Visualization of Endogenous Hypochlorous Acid During Ferroptosis Based on A Rhodamine B Fluorescent Probe

Visualization of Endogenous Hypochlorous Acid During Ferroptosis Based on A Rhodamine B Fluorescent Probe | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 20 January 2026 V1 Latest version Share on Visualization of Endogenous Hypochlorous Acid During Ferroptosis Based on A Rhodamine B Fluorescent Probe Authors : Shida Ma , Shujie Zhang , Feifei Jiang , Zuoyong Shi , Yan Liu , Yuhang Ji , and Jin Zhou 0000-0001-6761-3408 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176887450.06292803/v1 Published Analytica Chimica Acta Version of record Peer review timeline 126 views 66 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Hypochlorous acid (HClO) plays critical roles in ferroptosis process and the specific molecular mechanism related to it are still obscure. To reveal HClO fluctuation in the ferroptosis cells, an activity-based fluorescence probe RDM-577 that can detect intracellular HClO was exquisitely designed. The probe responded to HClO with significant fluorescence increases ( F/F 0 =35), thus laying the foundation for the sensitive detection of HClO in in vitro and in vivo . Moreover, the probe also possessed some fascinating performances, including a rather low detection limit (5.8 nm). As a result, the probe can readily reflect the fluctuation of HClO content in ferroptosis cells and by imaging the changes in HClO levels in cells undergoing ferroptosis, we found that GSH can resist the degradation of GPX4 caused by ferroptosis initiators. More importantly, RDM-577 could accurately indicated the site of inflammation in vivo and exhibits strong fluorescent signals in hepatitis mouse model. This work afforded a specific activity-based probe for the intravital imaging of HClO in the ferroptosis cells, which could be further extended to the specific molecular mechanism in ferroptosis process. Cite this paper: Chin. J. Chem. 2021 , 39 , XXX—XXX. DOI: 10.1002/cjoc.202100XXX Visualization of Endogenous Hypochlorous Acid During Ferroptosis Based on A Rhodamine B Fluorescent Probe Shida Ma, a, † Shujie Zhang, a, † Feifei Jiang, a Zuoyong Shi, a Yan Liu, a Yuhang Ji, a and Jin Zhou a,* a School of Pharmacy, School of Public Health, School of Nursing, Shandong Second Medical University, Weifang 261053, China. E-mail: [email protected] . . † These authors contributed equally to this work. Ferroptosis| fluorescence imaging | hypochlorous acid | hepatitis Hypochlorous acid (HClO) plays critical roles in ferroptosis process and the specific molecular mechanism related to it are still obscure. To reveal HClO fluctuation in the ferroptosis cells, an activity-based fluorescence probe RDM-577 that can detect intracellular HClO was exquisitely designed. The probe responded to HClO with significant fluorescence increases ( F/F 0 =35), thus laying the foundation for the sensitive detection of HClO in in vitro and in vivo . Moreover, the probe also possessed some fascinating performances, including a rather low detection limit (5.8 nm). As a result, the probe can readily reflect the fluctuation of HClO content in ferroptosis cells and by imaging the changes in HClO levels in cells undergoing ferroptosis, we found that GSH can resist the degradation of GPX4 caused by ferroptosis initiators. More importantly, RDM-577 could accurately indicated the site of inflammation in vivo and exhibits strong fluorescent signals in hepatitis mouse model. This work afforded a specific activity-based probe for the intravital imaging of HClO in the ferroptosis cells, which could be further extended to the specific molecular mechanism in ferroptosis process. Background and Originality Content Iron-dependent regulated cell death, known as ferroptosis, participates in numerous vital physiological and pathological processes, including development, immunity, tumor suppression and neurodegeneration. [1-3] The ferroptosis is driven by the deactivation of the antitransporter protein (system XC−) or glutathione peroxidase 4 (GPX4), accompanied by the production of large amounts of lipid reactive oxygen species (ROS). 4 As a highly reactive ROS, hypochlorous acid (HClO) can be produced from hydrogen peroxide and chloride ions (Cl−), with myeloperoxidase (MPO) as the catalyst. 5 Under physiological conditions, abnormal changes in HClO levels can affect many normal physiological processes, such as inflammation, Alzheimer’s disease, ferroptosis, cancer, etc. [6-8] At present, numerous studies have found that intracellular HClO is tightly linked to the process of ferroptosis and may increase in cells during ferroptosis. [9-11] Consequently, accurate tracking of the dynamic changes in HClO is especially meaningful for understanding the latent mechanisms of ferroptosis in some diseases. At present, multiple analytical methods have been developed for HClO detection, [12] including those based on electrochemical methods, [13] chemiluminescence [14] and test strip. [15] Moreover, fluorescent imaging technology has drawn considerable focus in recent years, due to its high sensitivity and high selectivity. [16-19] Compared to these methods such as electrochemical methods and test strip, fluorescence probe has possessed the advantages of non-invasiveness and real-time imaging. To date, numerous fluorescent probes have been developed to detect HClO variations by integrating diverse sensing mechanisms. However, the specificity of many traditional HClO probes are often interfered by other ROS, the high detection limit results in poor sensitivity of these probes, and more importantly, these probes are relatively few used for imaging HClO in ferroptosis cells. Therefore, it is crucial to develop a probe with a low detection limit that can detect HClO during the ferroptosis process. In this work, we report such a HClO fluorescent probe RDM-577 , which can be used to image HClO level fluctuation during ferroptosis. As illustrated in Scheme 1A, RDM-577 is built upon a stable and highly fluorescent Rhodamine B core, with the dihydroxybenzaldehyde hydrazone group seving as recoginition unit for HClO, [20] synthesized through a facile method (Scheme S1). RDM-577 exists in the colorless, nonfluorescent spirocyclic form in aqueous solution. After reaction with HClO, the probe RDM-577 transformed into the fluorescent group Rhodamine B. RDM-577 has been utilized to track HClO fluctuations in the cell throughout the oxidative stress progress. More importantly, it’s found that GSH can alleviate the decrease of intracellular GPX4 protein levels caused by ferroptosis initiators. Besides, RDM-577 could accurately indicated the site of inflammation in vivo and exhibits strong fluorescent signals in inflammatory site. Results and Discussion Probe Design and Synthesis RDM-577 and its associated intermediates were synthesized following the protocol outlined in Scheme S1 and subjected to characterization via 1 H NMR, 13 C NMR and high-resolution mass spectrometry (HRMS) (Figure S1-3). In simple terms, RDM-577 was readily prepared from Rhodamine B via a two-step procedure. Initially, Rhodamine B hydrazide was generated by reacting rhodamine B with hydrazine hydrate in dichloromethane. [21] Then, under the catalysis of trifluoroacetic acid, the intermediate of Rhodamine B hydrazide reacted with 3,4 dihydroxybenzaldehyde in methanol solution via reductive amination to obtain RDM-577 . Scheme 1 Scheme 1 (A) Molecular structure and fluorescence detection mechanism of RDM-577 . (B) The application of RDM-577 in imaging of ferroptosis. Fluorescence Response Properties to HClO in Vitro The spectroscopic characteristics of RDM-577 upon HClO exposure were examined in phosphate-buffered solutions (10 mM, pH 7.4). As depicted in Figure 1A, in the absence of HClO, RDM-577 had a weak absorption peak at around 500 nm. After reaction with HClO (30 μM), a significant rise around 560 nm was detected, with a simultaneous color transition from colorless to bright pink (see inset in Figure 1A). Conversely, RDM-577 exhibited a substantial fluorescence increase of approximately 35-fold at 574 nm (Figure 1B) when excited at 547 nm following HClO reaction. Subsequently, the influence of detection conditions (e.g., pH and temperature) was examined. Figure S6A-B demonstrate that the fluorescence intensity of RDM-577 is almost unaffected in the PBS solution of pH range 6.07−8.91, indicating that it has better performance under physiological pH range. In Figure S6C, RDM-577 exhibits minimal alteration in fluorescence intensity under temperature range 25-40°C. The above results demonstrated that the RDM-577 could keep stability in the physiological temperature and pH conditions. Alternatively, reaction time and kinetic behavior were key factors for the quantitative utilization of reaction-based probes. As depicted in Figures S6D (ESI†), the fast fluorescence increase reached a plateau within 30 min. Furthermore, at varying concentrations of HClO, the fluorescence response of RDM-577 exhibited an approximately linear concentration dependence. The linear equation was determined as F = 32.12 C (μM) + 113.62 (Figure 1C), with a low detection limit (S/N = 3) of 5.8 nM, enabling quantitative analysis of HClO (Figure 1C). However, when the HClO concentration surpassed 30 μM, the fluorescence intensity gradually declined (Figure S7), perhaps the high concentration of HClO rapidly oxidizes and destroys the structure of the probe so that affecting its response performance to HClO. To evaluate the reaction specificity, the fluorescence response of RDM-577 to various frequently coexisting substances in biological systems was simultaneously tested under identical conditions. The substances examined encompassed common ions, amino acid and the other ROS. As shown in Figure 1D and Figure 1E, only HClO induces a significant fluorescence enhancement, while other substances exhibit no such response. Additionally, the interference effects of these substances on the HClO assay were examined (Figure 1F), demonstrating that even at high concentrations, they minimally interfere with the assay. These findings confirm the probe’s excellent selectivity for HClO detection. Figure 1 The photophysical properties of RDM-577. Changes in (A) absorption and (B) fluorescence emission spectra of RDM-577 (10 μM) in PBS, (a) without and (b) with HClO (30 μM) in the pH 7.4 phosphate buffer for 30 min. (C) Chart of fluorescent emission intensity at 574 nm for RDM-577 (10 μM) compared to HClO with different concentration levels. (D) Fluorescence responses of RDM-577 to HClO (30 μM) and other common ROS (30 μM) in phosphate buffer (pH 7.4). (E) Fluorescence responses of RDM-577 to HClO (30 μM) and other common substances (30 μM) in phosphate buffer (pH 7.4). (F) The fluorescence intensity of RDM-577 (10 μM) and commonly coexisting substances (30 μM) in Figure 1E and HClO (30 μM) in phosphate buffer, common ROS (30 μM) in phosphate buffer (pH 7.4). λ ex/em = 548/574 nm, Slits: 5/5 nm. Error bars indicate relative standard deviations (RSD), n = 3. Fluorescence Imaging of HClO in Live Cells Motivated by the outstanding performance in vitro, the ability of RDM-577 to perform fluorescence imaging of HClO in cells was assessed. Prior to this, the cytotoxicity of RDM-577 underwent testing via standard MTT [(3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide] assays. The data (Supplementary Figure S8) revealed that cell viability remained largely unaffected after treatment, even at 10 μM for 24 h at 37 °C, demonstrating the probe’s low cytotoxicity and excellent biocompatibility. According to the cell viability test, the concentration of RDM-577 applied in this investigation was set at 10 μM. Following this, its capacity to sense intracellular HClO was examined. Figure S9 demonstrates that in the group treated with the probe but without exogenous HClO (Figure S9a), cells exhibited only faint fluorescence. Conversely, after exposure to HClO concentrations spanning 5–30 μM (Figure S9b-e), a dose-dependent amplification of the fluorescence signal was recorded. Next, in order to observe the endogenous generation of HClO, the cells underwent pretreatment with LPS (lipopolysaccharide), when LPS was co-incubated with cells, the MPO was overexpressed and oxidation of chloride ions to HClO, increasing in the intracellular HClO level. [22] As expected, the LPS treatment (Figure S10b-c) could result in significant, fluorescence enhancement in HeLa cells relative to the untreated cells (Figure S10a). Furthermore, the elevated fluorescence signals generated by endogenous HClO were effectively removed upon the addition of the HClO scavenger NAC (N-acetylcysteine) (Figure S10d). These results suggest that RDM-577 could be used to monitor the endogenous HClO level change in the cells. Figure 2 Intracellular HClO imaging during ferroptosis. (A) Fluorescence imaging of RDM-577 in HeLa cells treated by 10 μM ML210 and 1000 μM GSH. (a-b) Cells treated with ML210 (10 μM) for (a) 30 min and (b) 60 min. (c–e) Cells were pretreated with ML210 (10 μM) for 60 min, followed by exposure to GSH (1000 μM) for (c) 30 min, (d) 60 min, or (e) 90 min, and subsequently incubated with 10 μM RDM-577 for 60 min (B) Fluorescence imaging of RDM-577 in HeLa cells treated by ML210 (10 μM) and Fer-1 (10 μM). (f–h) Cells pretreated with ML210 (10 μM) for 60 min and then treated with Fer-1 (10 μM) for (f) 30 min, (g) 60 min, (h) 90 min and then exposed to 10 μM RDM-577 for 60 min. λ ex = 561 nm; λ em = [579nm - 707nm] nm. Scale bar: 20 μm. (C) WB analysis of the GPX4 level in different group cells. I: Control, II: Cells treated with ML210 (10 μM) for 6 h, III: Cells pretreated with ML210 (10 μM) for 6 h and then treated with Fer-1 (10 μM) for 90 min, IV: Cells pretreated with ML210 (10 μM) for 6 h and then treated with GSH (1000 μM) for 90 min (D) Fluorescent imaging of ROS in (A) cells. λ ex = 488 nm; λ em = 525 nm. Scale bar: 20 μm. Owing to the excellent HClO imaging capability of RDM-577 , we subsequently utilized it to track HClO fluctuations during ferroptosis. To achieve this, the HeLa cells underwent pretreatment with two distinct ferroptosis initiators out of two distinct mechanisms, Erastin [23] and ML210 [24] ([4-[Bis[4-Chlorophenyl] Methyl]Piperazin-1-Yl][5-Methyl-4-Nitroisoxazol-3-Yl]Methanone) to induce ferroptosis and then stained with RDM-577 for fluorescence imaging. Erastin exerts its effect by inhibiting cystine uptake through the blockade of the cystine/glutamate antiporter. This creates a deficiency in the cell’s antioxidant defenses, culminating in iron-dependent oxidative cell death. Consequently, cells exposed to Erastin experience LPO accumulation and ultimately undergo ferroptosis. [25] Initially, it was observed that a 30 min or 60 min incubation with Erastin resulted in a 5.36-fold and 10.25-fold increase in fluorescence intensity in HeLa cells, respectively (Figure S11b-c). On the flip side, ML210 is an inhibitor of GPX4 that can reduce the intracellular GPX4 protein content and leading to the termination of intracellular GPX4-GSH circulation, caused LPO accumulation and ultimately trigger ferroptosis. Likewise, exposure to ML210 for 30 min or 60 min also produced a 5.24- or 10.26-fold fluorescence enhancement, respectively (Figure 2Aa-b). These findings demonstrated that both Erastin and ML210-induced ferroptosis led to a significant elevation in HClO levels. Notably, co-incubation with GSH inhibited the intracellular fluorescence signals in cells treated with either compound (Figure S11d-f and Figure 2Ac-e). Besides, we also explored the intracellular HClO levels after incubated with different ferroptosis inhibitor. Fer-1 acts as a lipid ROS scavenger and prevents ferroptosis by reducing LPO, without affecting the intrinsic antioxidant capacity of cells. [26] As shown in the Figure 2B, after incubated with ML210 and Fer-1, the intracellular fluorescence has decreased when incubated with Fer-1 for 30 min, but as shown in Figure 2Bg-h, the intracellular fluorescence intensity cell remains almost unchanged after incubated with Fer-1 for 60 min and 90 min. As shown in Figure S12, the fluorescence intensity comparison chart can more intuitively reflect the differences. This may indicate that the GPX4-GSH circulation in cells was restored after incubation with GSH, while the circulation in cells incubated with Fer-1 was not restored. To investigate the effects of GSH and Fer-1 on GPX4 in ferroptosis cells, the expression level of GPX4 was assessed using Western blotting (WB) to evaluate the impact of GSH and Fer-1 on GPX4 expression, as shown in the Figure 2C, these results suggest that GSH can resisted the degradation of GPX4 caused by ML210. We also measured the content of intracellular ROS after treated with ML210 and GSH, and found that the content of intracellular ROS decreased after GSH treatment (Figure 2D and Figure S13). Intravital imaging of HClO in hepatitis mouse Finally, in vivo imaging experiments were conducted using the probe for detecting drug-induced hepatitis in animals. We induced a mouse inflammatory model by intraperitoneal injection of LPS [27] for 24 h, the specific experimental methods were shown in Figure 3A. As shown in Figure 3B-C, a substantial increase in fluorescence in the liver region of the model group but no obvious fluorescence in other organs, suggesting that inflammation may have occurred in the liver region of mouse. The subsequent TUNEL staining (Figure 3D) also suggested this result that the red fluorescence indicates inflammation in the liver tissue, while the normal liver in the control group did not. Further in vivo biocompatibility was evaluated (Figure 3E), after sacrifice, the heart, liver, spleen, lungs, and kidneys were harvested for hematoxylin and eosin (H&E) staining, which suggested that the probe RDM-577 showed no significant damage to mouse organs, confirming desirable biocompatibility and thus supporting its potential for live-animal imaging. Figure 3 Intravital imaging of HClO in hepatitis mouse. (A) Experimental schematic for HClO detection in inflammatory mouse models. (B) In vivo fluorescence imaging of RDM-577 (10 μM) in different group mice: Control group and Intraperitoneal injection of LPS group (C LPS =10 mg/Kg, 100 μL). λ ex/em = 550/585 nm. (C) In vitro organ imaging of different groups of mice. (D) Tunel staining of liver sections in different group mouse. (E) HE staining of various tissues of different groups of mice. Probe group: tail vein injection of RDM-577 for 12 h, control group: tail vein injection of saline. λ ex = 561 nm; λ em = [579nm - 707nm] nm. Scale bar: 200 μm. Conclusions In summary, we created a highly sensitive and selective fluorescent probe for HClO detection. Upon HClO exposure, the signal (F574) of the probe changed by 35-fold. With sufficient sensitivity to serve as a signaling molecule, the probe can detect exogenous HClO and endogenous HClO in living HeLa cells during LPS and ferroptosis inducer treatment. This allows quantification of drug-related adverse effects, including oxidative stress. The combination of real-time imaging, elevated sensitivity, robust specificity, and low biological toxicity positions the probe as a versatile tool with broad potential applications. Further structural optimization may yield an even superior probe. Experimental Regents and instruments Details are presented in the Supporting Information. Procedures for spectroscopic measurements Unless otherwise indicated, all absorption and fluorescence spectra were measured in 20 mM pH 7.4 phosphate buffers at room temperature. The probes were dissolved in DMSO as 1 mM stock solutions, which were diluted into the phosphate buffers to a final concentration of 10 μM. Then, appropriate amounts of different analytes (H 2 O 2 other ROS and physiologically active species) were added to the above solutions. The mixtures were kept at room temperature for the indicated time (typically 30 min) for spectroscopic measurements. All spectroscopic tests were performed 3 times in parallel and the results were expressed as mean ± standard deviation (SD). Cell culture and imaging HeLa cells were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences and cultured in DMEM supplemented with 100 μg/mL streptomycin, 100 U/mL penicillin, 10% FBS, and stayed in an incubator with an atmosphere of 10% CO 2 /90% air at 37. 1 °C. Intravital imaging of hepatitis mouse Normal KM mice (6 weeks old, 25 ± 1.0 g) were raised under standard conditions with enough food and water. 24 h before the experiment, intraperitoneal injection of LPS was performed to establish a hepatitis model. For imaging, the mice were anesthetized by a gas anesthesia machine and administrated with 100 μL 0.5 mM RDM-577 through tail vein injection. Subsequently, the mice were imaged and recorded by IVIS imaging system (PerkinElmer) in 0-2 h. Supporting Information The supporting information for this article is available on the WWW under https://doi.org/10.1002/cjoc.2021xxxxx. Acknowledgement We are grateful for the financial support from the National Natural Science Foundation of China (No. 21705120), the Project of Shandong Province Higher Educational Outstanding Youth Innovation Team (No. 2019KJM008), the Natural Science Foundation of Shandong Province, China (Nos. ZR2023MB001 and ZR2017LB016), Special Fund for Taishan Scholar Project (No. tsqn202211231), and Foundation of Yuandu Scholar. References 1. X, Jiang.; B, R. Stockwell.; M, Conrad. Ferroptosis: mechanisms, biology and role in disease. Nat. Rev. Mol. Cell Biol . 2021 , 22, 266-282. 2. B, R. Stockwell.; J, P. Friedmann Angeli.; H, Bayir.; A, I. Bush.; M, Conrad.; S, J. Dixon.; S, Fulda.; S, Gascón.; S, K. Hatzios.; V, E. Kagan.; K, Noel.; X, Jiang.; A, Linkermann.; M, E. Murphy.; M, Overholtzer.; A, Oyagi.; G, C. Pagnussat.; J, Park.; Q, Ran.; C, S. 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DOI: 10.1002/cjoc.202100XXX Ferroptosis, an iron-dependent regulated cell death, involves a variety of important physiological and pathological processes, intracellular HClO is closely related to the process of ferroptosis and may increase in cells during ferroptosis. Therefore, to further understand the relevant molecular mechanisms involved in the ferroptosis process, we constructed a fluorescent probe ( RDM-577 ), which could specifically enable real-time detection of HClO in cells during ferroptosis cells. Information & Authors Information Version history V1 Version 1 20 January 2026 Peer review timeline Published Analytica Chimica Acta Version of Record 1 Apr 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords ferroptosis fluorescence imaging hepatitis hypochlorous acid Authors Affiliations Shida Ma Shandong Second Medical University View all articles by this author Shujie Zhang Shandong Second Medical University View all articles by this author Feifei Jiang Shandong Second Medical University View all articles by this author Zuoyong Shi Shandong Second Medical University View all articles by this author Yan Liu Shandong Second Medical University View all articles by this author Yuhang Ji Shandong Second Medical University View all articles by this author Jin Zhou 0000-0001-6761-3408 [email protected] Shandong Second Medical University View all articles by this author Metrics & Citations Metrics Article Usage 126 views 66 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Shida Ma, Shujie Zhang, Feifei Jiang, et al. Visualization of Endogenous Hypochlorous Acid During Ferroptosis Based on A Rhodamine B Fluorescent Probe. Authorea . 20 January 2026. 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