NO2 fluorescent probe is capable of imaging OCl− in the living cells and tissues.

development of nanotechnology. In the structure of probes, the recognition moiety defines the specificity and sensitivity. Double carbon bond, thioether, and selenide are announced as the OCl− recognition groups,24,36,37 while controversially, similar structures are also reported to show response to other ROS, including H2O2 and ONOO−,38−40 which indicates lack of specificity of these molecules for detection of OCl−. Fluorescent probes containing dithiolane or thiohydrazide moiety as recognition groups show poor linear relationship.41,42 Thus, an easy-to-fabricate fluorescent probe with excellent selectivity for OCl− remains highly desirable. Herein, we propose the application of aryl dihydrazideincorporated fluorescent probes for highly specific detection and biological imaging of OCl− in living cells and tissues (Figure 1). Long et al. previously reported a fluorescence

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RESULTS AND DISCUSSION Design of Aryl Dihydrazide-Containing OCl− Fluorescent Probes. We first designed and synthesized a series of OCl−-responsive aryl dihydrazide-containing coumarin-based fluorescent probes (Figure S1). The absorbance and fluorescence spectra of the synthetic fluorescent molecules were determined. A bathochromic-shift phenomenon was observed in both absorbance and fluorescence spectra of the molecules compared to Cou-COOH as expected (Figure 2a and Figure S2). This red shift is speculated to result from the conjugation of the benzene ring to the conjugated π-system of coumarin,45,46 although the molecules may not be in a complete coplanar conformation. For the fluorescence variation, the conjugation of the benzene ring led to enhancement on the fluorescence intensity of the Cou-dhzPh molecule, compared to that of Cou-COOH, which is a further evidence of the formation of a larger conjugation system. It is well established that the fluorescence properties are closely related to the substituents, which can interfere with the conjugation system. After modification with different substituted benzene groups, nine synthesized fluorescent probes exhibited distinctive fluorescence characteristics with different fluorescence intensities (Figure 2b). In comparison to Cou-dhz-Ph as control, both NH2 and OCH3 groups serve as the electron-donating parts, which resulted in the increased fluorescence intensity of Cou-dhz-Ph-NH2 and Cou-dhz-PhOCH3. In sharp contrast, the fluorescence molecules modified with the electron-withdrawing groups presented varying degree

Figure 1. Schematic illustration of Cou-dhz-Ph-NO2 for highly specific detection of OCl− in the living cells and pathological tissues.

ratiometric sensor probe containing dihydrazide linker for detection of OCl−.43,44 However, the probes are greatly limited by the complicated synthesis and changeless spirocyclic rhodamine structure, which undermines its universality. Using a different strategy, we envision that the influence of the substituent groups with disparate electron cloud distribution on the fluorescence property is a significant rationale for the design of OCl− fluorescent probe. Coumarin is selected as

Figure 2. (a) Fluorescence spectra of the obtained coumarin-based fluorescence molecules (10 μM) in the aqueous solution [10% dimethyl sulfoxide (DMSO), v:v] upon the excitation at 430 nm. (b) Fluorescence intensity (FI) of the fluorescence molecules at 478 nm upon the excitation at 430 nm. Slit width: 5 nm. B

DOI: 10.1021/acs.bioconjchem.8b00430 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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of 478 nm and the tested concentration of OCl− was further examined. A good linear correlation for both NO2-containing molecules was determined with the coefficient of correlation, R2 higher than 0.999 (Figure S6), which showed higher linearity than that of several previously reported OCl− fluorescent probes.39,40 Next, we evaluated the selectivity of the Cou-dhz-Ph-NO2 and Cou-dhz-Ph-(NO2)2 probes for detection of OCl− (Figure 3b and Figure S7). The fluorescence variation of the molecules treated with different ROS was detected. No significant difference in the fluorescence intensity between the untreated control group and other ROS-treated ones was observed. Except OCl−, all ROS were unable to activate the fluorescence recovery of the molecules, which indicates that the aryl dihydrazide linker can be highly specifically cleaved by OCl−, conferring favorable selectivity to Cou-dhz-Ph-NO2 and Coudhz-Ph-(NO2)2 for specific detection of OCl−. To elucidate the mechanism of the OCl−-induced cleavage of the aryl dihydrazide linker, the resultant reaction products were separated and characterized by the mass spectrometer after exposing Cou-dhz-Ph-NO2 to OCl−. On the basis of the molecular weight confirmation, we infer that after reacting with OCl−, Cou-dhz-Ph-NO2 converts to the intermediate, 3(diazenecarbonyl)-7-(diethylamino)coumarin, and further to the end product, Cou-COOH (Figure S8). Optimization for Detection of OCl−. The fluorescence intensity of Cou-dhz-Ph-NO2 and Cou-dhz-Ph-(NO2)2 was promptly and dramatically enhanced after addition of OCl−, which is indicative of a fast response of the probes to the surrounding OCl− (Figure 4a). We further evaluated the effect

of reduction in the fluorescence intensity. It is noteworthy that the order of decrease degree of halogen-decorated molecules is found as follows: Cou-dhz-Ph-Br > Cou-dhz-Ph-Cl > Cou-dhzPh-F, which is mainly attributed to the intramolecular heavyatom effect rather than the electron withdrawing effect. The heavy-atom effect has been demonstrated to induce enhancement of phosphorescence but attenuation of fluorescence.47−49 By comparison, Cou-dhz-Ph-NO2 and Cou-dhz-Ph-(NO2)2 showed nearly complete fluorescence quenching due to the impairment of the conjugation system by the powerful electron-withdrawing capacity of the NO2 group, which lays a good foundation for exploration of an “Off-On” fluorescent probe with negligible background fluorescence. Responsiveness and Selectivity for Detection of OCl−. To demonstrate the OCl−-responsiveness of the “turnon” fluorescent probes, Cou-dhz-Ph-NO2 and Cou-dhz-Ph(NO2)2, the change in the fluorescence intensity after exposure to OCl− was monitored. A concomitant increase in the fluorescence intensity of Cou-dhz-Ph-NO2 was apparently observed with the increase of the OCl− concentration within a short reaction period of 30 min (Figure 3a). Similar

Figure 3. (a) Fluorescence spectra of Cou-dhz-Ph-NO2 (10 μM) in aqueous solution (10% DMSO, v:v) after incubation with different concentrations of OCl− for 30 min upon the excitation at 430 nm. FI indicates the fluorescence intensity. (b) Fluorescence change of Coudhz-Ph-NO2 (10 μM) after incubation with different ROS (OCl−, H2O2, OH·, 1O2, and NO) for 30 min. F/F0 is the ratio of the fluorescence intensity of Cou-dhz-Ph-NO2 at 478 nm upon the excitation at 430 nm after treatment (F) to that before treatment (F0).

phenomenon was observed in Cou-dhz-Ph-(NO2)2 after treatment with OCl− (Figure S3). It is generally accepted that the fluorescence property of a compound depends on the conjugation situation in its structure, essentially as the electron arrangement. In this case, the conjugation in the NO2containing molecules is anticipated to be reinforced after the removal of the NO2 group by the OCl−-induced degradation of the aryl dihydrazide linker, which leads to the fluorescence recovery of the coumarin molecule. As a comparison, the ester linker in Cou-ester-Ph-NO2 is supposed to be blunt to OCl−, as confirmed by the fluorescence intensity of Cou-ester-PhNO2 remaining almost unchanged in the presence of OCl− (Figure S4). These data suggest that both Cou-dhz-Ph-NO2 and Cou-dhz-Ph-(NO2)2 possess excellent OCl−-responsiveness, which can be applied as the “turn-on” fluorescent probes for ultrarapid detection of OCl−. In addition, treatment with OCl− caused the fluorescence regression of either Cou-dhz-PhNH2 or Cou-dhz-Ph-OCH3 (Figure S5), which further confirms the OCl−-mediated cleavage of the aryl dihydrazide linker along with the removal of the electron-donating moieties. The correlation between the fluorescence intensity of Cou-dhz-Ph-NO2 or Cou-dhz-Ph-(NO2)2 at the wavelength

Figure 4. (a) Fluorescence intensity (FI) of Cou-dhz-Ph-NO2 and Cou-dhz-Ph-(NO2)2 (10 μM) after incubation with OCl− within 1 h. (b) Fluorescence change of Cou-dhz-Ph-NO2 (10 μM) after incubation with OCl− at different pH values for 30 min. F/F0 is the ratio of the fluorescence intensity of Cou-dhz-Ph-NO2 at 478 nm upon the excitation at 430 nm after treatment (F) to that before treatment (F0).

of temperature or pH on the OCl− detection capability of Coudhz-Ph-NO2 and Cou-dhz-Ph-(NO2)2. At either 25 °C (room temperature) or 37 °C (physiological temperature), both Coudhz-Ph-NO2 and Cou-dhz-Ph-(NO2)2 could be applied for detection of OCl−, as evidenced by increased fluorescence signal after exposure to OCl− (Figure S9). On the other hand, the detection of OCl− using both Cou-dhz-Ph-NO2 and Coudhz-Ph-(NO2)2 showed pH-dependence (Figure 4b and Figure S10b). In acidic condition, the fluorescence intensity of Coudhz-Ph-NO2 and Cou-dhz-Ph-(NO2)2 did not show any significant change after addition of OCl− at the studied short period of time. In stark contrast, under alkaline condition (pH C

DOI: 10.1021/acs.bioconjchem.8b00430 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry ≥ 10), the presence of OCl− produced more than 7-fold and 3fold increase in fluorescence intensity of Cou-dhz-Ph-NO2 and Cou-dhz-Ph-(NO2)2, respectively. Such remarkable enhancement may result from the alkaline environment, in which OCl− is much more oxidative. We speculated that OH− can accelerate the OCl−-mediated degradation of the acryl dihydrazide linker. Of note, Cou-dhz-Ph-NO2 preserved the OCl− responsiveness under the neutral condition (Figure 4b) compared to Cou-dhz-Ph-(NO2)2 (Figure S11b). These results indicate that Cou-dhz-Ph-NO2 is preferable to Cou-dhz-Ph(NO2)2 for detection of OCl− at body temperature and physiological pH with fast response speed, which is more favorable for biomedical applications. Detection of OCl− in Cell Models. To assess the ability of Cou-dhz-Ph-NO2 to detect OCl− inside the cells, the human breast carcinoma (MDA-MB-231) cells were first selected as a cell model, and the diffusion of Cou-dhz-Ph-NO2 into the MDA-MB-231 cells was explored (Figure S11a). The intracellular quantity of Cou-dhz-Ph-NO2 significantly increased as the incubation time extended, which suggests that Cou-dhzPh-NO2 can easily permeate into the cells for subsequent detection of intracellular OCl−. Next, we investigated the capability of Cou-dhz-Ph-NO2 to detect the exogenous OCl− inside the MDA-MB-231 cells. After the cells were preincubated with Cou-dhz-Ph-NO2 for 2 h, the OCl− was added, and after 0.5 h of incubation, the cells were observed using the confocal laser scanning microscope (CLSM) (Figure 5a). After incubation with Cou-dhz-Ph-NO2, the cells showed weak fluorescence signal due to the self-quenching effect of Cou-dhz-Ph-NO2, while strong fluorescence signals were observable in the cells incubated with OCl−. The quantitative analysis results showed that the fluorescence intensity in the cells after treatment with 100 μM and 1 mM OCl− was 1.6-fold and 2.5-fold that of the untreated cells, respectively (Figure 5b). These data suggest that Cou-dhz-Ph-NO2 is a qualified probe for detection of exogenous OCl− in the cells. To further demonstrate the quality of Cou-dhz-Ph-NO2 for detection of endogenous OCl−, the fluorescence variation of Cou-dhz-Ph-NO2 was investigated in the mouse monocytemacrophage (RAW 264.7) cells and the mouse bone marrowderived neutrophils after activation, respectively, given that these immune cells can be stimulated to generate a high level of intracellular ROS. Consistent with the cellular uptake results obtained in the MDA-MB-231 cell model, Cou-dhz-Ph-NO2 was able to efficiently diffuse into the RAW 264.7 cells and the neutrophils (Figure S11b,c). To produce a high concentration of ROS in the RAW 264.7 cells, lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) were used for pretreating these cells prior to incubation with Cou-dhz-Ph-NO2. Coudhz-Ph-NO2 showed elevated fluorescence signal in the LPS&PMA-treated RAW 264.7 cells as expected, compared to that in the untreated cells (Figure 5c). The fluorescence intensity of the coumarin signal in the treated cells rose significantly compared to that in the untreated cells (Figure 5d). Furthermore, we used the neutrophils isolated from the mouse bone marrow as primary cells to reflect the OCl−detecting capacity of Cou-dhz-Ph-NO2 in the body, which is more appropriate than utilization of the commonly used cell models. For the neutrophils, treatment of PMA caused increased level of OCl− that is mainly produced by the myeloperoxidase-catalyzed reaction between H2O2 and Cl−, which resulted in the elevated fluorescence signal, as

Figure 5. (a) CLSM images of the MDA-MB-231 cells before and after addition of OCl−. (b) Fluorescence intensity of the fluorescence signal in MDA-MB-231 cells before and after addition of OCl−. *P < 0.05. (c) CLSM images of untreated and treated RAW 264.7 cells after incubation with Cou-dhz-Ph-NO2. (d) Fluorescence intensity of the fluorescence signal in the treated RAW 264.7 cells after incubation with Cou-dhz-Ph-NO2. *P < 0.05, **P < 0.01. (e) CLSM images of the untreated and treated neutrophils after incubation with Cou-dhzPh-NO2. (f) Fluorescence intensity of the fluorescence signal in the treated neutrophils after incubation with Cou-dhz-Ph-NO2. **P < 0.01. FI/P indicates the ratio of fluorescence intensity to cell protein. Scale bars represent 10 μm. For CLSM observation, the excitation wavelength was 405 nm, and the fluorescence signal was collected at the emission wavelength ranging from 440 to 800 nm. For quantitative analysis, the excitation wavelength was 430 and the emission wavelength 478 nm.

apparently visualized by CLSM (Figure 5e). The quantitative result showed that the coumarin signal in the PMA-treated neutrophils was 2.2-fold that in the native neutrophils (Figure 5f). These findings support that Cou-dhz-Ph-NO2 is competent of imaging both exogenous and endogenous OCl− at the cellular level. Detection of OCl− in Animal Models. To demonstrate the OCl−-detecting capacity of Cou-dhz-Ph-NO2 in the animal models, the MDA-MB-231 tumor xenograft mouse model was first employed for detection of exogenous OCl− by Cou-dhzPh-NO2. The MDA-MB-231 tumor-bearing mice were intratumorally injected with the Cou-dhz-Ph-NO2 solution, followed by intratumoral injection of OCl− as previously reported.50−52 After 2 h, the tumor was withdrawn and prepared for cryosection. The frozen tumor section was monitored using CLSM (Figure S12a). The CLSM images showed that significantly stronger fluorescence signal was observable in the tumor of the mice receiving an injection of OCl−. The mean fluorescence intensity in the OCl−-treated tumor was 2.7-fold higher than that in the untreated tumor (Figure S12b). These data imply that Cou-dhz-Ph-NO2 manages to image exogenous OCl− in the tumor tissue. D

DOI: 10.1021/acs.bioconjchem.8b00430 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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perfused alcohol-treated mice was remarkably higher than that of the Cou-dhz-Ph-NO2-perfused normal mice (Figure 6b) and that of the Cou-ester-Ph-NO2-perfused alcohol-treated mice (Figure S14b). We further employed intraperitoneal injection of LPS together with D-galactosamine (D-GalN) to build a mouse model of acute liver failure.54 The histological examination on the liver and the relative activities of ALT and AST in the serum of the LPS/D-GalN-treated mice compared to the normal mice showed the successful establishment of the model (Figure S15). The LPS/D-GalN-treated mice were intravenously injected with Cou-dhz-Ph-NO2. After 2 h, the liver was harvested and treated by cryotomy. The CLSM images of the liver sections showed that the fluorescence signal in the liver of the LPS/D-GalN-treated mice was markedly stronger than that in the liver of the normal mice (Figure 6c), as further validated by the quantitative data (Figure 6d). In sharp contrast, the fluorescence signal was quite weak in the liver of the LPS/D-GalN-treated mice receiving intravenous injection of Cou-ester-Ph-NO2 (Figure S16). Taken together, Cou-dhzPh-NO2 as a “turn-on” fluorescent probe reveals a prominent capacity of detecting the generation of OCl−, which would be a promising tool to diagnose the onset of the ROS-related disease.

To further evaluate the feasibility of Cou-dhz-Ph-NO2 to monitor the change in the endogenous OCl− level upon the initiation of disease, the acute liver injury mouse models were established, followed by monitoring the OCl− variation using the Cou-dhz-Ph-NO2 probe. Intragastric administration of a high dosage of alcohol was first applied to induce the acute liver injury with an accelerated generation of a high level of ROS in the liver.53 The alcohol-treated mice showed severe damage in the liver with massive hepatocyte necrosis, edema, and inflammatory cell infiltration, as characterized by the histological examination using the hematoxylin and eosin (H&E) staining (Figure S13a). Additionally, the serum levels of both alanine transaminase (ALT) and aspartate transaminase (AST) in the alcohol-treated mice were significantly higher than that in the normal mice (Figure S13b). These results are indicative of a successful establishment of the acute liver injury mouse model. Next, the liver of the mice under anesthesia were in situ perfused with Cou-dhz-Ph-NO2 and were subsequently harvested after perfusion. A notably higher fluorescence signal was visualized in the liver collected from the alcohol-treated mice with the acute liver injury than that in the untreated mice (Figure 6a). On the contrary, there was no

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CONCLUSIONS In summary, we have developed a specific “turn-on” OCl− probe with the aryl dihydrazide linker, in which the electronwithdrawing nitro group was employed to quench fluorescence of coumarin. The Cou-dhz-Ph-NO2 probe with excellent linear relationship for the OCl− detection showed great potential in imaging the exogenous and endogenous OCl − . The upregulation of the endogenous OCl− in the alcohol-induced acutely injured liver was successfully visualized by the Coudhz-Ph-NO2 probe. In addition, the OCl−-responsive mechanism of the aryl dihydrazide linker was confirmed to be cleaved by OCl− to form a diimide group, which further converts to carboxylic acid. We acknowledge that Cou-dhz-Ph-NO2 is not suitable for in vivo deep tissue imaging, arising from the relatively short maximum emission wavelength (