A dual-response fluorescent probe and a preparation method and detection application thereof

Abstract

The application discloses a dual-response fluorescent probe and a preparation method and detection application thereof, and belongs to the technical field of fluorescent probes. The probe is prepared by condensation reaction of 4-methyl-7-hydroxy-8-aldehyde coumarin and 4-morpholine aniline, and can simultaneously and specifically respond to peroxynitrite and viscosity. ‑ The probe specifically reacts with ONOO to generate a concentration-dependent fluorescence enhancement, has high selectivity, fast response and good linearity, and the fluorescence is significantly enhanced with the increase of viscosity. The probe has lysosome targeting properties, and can realize real-time imaging and dynamic monitoring of endogenous and exogenous ONOO ‑ in living cells, and presents a high-contrast fluorescent signal in a non-alcoholic fatty liver model mouse liver tissue slice, so that ONOO ‑ overproduction and viscosity increase in a pathological state can be accurately identified.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, and specifically relates to a dual-response fluorescent probe, its preparation method, and its detection application. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) has become one of the most common chronic liver diseases worldwide, closely associated with obesity, insulin resistance, and metabolic syndrome. Its pathological process encompasses a complete spectrum, from simple steatosis to non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, and ultimately hepatocellular carcinoma. Notably, early NAFLD often presents with no obvious symptoms, but significant metabolic reprogramming and subcellular functional abnormalities have already occurred at this stage. Therefore, a highly sensitive detection method is urgently needed to capture molecular events in the early stages of the disease. Among the many pathogenic mechanisms, oxidative stress and nitrification stress are widely considered to be the core drivers of NAFLD development. However, the specific molecular mediators connecting metabolic disorders and oxidative / nitrification damage, as well as effective strategies for achieving real-time visualization of these mediators, remain poorly understood.

[0003] Among reactive nitrogen species, peroxynitrite (ONOO) - ) is composed of nitric oxide (NO) and superoxide anion (O2• - ONOO is produced through a diffusion-controlled reaction and is a highly active oxidant with strong nitrifying and oxidizing capabilities. Previous studies have shown that ONOO... - It plays a crucial role in the development and progression of NAFLD by inducing mitochondrial dysfunction, lipid peroxidation, and inflammatory signaling cascades, and its overproduction can occur in the early stages of fatty degeneration. Although ONOO - It has significant pathological value, but due to its extremely short half-life and high reactivity, it is difficult to achieve real-time, in-situ detection in vivo, leading to its long-term underestimation in NAFLD research. Furthermore, existing research largely focuses on downstream oxidative damage, while neglecting its role in regulating ONOO. - The upstream mechanisms underlying the excessive generation remain unclear.

[0004] Along with changes in chemical properties, the physical properties of the intracellular microenvironment have recently been recognized as important indicators of metabolic disorders, with intracellular viscosity being highly sensitive to lipid accumulation and organelle remodeling. In the progression of NAFLD, excessive lipid deposition leads to macromolecular crowding, restricting molecular diffusion and consequently increasing viscosity. This increased viscosity further affects enzyme kinetics, signal transduction, and redox homeostasis. Recent studies have shown a close interaction between increased viscosity and amplified oxidative stress in fatty liver disease. However, direct visualization and quantitative assessment of viscosity dynamics in NAFLD models remain limited, particularly regarding the lack of integration with ONOO (non-alcoholic) technology. - Joint detection of key active species.

[0005] Fluorescent probes, due to their high sensitivity, real-time response, and excellent spatiotemporal resolution, have become powerful tools for monitoring disease-related biomarkers. Although currently in ONOO... - While some progress has been made in the development of reactive fluorescent probes for detection and molecular rotor probes for viscosity detection, most existing probes can only achieve single-parameter detection and cannot reflect the complex interactions between changes in chemical and physical properties in the NAFLD microenvironment. Therefore, it is necessary to develop a probe that can simultaneously monitor ONOO. - Dual-responsive fluorescent probes with varying viscosity are of great significance for a more comprehensive understanding of changes in the disease-related microenvironment. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a dual-response fluorescent probe, its preparation method, and its detection application.

[0007] This invention is achieved through the following technical solution: A dual-response fluorescent probe has the following structural formula: .

[0008] A second objective of this invention is to provide a method for synthesizing the dual-response fluorescent probe, comprising the following steps: Add 4-methyl-7-hydroxy-8-aldehyde coumarin and toluene to the reaction vessel, stir and heat to 60°C, add 4-morpholinoaniline, continue heating to 85°C until reflux, the reaction solution becomes clear, adjust the pH of the reaction solution to 4-5 with 3 mol / mL hydrochloric acid solution, reflux the reaction, monitor the reaction by TLC, stop the reaction after the starting material spot disappears, cool to room temperature, let stand, a light yellow solid precipitates, filter, and recrystallize the solid with ethanol.

[0009] Furthermore, the mass ratio of 4-methyl-7-hydroxy-8-aldehyde coumarin to 4-morpholinoaniline is 1:1, and the amount of toluene added is limited to 0.1 g of 4-methyl-7-hydroxy-8-aldehyde coumarin corresponding to 25 mL of toluene.

[0010] A third objective of this invention is to provide the application of the dual-response fluorescent probe in the detection of peroxynitrite.

[0011] A fourth objective of this invention is to provide the application of the dual-response fluorescent probe in viscosity detection.

[0012] This invention also provides the application of the dual-response fluorescent probe in the simultaneous detection of viscosity and peroxynitrite in cells and organisms.

[0013] Beneficial technical effects of the present invention: The probe of this invention can simultaneously and specifically respond to peroxynitrite (ONOO). - The probe detects two key pathophysiological parameters: α and β, and viscosity. In in vitro experiments, this probe was used to detect ONOO... - It exhibits high selectivity, rapid response, and a good linear relationship between fluorescence intensity and concentration, and shows significant fluorescence enhancement with increasing viscosity.

[0014] Secondly, the CMN probe has been successfully applied to complex biological systems, enabling visualized monitoring from the cellular to the tissue level. The probe itself possesses lysosomal targeting properties, allowing it to focus on specific subcellular structures. At the living cell level, the probe has successfully achieved both exogenous and endogenous ONOO. - It enables real-time fluorescence imaging and dynamic monitoring of changes in intracellular viscosity. This confirms its reliability and practicality in a living environment.

[0015] Finally, the CMN probe demonstrated clear potential for disease diagnosis and application. In liver tissue sections from mice with non-alcoholic fatty liver disease (NAFLD), the probe produced high-contrast fluorescence signals, accurately identifying ONOO under pathological conditions. - The excessive formation of [a substance] and the increase in viscosity [are observed]. This result provides an efficient visualization method for the early diagnosis of NAFLD and opens up new avenues for studying the mechanisms of oxidative / nitrification stress and abnormal microenvironment viscosity. Attached Figure Description

[0016] Figure 1 This is the mass spectrum of the fluorescent probe.

[0017] Figure 2 This is the NMR H-spectrum of the fluorescent probe.

[0018] Figure 3 For CMN probe to identify ONOO - The mechanism of action and the mass spectrum of the reaction products; among which: Figure 3 A represents peroxynitrite (ONOO). - A schematic diagram illustrating the working mechanism of the responsive probe CMN; Figure 3 B represents probes CMN and ONOO. - Mass spectrum of the hydroxycoumarin derivative obtained after the reaction.

[0019] Figure 4 The absorption spectrum of probe CMN (10 μM) after the addition of peroxynitrite (0-30 μM) is shown.

[0020] Figure 5 The fluorescence spectrum of probe CMN (10 μM) after the addition of peroxynitrite (0-10 μM) is shown.

[0021] Figure 6 The graph shows the linear relationship between the fluorescence intensity of the probe CMN and the concentration of peroxynitrite (0-10 μM).

[0022] Figure 7 The bar chart shows the fluorescence response of the probe CMN to various reactive oxygen species / nitrogen species and interfering substances (1: peroxynitrite, 2: hydrogen peroxide, 3: hydroxyl radical, 4: singlet oxygen, 5: hypochlorous acid, 6: nitric oxide, 7: superoxide anion, 8: nitrite, 9: tert-butyl hydroperoxide, 10: glutathione, 11: cysteine, 12: homocysteine, 13: potassium ion, 14: sodium ion, 15: magnesium ion).

[0023] Figure 8 The response time diagram of probe CMN to peroxynitrite (10 μM) is shown.

[0024] Figure 9 The graph shows the stability of the probe CMN and the solution after CMN reacts with peroxynitrite under different pH conditions.

[0025] Figure 10 The fluorescence spectra of probe CMN (10 μM) in water / glycerol mixtures with different volume ratios are shown.

[0026] Figure 11 For the probe CMN in a water / glycerol mixture, log(I 563 / 485 A graph showing the linear relationship between viscosity and log(viscosity).

[0027] Figure 12 Cell survival rates after treatment with CMN probes at different concentrations (10, 20, 40, 60 μM).

[0028] Figure 13The image shows the co-staining fluorescence of the fluorescent probe and the commercial lysosomal localization dye Lyso-Tracker Green in RAW264.7 cells. RAW264.7 cells were co-cultured with 10 μM of the fluorescent probe and Lyso-Tracker Green for 30 minutes, and fluorescence imaging was performed using a laser confocal microscope. Figure 13 A represents the green channel: a fluorescence image of the lysosomal localization dye Lyso-Tracker Green; Figure 13 B represents the red channel: a fluorescence image of the fluorescent probe; Figure 13 C represents the image resulting from the overlay of the green and red channels; Figure 13 D is a scatter plot of fluorescence intensity in the green and red channels. Scale bar = 20 μm.

[0029] Figure 14 To determine the exogenous ONOO fluorescent probe in RAW264.7 cells. - The fluorescence imaging pattern of the response; where: Figure 14 A1 is a fluorescence imaging image of RAW264.7 cells after incubation with a 10 μM fluorescent probe for 30 minutes; Figure 14 A2 is a fluorescent RAW264.7 cell line treated with a 10 μM fluorescent probe and 20 μM ONOO. - Fluorescence imaging image after co-incubation for 30 minutes; Figure 14 A3 cells were RAW264.7 cells, first treated with a 10 μM fluorescent probe and 20 μM ONOO. - Fluorescence imaging image after co-incubation for 30 minutes, followed by co-incubation with 1mM uric acid for another 30 minutes. Figure 14 A4 Figure 14 A5 Figure 14 A6 corresponds to respectively Figure 14 A1、 Figure 14 A2, Figure 14 Bright-field images of cells in A3. Scale bar for all images = 100 μm.

[0030] Figure 15 To investigate the effect of fluorescent probes on endogenous ONOO in RAW264.7 cells. - The fluorescence imaging pattern of the response; where: Figure 15 B1 is a fluorescence imaging image of RAW264.7 cells after being pretreated with 2 μg / mL lipopolysaccharide (LPS) for 1 hour and then incubated with a 10 μM fluorescent probe for 30 minutes. Figure 15 B2 is a fluorescence imaging image of RAW264.7 cells after pretreatment with 2 μg / mL lipopolysaccharide (LPS) for 5 hours and incubation with a 10 μM fluorescent probe for 30 minutes. Figure 15B3 is a fluorescence imaging image of RAW264.7 cells after pretreatment with 2 μg / mL lipopolysaccharide (LPS) for 12 hours, followed by incubation with a 10 μM fluorescent probe for 30 minutes. Figure 15 B4 is a fluorescence imaging image of RAW264.7 cells after being pretreated with 2 μg / mL lipopolysaccharide (LPS) for 12 hours and then incubated with 10 μM fluorescent probe and 1 mM uric acid (UA) for 30 minutes. Figure 15 B5 Figure 15 B6 Figure 15 B7 Figure 15 B8 corresponds to Figure 15 B1, Figure 15 B2, Figure 15 B3 Figure 15 Bright-field image of B4 cells. Scale bar for all images = 100 μm.

[0031] Figure 16 The image shows a fluorescence image of the fluorescent probe's response to cell viscosity in RAW264.7 cells; where: Figure 16 C1 is a fluorescence imaging image of RAW264.7 cells after incubation with a 10 μM fluorescent probe for 30 minutes; Figure 16 C3 is a fluorescence image of RAW264.7 cells after treatment with 20 μM nystatin and then the addition of 10 μM fluorescent probe; Figure 16 C2, Figure 16 C4 corresponds to Figure 16 C1, Figure 16 Bright-field images of C3 cells, all images with a scale bar of 100 μm.

[0032] Figure 17 To detect the presence of ONOO in normal liver tissue and liver tissue of mice with non-alcoholic fatty liver disease, fluorescent probes were used. - The development diagram of viscosity; where: Figure 17 D1 represents normal mouse liver tissue, containing a fluorescent probe and endogenous ONOO. - Fluorescence imaging pattern; Figure 17 D2 is a fluorescence imaging diagram of viscosity of a fluorescent probe in normal mouse liver tissue; Figure 17 D4 represents the liver tissue of a mouse model of non-alcoholic fatty liver disease, containing a fluorescent probe and endogenous ONOO. - Fluorescence imaging pattern; Figure 17 D5 shows the fluorescence imaging of the fluorescent probe on viscosity in the liver tissue of a mouse model of non-alcoholic fatty liver disease. Figure 17 D3、 Figure 17 D6 are bright-field images of normal mouse liver tissue and non-alcoholic fatty liver model mouse liver tissue, respectively. All images have a scale bar of 20 μm. Detailed Implementation

[0033] Example 1: Synthesis of Probe CMN

[0034] Add 0.1 g (0.49 mmol) of 4-methyl-7-hydroxy-8-aldehyde coumarin and 25 mL of toluene to a 50 mL flask. Stir and heat to 60 °C, then add 0.1 g (0.59 mmol) of 4-morpholinoaniline. Continue heating to 85 °C and reflux. The reaction solution becomes clear. Adjust the pH of the reaction solution to 4–5 with 3 mol / mL hydrochloric acid solution and reflux. Monitor the reaction by TLC. After the starting material spot disappears, stop the reaction, cool to room temperature, and allow to stand. A large amount of pale yellow solid precipitates. Filter the solid and recrystallize it from ethanol to obtain the probe CMN. 1 HNMR(400MHz, DMSO) δ 9.17 (s), 7.72 (s), 7.43 (d, J = 8.1Hz), 7.01 (d, J = 8.0Hz), 6.86 (d, J = 8.6 Hz), 6.64 (d, J = 8.0 Hz), 6.19 (s), 3.71 (s), 3.15 (s), 2.37 (s).

[0035] The mass spectrum of the probe CMN is as follows: Figure 1 As shown, the molecular formula of the probe CMN is C 21 H 20 N2O4, its protonated molecular ion peak (M+H). + The theoretical molecular formula is C 21 H 21 N₂O₄ corresponds to a theoretical molecular weight of 365.1501. In positive ion mode mass spectrometry, the molecular ion peak mass-to-charge ratio of the probe CMN was measured to be 365.1530. The deviation between the measured value and the theoretical value was only 0.0029, which is within the allowable error range of high-resolution mass spectrometry. This indicates that the molecular weight of the synthesized probe CMN is in high agreement with the theoretical value, confirming the successful synthesis of the target compound.

[0036] The NMR spectrum of the probe CMN is as follows: Figure 2 As shown, the spectrum clearly identifies and assigns the chemical shift signals corresponding to each hydrogen atom in the molecule, further corroborating the molecular structure of the probe CMN.

[0037] To evaluate the sensing performance of the probe CMN, its application to ONOO was investigated. - The spectral response was systematically studied. As shown in 3A, the probe CMN is based on ONOO. -Designed to specifically react with the recognition unit, it can produce significant changes in optical signal. In the probe CMN molecule, the coumarin fluorophore and the 4-morpholinobenzylimine structure are linked by a C=N double bond, forming a conjugated system with intramolecular charge transfer (ICT) properties. At this time, the fluorescence of the coumarin fluorophore is effectively quenched due to the cis-trans isomerism of the C=N double bond and the photoinduced electron transfer (PET) effect, and the system is in a fluorescence "off" state; when ONOO is present in the system... - At that time, ONOO - As a highly reactive nitrogen species with strong oxidizing properties, it can specifically cleave the C=N double bond in the probe molecule, oxidizing and hydrolyzing it into an aldehyde group, while releasing a hydroxycoumarin derivative with strong fluorescence emission. During this reaction, the conjugated system of the probe molecule is disrupted, the PET effect disappears, and the intrinsic fluorescence of the coumarin fluorophore is restored, achieving a significant fluorescence signal change from "off" to "on," thus constructing a signal for ONOO. - A "turn-on" fluorescence sensing detection system. This specific reaction mechanism endows the probe CMN with ONOO... - Its high selective recognition capability allows for the identification of ONOO by monitoring changes in the fluorescence intensity of the system. - Quantitative detection. Probes CMN and ONOO - The mass spectrum after the reaction is as follows Figure 3 As shown in B, the obtained structure is a hydroxycoumarin derivative with a molecular weight of 205.0768.

[0038] 0-30 μM ONOO was gradually added to a 10 μM CMN solution. - Subsequently, the ultraviolet-visible absorption spectrum showed significant changes. (From...) Figure 4 It can be seen that with ONOO - With increasing concentration, the absorption peak of CMN changed significantly, indicating that the probe interacted with ONOO. - Effective interactions occurred between them. The fluorescence emission spectrum of CMN in ONOO - The concentration-dependent enhancement was observed after treatment. Figure 5 ). When ONOO - As the concentration gradually increases within the range of 0–10 μM, the fluorescence intensity gradually increases, indicating that CMN can inhibit ONOO through a fluorescence "on" mechanism. - Produces a sensitive response. In the range of 0~10μM, ONOO... - Within the concentration range, fluorescence intensity is related to ONOO - The concentration showed a good linear relationship. Figure 6 ), correlation coefficient (R) 2 The value is 0.99, proving that CMN has the capability to handle ONOO. -The quantitative detection capability of CMN was compared with that of other reactive oxygen species (including hypochlorous acid (HClO), hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and singlet oxygen (…). 1 O2), nitric oxide (NO), superoxide anion (O2• - ), nitrite (NO2) - ), tert-butyl hydroperoxide (TBHP), glutathione (GSH), cysteine ​​(Cys), homocysteine ​​(Hcy), potassium ions (K) + Sodium ions (Na) + ) and magnesium ions (Mg 2 + The fluorescence response of ) was further evaluated to assess its effect on ONOO. - The selectivity. For example... Figure 7 As shown, only ONOO - It can induce a significant increase in fluorescence intensity, while other competing species only cause weak signal changes, confirming that CMN inhibits the fluorescence intensity of ONOO. - The detection has high selectivity.

[0039] The influence of CMN on ONOO was also examined. - The response dynamics. For example... Figure 8 As shown, add ONOO - Subsequently, the fluorescence intensity rapidly increased and reached a stable state within a short time, indicating that the probe has a fast response speed and is suitable for ONOO. - Real-time monitoring was conducted. Furthermore, to evaluate the stability of the probe under physiological conditions, the stability of the CMN and the probe via ONOO was investigated. - The fluorescence behavior of CMN after treatment changes with pH value. For example... Figure 9 As shown, CMN exhibits a stable fluorescence signal over a wide pH range, while CMN+ONOO - The system maintained a significant fluorescence response even under near-physiological pH conditions, indicating that the probe has good stability and is suitable for detection applications in biological environments.

[0040] Furthermore, since CMN contains viscosity-sensitive fluorophore units, its fluorescence emission can be modulated by environmental viscosity, thereby enabling the control of ONOO. - Simultaneous sensing with viscosity changes. As the viscosity of the water / glycerol mixture increases, the fluorescence emission intensity of the probe gradually decreases at 485 nm, while the fluorescence intensity significantly increases at 563 nm (Figure 10). Further quantitative analysis shows that log(I 563 / I 485 There is a good linear relationship between (r=0.99) and log(viscosity). Figure 11 This dual-response characteristic lays a solid foundation for the application of CMN in complex biological systems, and is especially suitable for ONOO.- Early diagnosis of NAFLD in scenarios where abnormalities occur simultaneously with changes in microenvironment viscosity.

[0041] Before performing cell functional imaging, the biosafety of the probe CMN was first examined, such as... Figure 12 As shown, the cell viability of the probe CMN was above 80% in the concentration range of 10-60 μM, indicating that it has good biocompatibility. Subsequently, the subcellular localization of the CMN probe was investigated to clarify its intracellular distribution. Co-localization experiments were conducted in RAW264.7 cells using the commercially available lysosome-specific dye Lyso Tracker Green (a lysosomal green fluorescent probe). Figure 13 As shown, the fluorescence signals of CMN and Lyso Tracker Green exhibit strong overlap, and the fused image shows a high degree of co-localization (overlap coefficient of 0.95), indicating that CMN is mainly enriched in lysosomes. Since lysosomes are closely related to oxidative stress, inflammatory signal transduction, and microenvironmental changes in activated macrophages, this lysosomal targeting characteristic provides an important foundation for subsequent cell imaging studies.

[0042] To evaluate the detection of ONOO by probe CMN - To assess the ability to monitor changes in intracellular viscosity, fluorescence imaging experiments were conducted using RAW264.7 cells as the research subject. Figure 14 As shown, only cells incubated with CMN exhibited a weak fluorescence signal ( Figure 14 A2) indicates that the probe has a low background signal; when using 20μM exogenous ONOO - After cell treatment, a significant increase in fluorescence intensity was observed. Figure 14 A4), indicating that the probe is for ONOO - It exhibits high sensitivity; furthermore, the addition of 1 mM uric acid (UA, known as ONOO) provides better sensitivity. - After the removal agent was applied, the fluorescence intensity decreased significantly. Figure 14 A6), Figure 14 A1、 Figure 14 A3 Figure 14 A5 is a bright-field image, and the comparison further confirms the specificity of CMN for the detection of peroxynitrite.

[0043] Targeting endogenous ONOO - To detect the effect, RAW264.7 cells were pretreated with 2 μg / mL lipopolysaccharide (LPS) for different durations (1 h, 5 h, 12 h) to stimulate intracellular ONOO. - The cells were then incubated with 10 μM CMN for 0.5 h. (The sentence appears incomplete and lacks context.) Figure 15As shown in B1-B3, the fluorescence intensity increased in a dependent manner with increasing pretreatment time, indicating that intracellular ONOO - Gradually accumulates; conversely, cells simultaneously incubated with 1 mM UA ( Figure 15 B4) The fluorescence signal was significantly suppressed, further verifying the effect of CMN on ONOO in living cells. - Selective response. Bright-field image ( Figure 15 B5-B8 confirmed that the observed fluorescence changes were not caused by changes in cell morphology or cytotoxicity.

[0044] Except ONOO - Besides detection, CMN can also be used to monitor changes in intracellular viscosity. For example... Figure 16 As shown, the fluorescence signal of cells treated with CMN alone is weak ( Figure 16 C1); When cells were treated with 20 μM nystatin (a substance that can induce changes in cell membrane dynamics and intracellular viscosity), a significant increase in fluorescence intensity was detected. Figure 16 C3 indicates that the probe has a good response to viscosity changes. Bright-field image ( Figure 16 C2, Figure 16 C4) confirmed that the cell morphology remained intact throughout the experiment.

[0045] In summary, the above experimental results indicate that CMN can selectively detect exogenous and endogenous ONOO in RAW264.7 cells. - It can also monitor changes in intracellular viscosity, highlighting its potential as a multifunctional tool for studying reactive nitrogen species and dynamic changes in the microenvironment in living cells.

[0046] Based on the confirmed lysosomal targeting properties of the probe CMN and its effect on ONOO in RAW264.7 cells. - Based on the dual-response fluorescence behavior of viscosity, this invention uses a NAFLD mouse model to conduct tissue section imaging experiments to further evaluate the feasibility of CMN application in complex pathological environments. NAFLD is closely related to oxidative stress, nitrification stress, inflammation activation, and microenvironmental changes, making it an ideal disease model for validating the detection performance of CMN at the tissue level.

[0047] Liver tissue sections from NAFLD model mice and corresponding normal control mice were incubated with CMN under optimized staining conditions. Figure 17 As shown, normal mouse liver tissue sections exhibit only weak fluorescence signals, indicating extremely low background interference from the probe; conversely, NAFLD mouse liver tissue sections show significantly enhanced fluorescence intensity, suggesting the presence of ONOO in the diseased liver tissue. -Increased levels and / or viscosity were observed. In summary, tissue section imaging experiments based on a NAFLD mouse model demonstrate that CMN can effectively penetrate liver tissue and target disease-related ONOO. - Abnormalities and changes in microenvironment viscosity produce high-contrast fluorescence signals. The consistency between cellular and tissue-level imaging results highlights the stability and reliability of CMN, and also demonstrates its potential application in studying lysosomal oxidative stress and pathological progression in NAFLD.

Claims

1. A dual-response fluorescent probe, characterized in that, The structure is as follows: 。 2. A dual-response fluorescent probe as described in claim 1 for the preparation of a probe for detecting peroxynitrite (ONOO). - Applications in reagents.

3. The application of the dual-response fluorescent probe as described in claim 1 in the preparation of viscosity detection reagents.

4. A dual-response fluorescent probe as described in claim 1, used for the simultaneous detection of peroxynitrite (ONOO) in cells or organisms during preparation. - Applications in viscosity reagents.

Images