A fluorescent probe for dual-channel discrimination detection of hypochlorous acid and bisulfite in ferroptosis process, and a preparation method and application thereof

Abstract

The application discloses a fluorescent probe for dual-channel distinguishing detection of hypochlorous acid and bisulfite in ferroptosis, a preparation method and application thereof, and belongs to the technical field of fluorescent probe detection and analysis of HClO and HSO3 ‑ . The fluorescent probe is (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indol-3-ium iodide, and a structural formula thereof is shown in the description; the application further discloses a preparation method of the fluorescent probe and application of the fluorescent probe in simultaneous, rapid and high-selectivity identification of HClO and HSO3 ‑ and preparation of imaging detection reagents of HClO and HSO3 ‑ in organisms. The fluorescent probe can realize dual-channel distinguishing detection of HClO and HSO3 ‑ in pure PBS solution, has fast response, low detection limit and high selectivity, and has good imaging effect on endogenous HClO and HSO3 ‑ generated in the ferroptosis process.

Description

Technical Field

[0001] This invention pertains to hypochlorous acid (HClO) and bisulfite (HSO3). - This relates to the field of fluorescent probe detection and analysis technology, specifically to a fluorescent probe for the dual-channel differentiation and detection of hypochlorous acid and bisulfite during ferroptosis, its preparation method, and its application. Background Technology

[0002] In 2012, the Stockwell laboratory defined ferroptosis, ushering in a new era in ferroptosis research. It represents an iron-dependent, non-apoptotic form of programmed cell death characterized by aberrant lipid peroxidation (LPO), iron accumulation, and glutathione (GSH) depletion. The most direct driver of ferroptosis is the excessive accumulation of LPO. The two main regulatory pathways affecting LPO production are a significant increase in reactive oxygen species (ROS) and the depletion of reducing agents such as glutathione. Hypochlorous acid (HClO), as one of the key ROS, also plays a crucial role in ferroptosis, acting as a signaling molecule to regulate various physiological activities, including cell signaling, maintaining redox balance, and immune responses against invading pathogens. Normally, endogenous HClO in organisms is produced by myeloperoxidase in neutrophils through a redox reaction between hydrogen peroxide (H₂O₂) and chloride ions. During ferroptosis, GSH is metabolized and oxidized to SO₂ (primarily as bisulfite, HSO₃) by thiosulfate-transferase (TST). - ) and sulfites (SO3) 2- It exists in (form) and plays a crucial role in this process; its dynamic changes are also closely related to lipid peroxidation and mitochondrial dysfunction. Simultaneously, its self-oxidation generates a ROS burst, inducing oxidative stress and mitochondrial damage. Monitoring HClO and HSO3... - The concentration of HClO or HSO3 in ferroptosis can help us to understand the process and mechanism of ferroptosis more deeply, thereby significantly improving our understanding of the pathological mechanisms of ferroptosis-related diseases. Current literature reports on specific targeting of HClO or HSO3 in the ferroptosis process. - These are single-function fluorescent probes. However, these fluorescent probes cannot simultaneously or separately detect HClO and HSO3. - Therefore, little is known about the relationships between them, and there is an urgent need to design and synthesize methods to simultaneously detect HClO and HSO3. - A novel fluorescent probe.

[0003] The prior art patent document CN202010447385.1 discloses a bifunctional fluorescent probe for identifying hypochlorite and bisulfite, its preparation method, and its application. This fluorescent probe can identify hypochlorite and bisulfite separately under different excitation channels. At an excitation wavelength of 560 nm, the fluorescent probe exhibits specific selectivity for hypochlorite, with a detection limit of 15 nM and a fluorescence emission wavelength of 654 nm, achieving near-infrared detection. At an excitation wavelength of 470 nm, the fluorescent probe exhibits specific selectivity for bisulfite, with a detection limit of 16 nM. The fluorescent probe itself exhibits extremely weak fluorescence in the buffer system, but achieves dual recognition of hypochlorite and bisulfite under different wavelengths of fluorescence excitation, demonstrating strong anti-interference ability and low detection limits. Patent document CN202410112339.4 discloses a dual-channel ratiometric fluorescent probe for the simultaneous detection of HClO and H2S. The fluorescent probe exhibits good selectivity for HClO and H2S, enabling quantitative detection of HClO and H2S separately. The fluorescent probe shows a good linear relationship with the concentrations of HClO and H2S, has strong anti-interference ability, good cell membrane penetration, and a wide pH range, and can detect hypochlorous acid and hydrogen sulfide separately. Patent document CN202410504841.X discloses a dual-channel fluorescent probe for distinguishing and detecting hypochlorous acid and hydrazine, its preparation method, and its application. The fluorescent probe is 2-((10-butyl-2-methoxy-10H-phenthiazin-3-yl)methylene)-1H-indene-1,3(2H)-dione, which can be used for rapid and highly selective identification of hypochlorous acid and hydrazine, as well as for preparing imaging detection reagents for hypochlorous acid and hydrazine in cells, zebrafish, or Arabidopsis seedlings. It can achieve colorimetric and fluorescence detection of hypochlorous acid and hydrazine, with fast response, low detection limit, and high selectivity. It also enables imaging detection of hypochlorous acid and hydrazine in cells, zebrafish, and Arabidopsis seedlings. However, there are currently no records of (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE) as a fluorescent probe for the dual-channel differentiation detection of hypochlorous acid and bisulfite, nor are there any reports on its use as a fluorescent probe for the dual-channel differentiation detection of hypochlorous acid and bisulfite during ferroptosis. Summary of the Invention

[0004] The purpose of this invention is to provide a fluorescent probe for the dual-channel differentiation and detection of hypochlorous acid and bisulfite during ferroptosis, and a method for its preparation. The fluorescent probe prepared by this method can be used for rapid and highly selective dual-channel differentiation and recognition of HClO and HSO3. - And HClO and HSO3 in cells and zebrafish during iron death. - Dual-channel distinguishing detection and bioimaging.

[0005] To achieve the above objectives, this invention employs the following technical solution: a fluorescent probe for the dual-channel differentiation and detection of hypochlorous acid and bisulfite during ferroptosis, characterized in that: the fluorescent probe is (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide, with the structural formula HSO3. - .

[0006] The present invention describes a method for preparing a fluorescent probe for the dual-channel detection of hypochlorous acid and bisulfite during ferroptosis. The specific preparation process is as follows: using ethanol as the reaction medium, and 4-(methylthio)benzaldehyde and 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-onium iodide as reaction raw materials, the reaction is carried out at 70-90°C for 8-12 hours under inert gas protection. The solvent is then removed, and the fluorescent probe for the dual-channel detection of hypochlorous acid and bisulfite during ferroptosis is obtained by column chromatography.

[0007] Further specifying, the molar ratio of the 4-(methylthio)benzaldehyde and 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-onium iodide is 1:1.

[0008] The specific synthetic route in the preparation method of the fluorescent probe for the dual-channel differentiation and detection of hypochlorous acid and bisulfite during ferroptosis described in this invention is as follows: HSO3 - .

[0009] The fluorescent probe described in this invention, used for the dual-channel differentiation and detection of hypochlorous acid and bisulfite during ferroptosis, is characterized by its rapid and highly selective dual-channel differentiation and identification of hypochlorous acid and bisulfite. The specific process is as follows: the fluorescent probe shows no fluorescence in phosphate-buffered saline (PBS) under 365nm handheld UV light irradiation; after adding a solution containing hypochlorous acid to the mixture, it exhibits bright orange-yellow fluorescence under 365nm handheld UV light irradiation; after adding a solution containing sodium bisulfite to the mixture, it exhibits bright cyan fluorescence under 365nm handheld UV light irradiation.

[0010] The fluorescent probe described in this invention for the dual-channel differentiation detection of hypochlorous acid and bisulfite during ferroptosis is used in the preparation of a dual-channel imaging detection reagent for the differentiation of exogenous and / or endogenous hypochlorous acid and bisulfite in cells.

[0011] The fluorescent probe described in this invention for the dual-channel differentiation detection of hypochlorous acid and bisulfite during ferroptosis is used in the preparation of a dual-channel imaging detection reagent for the differentiation of endogenous hypochlorous acid and bisulfite in zebrafish.

[0012] The fluorescent probe described in this invention for the dual-channel differentiation detection of hypochlorous acid and bisulfite during ferroptosis is used in the preparation of a dual-channel imaging detection reagent for intracellular hypochlorous acid and bisulfite differentiation during ferroptosis.

[0013] The fluorescent probe described in this invention for the dual-channel differentiation detection of hypochlorous acid and bisulfite during ferroptosis is used in the preparation of a dual-channel imaging detection reagent for the differentiation of hypochlorous acid and bisulfite in zebrafish during ferroptosis.

[0014] Compared with existing methods for detecting HClO or HSO3 - Compared with field-specific fluorescent probe technology, the HClO and HSO3 prepared in this invention are used in the process of ferroptosis. - Dual-channel fluorescent probes for differential detection offer advantages such as simple preparation, high yield, fast response, low detection limit, and good selectivity. A single fluorescent probe can simultaneously detect different fluorescence colors in HClO and HSO3 in solution. - The distinguishing detection of HClO and HSO3 prepared using this invention is described. - The dual-channel fluorescent probe can differentiate between exogenous and endogenous HClO and HSO3 in cells. - Its dual-channel distinguishing detection and imaging can also be used to detect HClO and HSO3 in cells and zebrafish during ferroptosis. - Dual-channel distinguishing imaging. Attached Figure Description

[0015] Figure 1 The fluorescent probe ISE prepared in Example 1 was subjected to the addition of HClO and HSO3 to a solution. - The fluorescence spectrum after adding HClO and HSO3 is shown in the inset. - Photographs of the solution before and after observation under 365nm handheld ultraviolet irradiation.

[0016] Figure 2 The images show the UV absorption (a) and fluorescence spectra (b) of the fluorescent probe ISE prepared in Example 1 after adding different equivalents of HClO to the solution.

[0017] Figure 3 The fluorescent probe ISE prepared in Example 1 reacts with different equivalents of HSO3 in solution. - UV absorption (a) and fluorescence spectrum (b) after the reaction.

[0018] Figure 4 The fluorescent probe ISE prepared in Example 1 was subjected to the addition of different concentrations of HClO(a) and HSO3 to the solution. - (b) Linear fitting between the fluorescence intensity of the solution at 605 nm (a) and 478 nm (b) and the concentration of the additive.

[0019] Figure 5 The fluorescent probe ISE prepared in Example 1 was subjected to the addition of different concentrations of HClO(a) and HSO3 to the solution. - (b) The fluorescence intensity of the solution at 605 nm (a) and 478 nm (b) as a function of time.

[0020] Figure 6 The fluorescent probe ISE prepared in Example 1 reacts with HClO(a) and HSO3 in solutions of different pH values. - (b) Fluorescence intensity at 605 nm (a) and 478 nm (b) before and after the reaction.

[0021] Figure 7 The fluorescence intensities at 478 nm and 605 nm of the fluorescent probe ISE prepared in Example 1 after reacting with different interfering substances are (0-blank; 1-HClO; 2-H2O2; 3-t-BuOOH; 4-HO•; 5- 1 O2; 6-O2 •- ; 7-NO; 8-ONOO - ;9-Cys;10-Hcy;11-GSH;12-HS - ;13-HSO3 - ).

[0022] Figure 8 These are cell imaging images in the blue and yellow channels before and after the fluorescent probe ISE prepared in Example 1 reacts with different concentrations of HClO.

[0023] Figure 9 The fluorescent probe ISE prepared in Example 1 is reacted with different concentrations of HSO3. - Cell imaging images in the blue and yellow channels before and after the reaction.

[0024] Figure 10 The fluorescent probe ISE prepared in Example 1 reacts with endogenous HClO(a) and HSO3 in cells. - (b) Cell imaging images in the blue and yellow channels before and after the reaction.

[0025] Figure 11 This is a cell imaging image of the fluorescent probe ISE prepared in Example 1 in zebrafish before and after reacting with endogenous HClO in the blue and yellow channels.

[0026] Figure 12 The fluorescent probe ISE prepared in Example 1 reacts with endogenous HSO3 in zebrafish. - Cell imaging images in the blue and yellow channels before and after the reaction.

[0027] Figure 13 The fluorescent probe ISE prepared in Example 1 reacts with HClO and HSO3 produced during ferroptosis in cells. - Cell imaging before and after the reaction (a) and fluorescence intensity quantification maps of the yellow channel (b) and blue channel (c).

[0028] Figure 14 The fluorescent probe ISE prepared in Example 1 reacts with HClO and HSO3 produced during iron death in zebrafish. - Cell imaging before and after the reaction (a) and fluorescence intensity quantification maps of the yellow channel (b) and blue channel (c). Detailed Implementation

[0029] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0030] Example 1

[0031] Synthesis of the fluorescent probe (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE)

[0032] The fluorescent probe (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE) was synthesized via the following route:

[0033]

[0034] Under argon protection, 4-(methylthio)benzaldehyde and 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-onium iodide were dissolved in ethanol and reacted at 80°C for 10 hours. After returning to room temperature, the solvent was removed, and the product was then subjected to column chromatography with 200-300 mesh silica gel to obtain a dark red solid product with a yield of 87%.

[0035] The deep red solid product prepared in this embodiment has the following 1H NMR spectrum: 1H NMR (400MHz, CDCl3, 25℃, TMS): δ = 8.33 (d, J = 16.1 Hz, 1H, =H), 8.26 (d, J = 8.5Hz, 2H, Ar-H), 8.22 (d, J = 8.5 Hz, 1H, Ar-H), 8.13 (d, J = 8.9 Hz, 1H, Ar-H), 8.07 (d, J = 8.1 Hz, 1H, Ar-H), 7.86 (d, J = 16.1 Hz, 1H, =H), 7.73-7.79(m, 2H, Ar-H), 7.65-7.69(m, 1H, Ar-H), 7.34 (d, J = 8.6 Hz, 2H, Ar-H), 5.15(q, J = 7.3 Hz, 2H, NCH2), 2.52 (s, 3H, SCH3), 2.12 (s, 6H, CH3), 1.68 (t, J =7.3 Hz, 3H, CH3) ppm.

[0036] Its carbon NMR spectrum is: 13 C NMR (100 MHz, CDCl3, 25 °C, TMS) δ = 181.8,153.6, 148.6, 138.6, 137.7, 133.7, 131.9, 131.9, 130.4, 130.0, 128.7, 127.5,127.4, 125.6, 122.7, 112.1, 110.5, 53.9, 44.4, 27.0, 14.7, 14.6 ppm.

[0037] Its high-resolution mass spectrometry is: HRMS (ESI) m / z: calcd. for C 25 H 26 NS + [M + ]: 372.1780, found 372.1786.

[0038] This proves that the obtained dark red solid is (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE), and its structural formula is as follows:

[0039] .

[0040] Example 2

[0041] Fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE) with HClO and HSO3 - UV and fluorescence response

[0042] The fluorescent probe ISE prepared in Example 1 was dissolved in phosphate buffer (pH=7.4) to obtain a test solution with a concentration of 10 μM fluorescent probe ISE. Figure 2 The ultraviolet absorption spectrum (a) of the fluorescent probe ISE prepared in Example 1 after the addition of the detectant HClO and the fluorescence spectrum (b) at the excitation wavelength of 388 nm are given. After the addition of HClO, the absorption in the long band decreases rapidly and the absorption in the short band increases. Under the excitation of 388 nm, the fluorescence of the fluorescent probe ISE solution at 605 nm increases rapidly. Figure 3 The fluorescent probe ISE prepared in Example 1 was added to the analyte HSO3. - The ultraviolet absorption spectrum (a) and fluorescence spectrum at an excitation wavelength of 338 nm (b) after the addition of HSO3 - Subsequently, the absorption in the long wavelength range decreased rapidly, while the absorption in the short wavelength range increased. Under excitation at 338 nm, the fluorescence of the solution of the fluorescent probe ISE rapidly increased at 478 nm. Figure 4 Different equivalents of HClO(a) and HSO3 were added to the test solution of the 10 μM fluorescent probe ISE. - (b) Fluorescence intensity at 605 nm (a) and 478 nm (b) after the addition of HClO and HSO3 - The curves obtained by linear fitting of the concentrations were used to calculate the responsiveness of the fluorescent probe ISE to HClO and HSO3. - The detection limits were 23 nM and 7 nM (S / N=3), respectively. Figure 5 The fluorescence intensity of probe ISE at 605 nm and 478 nm was shown to correlate with different concentrations of HClO(a) and HSO3, respectively. - (b) The time change curve after the response. Under the action of 3 equivalents of HClO, the fluorescent probe ISE only needs 3 seconds to complete the reaction with HClO. Under the action of 3 equivalents of HSO3 - Under the action of the fluorescent probe ISE, it only takes 45 seconds to react with HSO3. - The reaction has ended. Figure 6 The fluorescent probe ISE was demonstrated to react with HClO(a) and HSO3 in buffer solutions at different pH values. - (b) Reaction conditions. Within physiological conditions (pH = 6-9), the fluorescent probe ISE can be used effectively for HClO and HSO3. - The detection.

[0043] Example 3

[0044] Selective detection of the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE).

[0045] Various interfering substances (O-blank; 1-HClO; 2-H2O2; 3-t-BuOOH; 4-HO•; 5-) were added to the test solution of the 10 μM fluorescent probe ISE prepared in the above examples. 1 O2; 6-O2 •- ; 7-NO; 8-ONOO - ;9-Cys;10-Hcy;11-GSH;12-HS - ;13-HSO3 - In addition to adding HClO at a concentration of 50 μM, HSO3 was also added. - Except for the concentration of [specific substance name], which was 100 μM, the concentrations of other interfering substances were all 1000 μM. For example... Figure 7 As shown, when the solution was excited at 388 nm, only the solution reacting with HClO showed a significant fluorescence enhancement at 605 nm, with no significant change compared to other interfering substances; under 338 nm excitation, only the fluorescent probe ISE reacted with HSO3. - The responsive solution showed a significant response at 478 nm, with no obvious change compared to other interfering substances. This further indicates that the fluorescent probe ISE responds to HClO and HSO3. - It offers good selectivity.

[0046] Example 4

[0047] The fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE) induced exogenous HClO and HSO3 in HeLa cells. - Imaging applications

[0048] HeLa cells were placed at 2 × 10⁻⁶ cells per coverslip. 4 Cells were seeded at a density of 10 μM on 14 mm glass coverslips. Then, 10 μM of the fluorescent probe ISE was added to the culture medium and the cells were incubated for half an hour, followed by washing three times with phosphate-buffered saline (PBS, pH 7.4). Subsequently, cells were seeded with different concentrations of HClO and HSO3. - Add the ingredients separately to the cells and incubate at 37°C for another 30 minutes. Rinse three times with PBS and image under a laser confocal microscope. Figure 8The results of dual-channel detection of the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE) in cells using HClO imaging are presented. The fluorescent probe ISE showed no fluorescence in the yellow and blue channels, but after the addition of HClO, significant fluorescence was observed in the yellow channel. Furthermore, the yellow fluorescence increased more significantly with increasing HClO concentration, while there was no significant change in the blue channel. Figure 9 The dual-channel detection method for the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE) in cells for the detection of HSO3- - The imaging results showed that the fluorescent probe ISE was non-fluorescent in the yellow and blue channels, but fluoresced when HSO3 was added. - Subsequently, it was observed that the fluorescent probe ISE exhibited significant fluorescence in the blue channel, and this fluorescence was further enhanced with the addition of HSO3. - As the concentration increases, the blue fluorescence becomes more significantly enhanced, while the yellow channel shows no obvious change.

[0049] Therefore, the dual-channel detection fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide prepared in this invention can effectively detect exogenous HClO and HSO3 in HeLa cells. - Dual-channel distinguishing imaging detection.

[0050] Example 5

[0051] The fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE) inhibits the production of endogenous HClO and HSO3 by cells. - Imaging applications

[0052] Figure 10 Image (a) shows the imaging results of endogenously produced HClO in cells using the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE). Endogenous HClO is produced by stimulation with PMA (Phorbol 12-myristate 13-acetate, also known as TPA) and LPS (lipopolysaccharide). Figure 10As shown in the first row of (a), cells not stimulated by PMA and LPS showed no obvious fluorescence signal in either the yellow or blue channels after treatment with the fluorescent probe ISE (10 μM). Cells treated with PMA (5 μg / mL) and LPS (1 μg / mL) were then imaged with the fluorescent probe ISE (10 μM), and a significant fluorescence signal was observed in the yellow channel, while no significant change was observed in the blue channel. Endogenous HClO generated by PMA (5 μg / mL) and LPS (1 μg / mL) stimulation was treated with NAC (1 mM) and then imaged with the fluorescent probe ISE (10 μM), and no fluorescence was observed in either the yellow or blue channels. Endogenous HSO3... - Produced by LPS stimulation. For example... Figure 10 As shown in the first row of (b), cells not stimulated by LPS showed no obvious fluorescence signal in either the yellow or blue channels after treatment with the fluorescent probe ISE (10 μM). However, when cells treated with LPS (1 μg / mL) were then imaged with the fluorescent probe ISE (10 μM), a significant fluorescence signal was observed in the blue channel, while no significant change was observed in the yellow channel. Endogenous HSO3 produced by LPS (1 μg / mL) stimulation... - Treatment with formaldehyde (FA, 25 μM) followed by the addition of the fluorescent probe ISE (10 μM) resulted in no fluorescence in either the yellow or blue channels. These results indicate that the fluorescent probe ISE can be used to target endogenous HClO and HSO3 in cells. - Imaging.

[0053] Example 6

[0054] The fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE) induced the reaction of endogenous HClO and HSO3 in zebrafish. - Imaging applications

[0055] Figure 11 Image (a) shows the imaging results of endogenous HClO in zebrafish using the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE). Figure 11As shown in (a), zebrafish treated with only the fluorescent probe ISE (10 μM) showed no obvious fluorescence signal in either the yellow or blue channels. When ISE (10 μM) was added to zebrafish incubated with PMA (5 μg / mL) and LPS (1 μg / mL), obvious fluorescence was observed in the yellow channel, but no obvious fluorescence signal was observed in the blue channel. If, after stimulation with PMA (5 μg / mL) and LPS (1 μg / mL), the generated endogenous HClO was consumed by NAC, no obvious yellow fluorescence appeared, and no fluorescence signal was observed in the blue channel either.

[0056] Figure 12 Figure (a) shows the effect of the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE) on endogenous HSO3 in zebrafish. - The imaging results, such as Figure 12 As shown in (a), zebrafish treated with only the fluorescent probe ISE (10 μM) showed no obvious fluorescence signal in either the yellow or blue channels. Adding the fluorescent probe ISE (10 μM) to zebrafish incubated with LPS (1 μg / mL) resulted in significant fluorescence in the blue channel, while no obvious fluorescence signal was observed in the yellow channel. If, after stimulation with LPS (1 μg / mL), the generated endogenous HSO3 was consumed using FA... - No obvious blue fluorescence appears, and there is no fluorescence signal in the yellow channel either.

[0057] Example 7

[0058] The fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE) inhibits the production of HClO and HSO3 by cells during ferroptosis. - Imaging applications

[0059] Figure 13 Figure (a) shows the effect of the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE) on the production of HClO and HSO3 during ferroptosis induced by the ferroptosis inducer Erastin in cells. -The imaging image shows that cells treated with the fluorescent probe ISE showed no fluorescence in either the yellow or blue channels. When ferroptosis was induced with the ferroptosis inducer Erastin (10 μM) followed by the addition of the fluorescent probe ISE (10 μM), yellow fluorescence continuously increased in the first 6 hours, followed by a significant blue fluorescence signal around 6 hours, after which the yellow fluorescence signal began to decrease. When inhibited with the ferroptosis inhibitor Ferrostatin-1 (Fer-1, 10 μM), neither yellow nor blue fluorescence was observed. This indicates that the fluorescent probe ISE can be used to treat HClO and HSO3 produced during ferroptosis. - Imaging.

[0060] Endogenous HClO and HSO3 produced in zebrafish during ferroptosis - The imaging results are as follows Figure 14 As shown in (a), zebrafish treated with only the fluorescent probe ISE (10 μM) showed no fluorescence in the yellow and blue channels. After incubation with the ferroptosis inducer Erastin (10 μM) followed by the addition of the fluorescent probe ISE (10 μM), enhanced fluorescence was observed in the yellow channel within the first 6 hours, while the fluorescence signal in the blue channel increased over time. If ferroptosis was inhibited by the ferroptosis inhibitor Fer-1 after stimulation with Erastin, no obvious yellow or blue fluorescence appeared.

[0061] As can be seen from the above, compared with existing fluorescent probes in HClO or HSO3 - Compared with existing detection technologies, the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide (ISE) prepared in this invention can effectively detect HClO and HSO3. - This dual-channel simultaneous detection method has the advantages of simple synthesis, and its good water solubility allows the fluorescent probe to be detected in pure PBS solution. It is also effective against HClO and HSO3. - The detection exhibits good selectivity and sensitivity, enabling the detection of both endogenous and exogenous HClO and HSO3 in cells. - It can also perform imaging and detect HClO and HSO3 produced in cells and zebrafish during ferroptosis. - The imaging detection provides a visual imaging tool for research on ferroptosis and the development of drugs for the treatment of related diseases.

[0062] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A fluorescent probe for the dual-channel detection of hypochlorous acid and bisulfite during ferroptosis, characterized in that: The fluorescent probe is (E)-3-ethyl-1,1-dimethyl-2-(4-(methylthio)styryl)-1H-benzo[e]indole-3-onium iodide, with the following structural formula: 。 2. A method for preparing a fluorescent probe according to claim 1 for the dual-channel detection of hypochlorous acid and bisulfite during ferroptosis, characterized in that... The specific preparation process is as follows: using ethanol as the reaction medium, 4-(methylthio)benzaldehyde and 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-onium iodide as the reaction raw materials, the reaction is carried out at 70~90℃ for 8~12 hours under inert gas protection. After removing the solvent, the fluorescent probe for dual-channel detection of hypochlorous acid and bisulfite ions in the process of ferroptosis is obtained by column chromatography.

3. The method for preparing the fluorescent probe for the dual-channel differentiation and detection of hypochlorous acid and bisulfite during ferroptosis according to claim 2, characterized in that: The molar ratio of 4-(methylthio)benzaldehyde to 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-onium iodide is 1:

1.

4. The method for preparing the fluorescent probe for dual-channel detection of hypochlorous acid and bisulfite during ferroptosis according to claim 2, characterized in that... The specific synthesis route is as follows: 。 5. The application of the fluorescent probe of claim 1 for the dual-channel differentiation and detection of hypochlorous acid and bisulfite during ferroptosis in rapid, highly selective dual-channel differentiation and identification of hypochlorous acid and bisulfite.

6. The application according to claim 5, characterized in that... The specific process is as follows: the fluorescent probe shows no fluorescence in phosphate buffer under 365nm portable UV light irradiation; after adding a solution containing hypochlorous acid to the mixture, it shows bright orange-yellow fluorescence under 365nm portable UV light irradiation; after adding a solution containing sodium bisulfite to the mixture, it shows bright cyan fluorescence under 365nm portable UV light irradiation.

7. The application of the fluorescent probe of claim 1 for the dual-channel differentiation detection of hypochlorous acid and bisulfite during ferroptosis in the preparation of a dual-channel differentiation imaging detection reagent for exogenous and / or endogenous hypochlorous acid and bisulfite in cells.

8. The application of the fluorescent probe of claim 1 for the dual-channel differentiation detection of hypochlorous acid and bisulfite during ferroptosis in the preparation of a dual-channel differentiation imaging detection reagent for endogenous hypochlorous acid and bisulfite in zebrafish.

9. The application of the fluorescent probe of claim 1 for the dual-channel differentiation detection of hypochlorous acid and bisulfite during ferroptosis in the preparation of a dual-channel imaging detection reagent for intracellular hypochlorous acid and bisulfite during ferroptosis.

10. The application of the fluorescent probe of claim 1 for the dual-channel differentiation detection of hypochlorous acid and bisulfite during ferroptosis in zebrafish during ferroptosis in the preparation of a dual-channel differentiation imaging detection reagent for hypochlorous acid and bisulfite in zebrafish during ferroptosis.

Images