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• Reactive oxygen species (ROS) are a group of highly reactive chemicals that are produced via normal cellular metabolic processes [1-3]. Normally, ROS play a critical role in cellular signaling and immune defense. However, when ROS are generated in excess or when there are problems with their removal mechanisms, oxidative stress is triggered, resulting in cellular damage that can lead to a variety of diseases, such as cardiovascular disease, diabetes, and neurodegenerative diseases [4-7]. Hypochlorous acid (HClO), an important type of ROS, is a powerful oxidizing agent that destroys cell membranes and proteins of microorganisms to achieve antimicrobial effects [8-11]. Nevertheless, if HClO is produced in excess or used improperly, it may cause damage to host cells, increasing the possibility of inflammatory reactions and tissue damage [12-14]. Therefore, HClO is a double-edged sword in vivo. On one hand, it is indispensable for life-sustaining activities, and on the other hand, it is a potential disease-causing factor [15,16]. The rational control of HClO production and removal is of great significance in the prevention and treatment of associated diseases [17]. Therefore, it is necessary to detect HClO fluctuations in real time in living organisms, which can serve the purpose of preventing or diagnosing related diseases, or even in order to better intervene in interrelated diseases.

  A series of methods have been developed for the qualitative and quantitative determination of HClO, such as chromatographic methods, electrochemical methods, and mass spectrometry [18-20]. These excellent methods can effectively quantify analysis of HClO content in samples, however, they require complex sample pretreatment, which is time-consuming or necessitates sample destruction. In recent decades, fluorescence imaging technology has been rapidly developed due to its good sensitivity and specificity, high temporal resolution, and the possibility of real-time and non-invasive detection of biological samples [21-24]. To date, a number of fluorescent probes have been proposed for detecting HClO in biological systems, primarily based on structures such as coumarins, fluoresceins, and rhodamines [25-27]. These probes perform well in terms of spectral properties but still suffer from slow response time and short emission wavelengths, which limit their application in organisms [28,29]. Near-infrared (NIR) fluorescent probes are able to penetrate biological tissues without much absorption, while reducing the effects of light scattering and background signals more suitable for biofluorescence imaging, making them particularly suitable for biological fluorescence imaging [30,31]. In contrast to the limitations of single-photon imaging in the visible light spectrum, NIR two-photon microscopy techniques can mitigate the effects of autofluorescence during imaging and reduce photodamage to tissues [32,33]. However, a significant challenge faced by many two-photon imaging probes is that they predominantly emit fluorescence at shorter wavelengths (<600 nm) [34]. The shorter wavelength fluorescence may struggle to effectively image biological tissues through the brain due to limitations in light scattering and penetration depth. Therefore, the development of rapid HClO detection fluorescence probes with NIR two-photon offer significant advantages. Such probes can not only provide more accurate detection results, but also reduce the damage to biological samples and improve the real-time and non-invasive detection, which has wide-ranging applications in the fields of biomedical research, clinical diagnosis and environmental monitoring.

  In this work, we successfully developed a novel NIR two-photon fluorescent probe that exhibits a rapid response to HClO based on the above challenges. Upon the presence of HClO, the probe triggers an apparent fluorescent signal at 660 nm within 5 s. Its limit of detection is as low as 17.2 nmol/L, exhibiting extremely high sensitivity. The probe not only demonstrates high reactivity and specificity for HClO, but also it is not interfered by other active species under the experimental conditions, which ensures the accuracy of the detection results. Utilizing this probe, we were not only able to achieve real-time monitoring of intracellular HClO levels, but also applied it to lipopolysaccharide (LPS)-induced inflammation models in mice and visual imaging of HClO in tumors. This innovative probe technology provides a powerful tool for in-depth study of the mechanism of action of HClO in organisms, as well as a new avenue for the diagnosis of related diseases. We are expected to gain a deeper understanding the role of HClO in the development of inflammation, tumors and other diseases, thus providing a scientific basis for disease prevention, diagnosis and treatment by monitoring HClO levels.

  The structure of the probe HDM-Cl-HClO and the mechanism of its reaction with HClO were performed as shown in Scheme 1. Dicyanoisophorone derivatives have a wide range of applications in biomedical and environmental sciences due to their favorable spectral properties and ease of synthesis. Despite the common use of DCI-OH fluorophores, there are still some limitations, the most important of which is their high pK_a value (9.02), which leads to their low protonation under physiological conditions, thus limiting their application for accurate imaging in biological systems [35,36]. To solve this problem, researchers found that introducing an electron-withdrawing group near the hydroxyl group of HDM-Cl can effectively reduce its pK_a value (7.60) [37]. Therefore, DCI-OH was selected as a fluorophore for the construction of probes with specific responsiveness. In addition, the N,N-dimethylthiocarbamoyl thiocarbamate (DMTT) moiety exhibits quick response, excellent sensitivity and selectivity to HClO, which makes it an ideal responsive moiety. By introducing the DMTT into the probe, the intramolecular charge transfer process of HDM-Cl-HClO can be inhibited so that it exhibits inert fluorescent properties. However, when HClO is present, the probe emits a strong fluorescent signal at 660 nm within 5 s. This design makes the probe highly sensitive and specific in response to HClO, opening up new possibilities for applications in biomedical and environmental sciences.

  Scheme 1

  

  Scheme 1.  (A) Structure of fluorophore DCI-OH and HDM-Cl. (B) Molecular design of the probe HDM-Cl-HClO and proposed response mechanisms for HClO.

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  We carried out an in-depth investigation of the spectral properties of the probe HDM-Cl-HClO, which was carried out in phosphate buffer saline (PBS) solution (PBS/dimethyl sulfoxide (DMSO) = 95/5, pH 7.4, 10 mmol/L). First, we analyzed the absorption spectrum of the probe in detail. As shown in Fig. 1A, the probe HDM-Cl-HClO exhibited a significant absorption peak in the wavelength region of about 400 nm. As the concentration of HClO increased, the absorption peak at this wavelength showed a decreasing trend, while the absorption peaks at 460 and 480 nm gradually enhanced. This phenomenon revealed that the probe HDM-Cl-HClO was able to interact with HClO and consequently generate the new compound HDM-Cl. Subsequently, we further examined the fluorescence intensity changes of HDM-Cl-HClO in the presence or absence of HClO. As shown in Fig. 1B, when the probe was independently present, it exhibited only weak fluorescence near 660 nm. A noteworthy phenomenon was that the intensity of the fluorescence emission peak of the probe at 660 nm was enhanced as the concentration of HClO increased. This finding confirmed the effectiveness of the probe for HClO detection. In addition, we observed a favorable linear relationship between the fluorescence intensity of the probe and the HClO concentration (0–40 µmol/L) (Fig. 1C), which was manifested by the equation Y = 6.9040[HClO] + 20.8849, where the R² value was 0.9978, showing a very high correlation. Based on the standard assay method of 3σ/k, we calculated that the detection limit of HClO was 17.2 nmol/L.

  Figure 1

  

  Figure 1.  Spectral properties of HDM-Cl-HClO for HClO. (A) The ultraviolet absorption spectrum and (B) fluorescence emission spectra of HDM-Cl-HClO toward HClO (0–70 µmol/L) for 5 s in PBS/DMSO (95:5) solution (pH 7.4, 10 mmol/L). (C) The linear relationship between the fluorescent signal of HDM-Cl-HClO to HClO (0–40 µmol/L). (D) The pH dependent fluorescence response of HDM-Cl-HClO to HClO. Data were presented as mean ± standard deviation (SD) (n = 3).

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  To explore the possibility of applying the probe (HDM-Cl-HClO) in biological systems, the effect of pH on the probe was evaluated. As shown in Fig. 1D, HDM-Cl-HClO displayed essentially no change in fluorescence signal between pH 4–9, indicating that HDM-Cl-HClO was essentially unaffected by pH value. However, in the presence of HClO, the probe showed a significant fluorescent signal enhancement between pH 6–9, indicating that there was a high probability that the probe could be used for the detection of HClO in organism. To further understand the responsiveness of the probe HDM-Cl-HClO to HClO, we performed kinetic experiments. As shown in Fig. S2 (Supporting information), when 10 µmol/L HClO was added, the fluorescence intensity of HDM-Cl-HClO rapidly rose to the peak within 5 s, demonstrating the rapid responsiveness of the probe to HClO. Subsequently, we added different concentrations of HClO (20 and 60 µmol/L), and we obtained similar experimental results, confirming the fast response of the probe to HClO. Next, we investigated the selectivity of HDM-Cl-HClO, and whether they could specifically respond to HClO was also a key consideration in the evaluation of the probe. For this purpose, we selected some common reactive oxygen/reactive nitrogen species (nitroxylic acid (HNO), superoxide anion (O₂^•−), hydroxyl radical (^•OH), hydrogen peroxide (H₂O₂), nitric oxide (NO), nitrite ion (NO₂⁻)), biothiols (homocysteine (Hcy), glutathione (GSH), cysteine (Cys), methionine (Met), glycine (Gly), lysine (Lys), tyrosine (Tyr), aspartate (Asp)), common anions and cations (Fe³⁺, Ca²⁺, Fe²⁺, Zn²⁺, K⁺, Na⁺, PO₄³⁻, CO₃²⁻, SO₄²⁻, Cl⁻, Br⁻, I⁻, F⁻). As shown in Fig. S3 (Supporting information), only HClO could cause a strong fluorescence signal change at 660 nm, whereas the presence of other species did not cause the probe to emit a strong fluorescence signal at 660 nm, which suggested that the probe HDM-Cl-HClO could specifically respond to HClO and effectively distinguish HClO from other substances. The above results indicated that the probe exhibited excellent spectral properties for HClO and had great potential for application in the monitoring of HClO in vivo.

  Next, we investigated whether the probe HDM-Cl-HClO could be used for two-photon detection of HClO. Therefore, we measured the two-photon absorption cross section (δ) of HDM-Cl in PBS (10 mmol/L, pH 7.4, 5% DMSO with 1 mmol/L hexadecyltrimethylammonium bromide (CTAB)). The maximal δ was measured to be 34 GM at 880 nm, indicating that HDM-Cl-HClO can track HClO via two-photon microscope.

  The probe HDM-Cl-HClO exhibited superior spectral performance for HClO, and we tried to apply it for detecting HClO in biological systems. Initially, we conducted a cytotoxicity study on the probe. The survival of the probe on RAW 264.7 and HeLa cells was assessed by cell-counting-kit-8 (CCK-8) assay. The probe maintained high activity even at higher concentrations of the probe (Fig. S4 in Supporting information), indicating its low cellular activity. Subsequently, we further investigated the ability of HDM-Cl-HClO for monitoring HClO in living cells. Here, we chose RAW 264.7 cells as the primary model cells for this study. After co-incubating the probe HDM-Cl-HClO with RAW 264.7 cells for 20 min, imaging was performed. As depicted in Figs. 2A and B, when the probe alone was used for co-incubation with the cells, we found only a weak fluorescent signal. Upon adding different levels of HClO (5–30 µmol/L) and then co-incubating with the probe, we found that the red fluorescence signal gradually intensified with increasing levels of HClO, suggesting that the probe HDM-Cl-HClO could be effectively applied for monitoring exogenous HClO levels.

  Figure 2

  

  Figure 2.  Imaging of exogenous and endogenous HClO in RAW 264.7 cells. (A) The RAW 264.7 cells were pre-treated with a series of concentrations of HClO (0–30 µmol/L) and then co-incubated with HDM-Cl-HClO (10 µmol/L) for 20 min at 37 ℃. (B) Relative fluorescent intensities in A. (C) Control group: Cells were imaged after incubation with the probe only for 20 min. For LPS/PMA group, LPS/PMA+NAC group, LPS/PMA+4-ABAH group, the cells were treated with LPS (1 µg/mL)/PMA (1 µg/mL), LPS (1 µg/mL)/PMA (1 µg/mL)/NAC (1 mmol/L), and LPS (1 µg/mL)/PMA (1 µg/mL)/4-ABAH (1 mmol/L) for 6 h, and then incubated with the probe for 20 min before imaging, respectively. For H₂O₂ group and SIN-1 group: the cells were pro-treated with H₂O₂ (50 µmol/L) and SIN-1 (50 µmol/L) for 0.5 h, and then incubated with the probe for 20 min before imaging, respectively. (D) Relative fluorescent intensities in C. Data were presented as mean ± SD (n = 3). λ_ex = 880 nm, λ_em = 630–690 nm. Scale bar: 50 µm.

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  Next, we investigated the potential of the probe HDM-Cl-HClO to detect endogenous HClO (Figs. 2C and D). Previous studies have shown that endogenous HClO could be generated by stimulating cells with LPS and phosphatidyl-12-monooleoyl-13-acetate (PMA) [38,39]. Here, we used LPS/PMA to treat with cells for 6 h, followed by the addition of the probe HDM-Cl-HClO for co-incubation for 20 min before imaging. Compared to control group, the fluorescence intensity was outstanding enhanced in LPS/PMA group, indicating that the probe could be applied for HClO detection. Subsequently, we further verified whether the signal was caused by HClO production by using a scavenger of ROS, N-acetyl-l-cysteine (NAC) [40]. When the RAW 264.7 cells were co-treated with LPS/PMA and NAC for 6 h, and then these cells were incubated with HDM-Cl-HClO for 20 min. A significantly reduced fluorescence signal was found, indicating that the probe could detect fluctuations in endogenous HClO levels. In biological systems, there is a correlation between HClO generation and the activity of myeloperoxidase (MPO), which catalyzes the reaction between H₂O₂ and Cl⁻ to ClO⁻ [41]. Therefore, we used MPO inhibitor, 4-aminobenzoic acid hydrazide (4-ABAH), to treat the cells and observe whether it could cause significant fluorescence signal changes [42]. When the RAW 264.7 cells were pretreated with 4-ABAH and LPS/PMA for 6 h and then incubated with HDM-Cl-HClO for 20 min, we observed a weak fluorescent signal. This should be attributed to the use of 4-ABAH, which inhibited the activity of MPO and further inhibited the production of HClO, which further suggested that HDM-Cl-HClO could be employed to specific detect HClO. Next, we treated the cells with H₂O₂ and 3-morpholinosydnonimine (SIN-1, a donor of ONOO⁻) for 30 min, we also found only weak fluorescence signals, which demonstrated that only the presence of HClO could cause a strong change in the fluorescence signal of the probe, further confirming the specific detection ability of the probe for HClO. The above experiments showed that the probe HDM-Cl-HClO could be used for the specific monitoring of fluctuations in intracellular HClO levels, providing an effective tool for the study of HClO in vivo.

  Considering the ability of HDM-Cl-HClO to detect changes in HClO levels at the cellular level, we set out to investigate its feasibility for HClO detection in living organisms. Zebrafish is an ideal animal model for imaging studies in living vertebrates due to its small size, rapid reproduction cycle, ease of handling, and excellent light transmission [43]. As shown in Figs. 3A and B, when the zebrafish were treated with the probe alone, we only captured a weak fluorescent response. However, we first treated zebrafish with 5 µmol/L HClO and subsequently co-incubated with the probe for 20 min, we observed a significant enhancement of red fluorescence, which confirmed the effectiveness of the probe in HClO detection. Next, we introduced different concentrations of HClO (15 and 30 µmol/L) and imaged the zebrafish after treatment with the probe again, and found that the red fluorescence signal was enhanced as the concentration of HClO elevated, which further demonstrated the potential of the probe’s application in monitoring changes in HClO levels within biological organisms. Given that HDM-Cl-HClO could achieve the detection of exogenous HClO in zebrafish, we further investigated its ability to detect endogenous HClO. Compared to the untreated group, the probe treated with LPS and co-incubated with HClO showed a significantly enhancement in red fluorescence signal (Figs. 3C and D). In contrast, the red fluorescent signal was weakened by the addition of NAC treatment, suggesting that the addition of NAC had an inhibitory effect on HClO levels. Together, these experimental results indicated that the probe possessed significant application in detecting the fluctuation of HClO levels in organisms.

  Figure 3

  

  Figure 3.  Imaging HClO in zebrafish. (A) Zebrafish were treated with no treatment and different levels of HClO (5, 15 and 30 µmol/L), and then imaged after incubation with HDM-Cl-HClO, respectively. (B) Relative fluorescent intensities in A. (C) Zebrafish were treated with no treatment, LPS, and LPS+NAC, and then imaged after incubation with the probe, respectively. (D) Relative fluorescent intensities in C. Data were presented as mean ± SD (n = 3). λ_ex = 880 nm, λ_em = 630–690 nm. Scale bar: 500 µm.

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  After confirming that the probe HDM-Cl-HClO could achieve HClO imaging in living cells and zebrafish, we attempted to monitor HClO changes in mice using HDM-Cl-HClO. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Hainan Medical University (Haikou, China). Inflammation is a complex biological process that is the body’s defense response to various injuries (infections, physical injuries, chemical stimuli, and so on) [44,45]. However, inflammation may lead to sustained tissue damage, such as joint damage in rheumatoid arthritis, neurodegenerative diseases, cardiovascular diseases [46]. Therefore, early identification and intervention of inflammation is essential. It is well documented that LPS stimulation induces localized inflammation [47]. Here, we constructed LPS-induced inflammation model mice to study the ability of HClO imaging in mice. Compared to the control group, the LPS group displayed obvious fluorescence signals (Fig. 4), indicating that the probe could be used to track fluctuations of HClO in inflamed mice.

  Figure 4

  

  Figure 4.  Fluorescence imaging of HClO in mice models of inflammation and tumor. (A) Schematic of inflammation and tumor mouse imaging. (B) Imaging of HClO in mice models of inflammation and tumor. Control group: healthy BALB/C mice; LPS group: LPS-induced inflammatory mice; LPS+(Sec)₂ group: (Sec)₂-treated the inflammatory mice; tumor group: tumor-bearing mice. (C) Relative fluorescent intensities in B. Data were presented as mean ± SD (n = 3). λ_ex = 490 nm, λ_em = 630–690 nm.

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  However, when LPS was incubated with selenocysteine ((Sec)₂, an important antioxidant that helps to combat cellular oxidative stress and maintain cellular redox balance), the red fluorescence signal decreased rapidly, which should be attributed to the presence of (Sec)₂, leading to a reduction in the level of HClO. These results demonstrated the probe’s capability for imaging of HClO in vivo. Unlike inflammation, the microenvironment at the tumor site is more complex, so we constructed corresponding tumor models for further research. As shown in Fig. 4, we also observed strong fluorescence signals at the tumor site. These results indicated that the probe could not only be applied for monitoring HClO levels in inflammatory environments, but also for the imaging of HClO at tumor sites, suggesting its great potential as a reliable tool for the detection of HClO levels in various disease models.

  In summary, we developed a NIR two-photon fluorescent probe HDM-Cl-HClO for the rapid detection of HClO in inflammatory and tumor environments. The excitation/emission wavelengths of this two-photon probe are in the NIR region, effectively increasing the imaging depth of the probe in biological systems. It is not only suitable for the detection of fluctuating HClO levels in cellular and zebrafish models, but its ability to detect HClO levels was also successfully validated in inflammatory and tumor mouse models. This discovery provides a reliable tool for HClO detection in disease models, and offers a new perspective for a deeper understanding of the association between HClO and related diseases. We firmly believe that the development of this probe will provide researchers with an efficient and precise means for the detection of HClO in different disease models, thereby helping to elucidate the role of HClO in the progression of diseases and providing a scientific basis for the prevention, diagnosis, and treatment of disease.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Xianzhu Luo: Writing – review & editing, Writing – original draft, Software, Investigation, Conceptualization. Feifei Yu: Writing – original draft, Software, Formal analysis. Rui Wang: Writing – original draft, Software, Resources, Funding acquisition, Formal analysis. Tian Su: Writing – original draft, Visualization, Software. Pan Luo: Validation, Formal analysis. Pengfei Wen: Writing – review & editing, Writing – original draft, Software, Resources. Fabiao Yu: Writing – review & editing, Writing – original draft, Resources, Funding acquisition, Formal analysis.

  Acknowledgments

  Thanks to the National Natural Science Foundation of China (No. 22264013), Hainan Province Clinical Medical Center (No. 2021), Hainan Province Science and Technology Special Fund (No. ZDYF2024SHFZ104).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110531.

  
  
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• 

  Scheme 1  (A) Structure of fluorophore DCI-OH and HDM-Cl. (B) Molecular design of the probe HDM-Cl-HClO and proposed response mechanisms for HClO.

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  Figure 1  Spectral properties of HDM-Cl-HClO for HClO. (A) The ultraviolet absorption spectrum and (B) fluorescence emission spectra of HDM-Cl-HClO toward HClO (0–70 µmol/L) for 5 s in PBS/DMSO (95:5) solution (pH 7.4, 10 mmol/L). (C) The linear relationship between the fluorescent signal of HDM-Cl-HClO to HClO (0–40 µmol/L). (D) The pH dependent fluorescence response of HDM-Cl-HClO to HClO. Data were presented as mean ± standard deviation (SD) (n = 3).

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  Figure 2  Imaging of exogenous and endogenous HClO in RAW 264.7 cells. (A) The RAW 264.7 cells were pre-treated with a series of concentrations of HClO (0–30 µmol/L) and then co-incubated with HDM-Cl-HClO (10 µmol/L) for 20 min at 37 ℃. (B) Relative fluorescent intensities in A. (C) Control group: Cells were imaged after incubation with the probe only for 20 min. For LPS/PMA group, LPS/PMA+NAC group, LPS/PMA+4-ABAH group, the cells were treated with LPS (1 µg/mL)/PMA (1 µg/mL), LPS (1 µg/mL)/PMA (1 µg/mL)/NAC (1 mmol/L), and LPS (1 µg/mL)/PMA (1 µg/mL)/4-ABAH (1 mmol/L) for 6 h, and then incubated with the probe for 20 min before imaging, respectively. For H₂O₂ group and SIN-1 group: the cells were pro-treated with H₂O₂ (50 µmol/L) and SIN-1 (50 µmol/L) for 0.5 h, and then incubated with the probe for 20 min before imaging, respectively. (D) Relative fluorescent intensities in C. Data were presented as mean ± SD (n = 3). λ_ex = 880 nm, λ_em = 630–690 nm. Scale bar: 50 µm.

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  Figure 3  Imaging HClO in zebrafish. (A) Zebrafish were treated with no treatment and different levels of HClO (5, 15 and 30 µmol/L), and then imaged after incubation with HDM-Cl-HClO, respectively. (B) Relative fluorescent intensities in A. (C) Zebrafish were treated with no treatment, LPS, and LPS+NAC, and then imaged after incubation with the probe, respectively. (D) Relative fluorescent intensities in C. Data were presented as mean ± SD (n = 3). λ_ex = 880 nm, λ_em = 630–690 nm. Scale bar: 500 µm.

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  Figure 4  Fluorescence imaging of HClO in mice models of inflammation and tumor. (A) Schematic of inflammation and tumor mouse imaging. (B) Imaging of HClO in mice models of inflammation and tumor. Control group: healthy BALB/C mice; LPS group: LPS-induced inflammatory mice; LPS+(Sec)₂ group: (Sec)₂-treated the inflammatory mice; tumor group: tumor-bearing mice. (C) Relative fluorescent intensities in B. Data were presented as mean ± SD (n = 3). λ_ex = 490 nm, λ_em = 630–690 nm.

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