Mitochondrial targeting two-photon fluorescent probe and application thereof in ClO <-> detection

Abstract

The invention discloses a mitochondrial targeting two-photon fluorescent probe and application thereof in ClO <-> detection. The structure of the two-photon fluorescent probe is shown as follows: # imgabs0 #. The mitochondria targeting two-photon fluorescent probe responds to ClO <-> with different concentrations, the reaction speed is high (30 s), the detection limit is as low as 0.32 mu M, the probe has the two-photon absorption property, and the maximum two-photon absorption cross section at 840 nm is 41.2 GM. A cytotoxicity test shows that the probe is good in biocompatibility, a confocal fluorescence microscopic imaging experiment shows that the probe is good in light stability, has the capacity of monitoring endogenous and exogenous ClO <-> concentration changes of cells, has the mitochondrial targeting capacity (R2 = 0.82), and can be used for detecting the mitochondrial targeting of the cells. A confocal fluorescence microscopic imaging experiment can detect that an exothermic reaction occurs after the probe responds to ClO <-> in cells to generate various active oxygen (OH., O2 and < 1 > O2), and the probe has a certain biological application value.

Description

Technical Field

[0001] The present invention belongs to the field of organic chemistry and specifically relates to a mitochondrial targeted two-photon fluorescent probe and its - Application in detection. Background Art

[0002] Mitochondria are multifunctional protein organelles with double membrane structures that can regulate intracellular physiology and signal transduction. They also have their own circular genome that can control the operation of the electron transport chain (ETC). Based on the interconnected functions of mitochondria, mitochondrial damage will undoubtedly lead to disorders in normal functions, resulting in the collapse of cellular homeostasis. Mitochondrial damage can affect ETC and ATP production and the loss of antioxidant effects. Studies have shown that the pathology of many diseases is related to the dysfunction of normal mitochondrial function, including many common cancers and nervous system damage. When the number of damaged mitochondria increases, cells will experience many phenomena such as mitochondrial autophagy to control the number of damaged mitochondria to reduce the occurrence of harm. Under normal circumstances, macrophages, as a mechanism for cells to balance normal functions, maintain homeostasis by decomposing and removing dysfunctional organelles and protein aggregates through liposomes. Therefore, the exploration of the mechanism of mitochondrial-related diseases is crucial.

[0003] Reactive oxygen species are signaling molecules that regulate the life activities of organisms, such as immune regulation, aging and anti-inflammation, and have a significant impact on various physiological processes. ROS contains many types, and common types include superoxide anion free radicals, hydroxyl free radicals, singlet oxygen, peroxyl free radicals, hydrogen peroxide, peroxynitrite, hypochlorous acid, etc. The existence of these reactive oxygen species plays a vital role in the human body. They are closely related to biological processes such as cellular redox homeostasis, signal transduction, and defense against pathogen invasion. Normal concentrations of ROS are conducive to the normal growth of normal cells. When the concentration of ROS is abnormal, it will affect oxidative stress in the biological system and induce cell apoptosis. This will lead to serious diseases such as cardiovascular disease, arthritis, kidney disease, inflammatory diseases, neurodegenerative diseases and cancer. Hypochlorous acid / hypochlorite is commonly found in organisms and is one of the most important ROS in biological systems. It is produced from hydrogen peroxide and chloride ions in the body, catalyzed by myeloperoxidase (MPO). Hypochlorous acid / hypochlorite plays a key role in many physiological and pathological processes, including anti-inflammation, immune regulation and aging. However, ClO - Excessive levels can cause redox imbalance, leading to many serious diseases, including cancer. Therefore, it is necessary to develop a method that can accurately detect ClO in biological systems. - The approach still faces huge challenges.

[0004] So far, ClO has been reported -The determination methods include nuclear magnetic resonance (NMR), absorption spectroscopy, surface enhanced Raman scattering (SERS), etc. The fluorescence imaging method based on fluorescent probes can eliminate the interference of other signal molecules and is suitable for ClO - It has attracted much attention due to its advantages such as specific response, high sensitivity and real-time performance. - Two-photon fluorescent probes will be used for intracellular ClO - It provides a powerful tool for real-time imaging studies of fluctuations.

[0005] In recent years, fluorescence imaging has been widely used to monitor biological processes, but the - There are still very few specific imaging methods, especially two-photon fluorescent probes. Therefore, it is necessary to develop a ClO - Fluorescent probes will be used for intracellular ClO - It provides a powerful tool for real-time imaging studies of fluctuations. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a mitochondrial target two-photon fluorescent probe and a ClO - The technical problem to be solved is to obtain a fluorescence-on response ClO through molecular design. - and mitochondrial targeting molecular structure, requiring that the molecular structure not only has the ability to target mitochondria, but also has the ability to - The fluorescence response has the advantages of high sensitivity and resolution, specific selectivity and good photostability.

[0007] The two-photon fluorescent probe of the present invention can monitor endogenous and exogenous ClO in living cells in real time. - The probe can target mitochondria, and cell confocal fluorescence imaging can also detect the probe response to ClO - Afterwards, a variety of active oxygen species are produced, which have certain biological application value.

[0008] The present invention uses carbazole with excellent performance as the fluorophore to design and synthesize a two-photon ClO - Fluorescent probe CMI-ClO. Carbazole and malononitrile in dicyanoisophorone form a D-π-A structure, which produces an intramolecular charge transfer (ICT) effect and reacts with ClO - After the reaction, the fluorescence at 560nm was enhanced. The optical properties and cytotoxicity tests showed that CMI-ClO has ClO - The specific response, fast reaction speed and good biocompatibility were evaluated by analyzing the cell confocal fluorescence imaging. - Feasibility of specific fluorescence imaging with probes responsive to ClO- After that, a variety of reactive oxygen species are produced, aiming to generate ClO in the mitochondria of living cells. - It provides a convenient chemical tool for visualization detection and subsequent biological application research.

[0009] The mitochondrial target two-photon fluorescent probe of the present invention is abbreviated as CMI-ClO, and its structure is shown below:

[0010] .

[0011] The preparation method of the two-photon fluorescent probe of the present invention comprises the following steps:

[0012] Step 1: Dissolve 14.63 g potassium hydroxide in 150 mL acetone and stir at room temperature for 30 min. Then add 14.65 g 3-iodocarbazole to continue the reaction. Add acetone containing ethyl bromide dropwise into the above solution, and the resulting system reacts overnight. Rotate the mixture to remove the solvent. The residue is purified by silica gel chromatography (eluent: petroleum ether / ethyl acetate = 6:1) to obtain intermediate 1.

[0013] Step 2: Under ice-water bath conditions, add 23.13 mL of phosphorus oxychloride to the 70 mL DMF solution stirred in the flask, and then place the reactants at room temperature to react. Add 12.84 g of intermediate 1 dissolved in 1,2-dichloroethane, stir and reflux for 11 h; after the reaction is completed, cool to room temperature, pour the mixture into water, extract with dichloromethane, wash the organic phase with water, dry with anhydrous magnesium sulfate, and remove the solvent by rotary evaporation. The residue is purified by silica gel chromatography (eluent is petroleum ether / ethyl acetate = 4:1) to obtain intermediate 2.

[0014] Step 3: 0.112 g of palladium acetate, 0.761 g of tri-o-tolylphosphine, 6.910 g of potassium carbonate and 3.50 g of intermediate 2 were added to a round-bottom flask, followed by the addition of 80 mL of N-methyl-2-pyrrolidone and 3.2 mL of 4-vinylpyridine as solvents, and the mixture was heated to 130 °C for 2 days under nitrogen protection to obtain a dark red suspension solution. The mixture was poured into water, extracted with dichloromethane, the organic phase was washed with water, dried over anhydrous magnesium sulfate, the solvent was removed by rotary evaporation, and the residue was purified by silica gel chromatography (eluent: petroleum ether / ethyl acetate = 2:1) to obtain intermediate 3.

[0015] Step 4: Dissolve 50 mg of intermediate 3 and 31.2 mg of dicyanoisophorone in 20 mL of anhydrous ethanol, add one or two drops of piperidine, heat to 80°C, reflux at this temperature for 12 hours, cool the reaction solution, extract the solution, evaporate the organic phase to obtain a crude product, and recrystallize from ethanol to obtain intermediate 4;

[0016] Step 5: Dissolve 190 mg of intermediate 4 in 7 mL of anhydrous tetrahydrofuran, add 544 mg of iodomethane, stir and react at room temperature for 12 hours, and finally recrystallize from ethyl acetate to obtain solid CMI-ClO.

[0017] The synthetic route is as follows:

[0018] .

[0019] The two-photon fluorescent probe of the present invention is used in the preparation of ClO - Application in detection reagents.

[0020] The detection reagent is in I 560 nm The fluorescence ratio at ClO - The concentration is linearly related.

[0021] The two-photon fluorescent probe of the present invention uses carbazole electron donor and dicyanoisophorone as electron acceptors to form a D-II-A structure, thereby emitting fluorescence at 560 nm. CMI-ClO has good cell compatibility and has the prospect of being used in cell confocal imaging. Since the CMI-ClO molecule is a positive ion molecule, CMI-ClO itself has the ability to enter mitochondria.

[0022] The detection method is as follows:

[0023] The CMI-ClO of the present invention was dissolved in DMSO to prepare a 2 mM stock solution, and 15 μL of the stock solution was taken to 3 mL of DMSO containing different concentrations of ClO. - The fluorescence and UV spectra of 10 μM CMI-ClO in different test solutions were obtained in a mixed solvent of PBS and DMF (PBS:DMF=1 / 1). - As the absorbance of CMI-ClO increases, the absorbance at 475 nm blue shifts. The fluorescence intensity at 560 nm gradually increases. - The fluorescence intensity after the reaction increased by more than 5 times, with open-type fluorescence detection capability, and its fluorescence ratio was similar to that of ClO - The concentration of 2 =0.99), CMI-ClO has a fast response time and can complete the reaction in 30 seconds. Fluorescence test shows that CMI-ClO and ClO - After the response, an exothermic reaction occurs to provide energy to produce a variety of reactive oxygen species. CMI-ClO can not only respond well to ClO in cells - , and the probe responds to ClO - Various reactive oxygen species were produced, and the mitochondrial imaging in the cells was good (Pr=0.82).

[0024] The two-photon fluorescent probe is used in the preparation of thermal therapy drug preparations and / or photodynamic therapy drug preparations.

[0025] During the experiment, it was found that CMI-ClO and ClO - After the reaction, an exothermic reaction occurs and the temperature rises, so it can be used to prepare thermal therapeutic drug preparations.

[0026] Cell confocal fluorescence imaging can also detect the probe response to ClO - Afterwards, a variety of reactive oxygen species are produced, which can be used for photodynamic therapy of hypochlorous acid-related diseases.

[0027] A method for detecting ClO in cells according to the present invention - A dual-functional two-photon fluorescent probe for mitochondrial imaging that responds to different concentrations of ClO - The minimum detection limit is as low as 0.32 μM. CMI-ClO responds to ClO - An exothermic reaction occurs when the CMI-ClO reacts to provide energy and generate a variety of reactive oxygen species. Cytotoxicity tests show that CMI-ClO has good biocompatibility, and confocal fluorescence microscopy experiments show that CMI-ClO has good photostability for HeLa cells and has the ability to monitor endogenous and exogenous ClO in cells. - concentration, and CMI-ClO has the ability to image mitochondria (R 2 =0.82), which is suitable for confocal fluorescence imaging of intracellular mitochondrial tracing. CMI-ClO responds to ClO - Afterwards, a variety of active oxygen species are produced, which have certain biological application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The difference between CMI-ClO and ClO at different reaction times - UV fluorescence and linear relationship of the response process and reaction time test. (a) CMI-ClO (10 μM) and ClO - (0-50 μM) UV absorption changes; (b) CMI-ClO (10 μM) and ClO - (0-100 μM) fluorescence emission peak changes; (c) CMI-ClO (10 μM) and different concentrations of ClO - After the reaction, a good linear relationship (R 2 =0.9995); (d) CMI-CLO (10 μM) and ClO - Response time at different concentrations.

[0029] Figure 2 (a) is CMI-ClO and different concentrations of ClO -Two-photon fluorescence titration diagram after the reaction; (b) is CMI-ClO (10 μM) and ClO - Two-photon performance verification diagram after reaction; (c) CMI-ClO and ClO - Two-photon absorption cross-section at different excitation wavelengths.

[0030] Figure 3 The selectivity test of CMI-ClO was investigated. The selectivity of CMI-ClO with common metal ions (100 µM), anions (100 µM), reactive oxygen species (100 µM), biothiols (100 µM) and ClO - (100 μM) after the reaction. Where: (1) only the probe, (2) Cu 2+ , (3) Cu + , (4) Ca 2+ , (5) Mg 2+ ,(6) Fe 3+ ,(7) Fe 2 + , (8) Na + , (9) K + , (10) SO3 2- , (11) SO4 2- , (12) HSO3 - , (13) HCO3 - , (14) CO3 2- , (15) S 2- , (16) H2PO4 - , (17) NH4 + , (18) GSH, (19) Hcy, (20) Cys, (21) H2O2, (22)ClO - .

[0031] Figure 4 CMI-ClO and ClO - Temperature changes after the reaction. (a) CMI-ClO (100 μM) and ClO - (a) Temperature change after reaction using infrared thermal imaging; (b) CMI-ClO (10 μM) and different concentrations of ClO - (10~50 μM) Temperature change after reaction detected by infrared thermal imaging; (c) CMI-ClO (10 μM) and ClO - (10 μM) after reaction using infrared thermography;

[0032] Figure 5 Is CMI-ClO responsive to ClO- Fluorescence and ESR graphs of reactive oxygen species produced after ionization. (a) CMI-ClO (10 μM) and ClO - (b) is the ESR spectra of the reaction system (a) at different temperatures (LT: 0℃; RT: 25℃; HT: 37℃); (c) CMI-ClO (10 μM) and ClO - (d) is the ESR spectra of the reaction system (c) at different temperatures (LT: 0℃; RT: 25℃; HT: 37℃); (e) CMI-ClO (10 μM) and ClO - (f) is the ESR spectrum of the reaction system (e) after reaction at different temperatures (LT: 0℃; RT: 25℃; HT: 37℃).

[0033] Figure 6 This is a mitochondrial imaging co-localization study of CMI-ClO. CMI-ClO and 1μg / mL mitochondrial dye (Mito-Tracker Red) were co-incubated in HeLa cells and then confocal imaging was performed to explore the mitochondrial imaging ability of CMI-ClO.

[0034] Figure 7 CMI-ClO is used to detect exogenous ClO - In the test, CMI-ClO (10 μM) was incubated with cells and then 20 μM, 30 μM and 40 μM ClO were added. - Confocal imaging was performed after 30 min of reaction to investigate the effect of CMI-ClO on ClO from outside the cell. - detection capabilities.

[0035] Figure 8 CMI-ClO is used to detect endogenous ClO - In the test, cells were induced with 1 μM lipopolysaccharide (LPS) and lipopolysaccharide (LPS) + NAC or ABH (antioxidant), and then CMI-ClO (10 μM) was added and incubated for 30 min. Confocal imaging was performed to explore the effect of CMI-ClO on ClO produced in cells. - monitoring capabilities.

[0036] Fig. 9 CMI-ClO and ClO - After the reaction, various reactive oxygen species were generated. -After incubation with 10 μM for 30 min, DCFH-DA, HPF, DHE and SOSG were used for confocal imaging for 15 min to investigate the relationship between CMI-ClO and ClO. - Ability to produce a variety of reactive oxygen species. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below through specific embodiments.

[0038] Example 1: Synthesis of CMI-ClO

[0039] 0.19 g of intermediate 4 was dissolved in 7 mL of anhydrous tetrahydrofuran, 0.54 mg of iodomethane was added, and the mixture was stirred at room temperature for 12 hours, and finally recrystallized from ethyl acetate to obtain the target product CMI-ClO. The yield was 90%.

[0040] 1 H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.55 (s, 2H), 8.50 (s, 1H), 7.84 (s, 2H), 7.70 (s, 4H), 7.58 (s, 2H), 7.52 (s, 1H), 7.29 (s, 1H), 6.88(d, J = 2.5 Hz, 1H), 4.50 (s,2H), 2.63 (s, 4H), 1.34 (s,3H), 1.06 (s,6H). 13 CNMR (101MHz, DMSO-d6) δ of 170.01, 156.51, 149.95, 144.66, 140.97, 140.36,139.21, 133.82, 127.93, 127.45, 126.89, 126.67, 125.84, 123.28, 122.82,122.70, 121.37, 120.51, 120.33, 119.32, 114.06, 113.33, 109.87, 74.69, 42.19,39.14, 37.37, 31.59, 27.42, 13.83.

[0041] Example 2: CMI-ClO responds to ClO in solvent - Optical titration, linearity and response time

[0042] In order to explore the response of CMI-ClO to ClO in solvent - Optical titration using ClO -(0 - 50 μM) UV absorption titration and ClO - (0 - 100 μM) fluorescence titration. Figure 1 (a) It can be seen that CMI-ClO increases with ClO - As the concentration increases, the ultraviolet absorption at 475nm gradually shifts to the blue. Figure 1 (b) It can be seen that as ClO - With the addition of CMI-ClO, the concentration of 100 μM ClO - The titration endpoint is reached at Figure 1 (c) It can be seen that the fluorescence signal of CMI-ClO is - It shows a good linear relationship, and the linear correlation coefficient R 2 =0.99, and the minimum detection limit is 0.32 μM. Figure 1 (d) Monitoring of CMI-ClO response to ClO in solvent - The reaction time of CMI-ClO and ClO - The basic reaction was completed within 30 s. - After the reaction, it can emit a good fluorescent signal and accurately monitor ClO in the solvent. - .

[0043] Example 3: CMI-ClO responds to ClO in solvent - Two-photon absorption performance

[0044] In order to explore the response of CMI-ClO to ClO in solvent - Two-photon absorption performance. Figure 2 (a) CMI-ClO in response to different concentrations of ClO - The two-photon fluorescence spectrum of ClO - As the concentration (0 - 100 μM) increases, the two-photon fluorescence gradually increases. Figure 2 (b), the slope is 2 calculated according to the two-photon verification formula, proving that the probe CMI-ClO is similar to ClO - After the response, it has two-photon absorption performance. Figure 2 (c) This shows that CMI-CO and ClO - The two-photon absorption cross section after response reaches 41.2 GM at 840 nm.

[0045] Example 4: CMI-ClO responds to ClO in solvent - Selective response experiment

[0046] In order to explore the response of CMI-ClO to ClO in solvent -In order to improve the specificity of CMI-ClO, we used CMI-ClO to detect common biological thiols, metal ions, anions, and reactive oxygen species in solvents. Figure 3 This indicates that CMI-ClO is not affected by other metal ions, enzymes, and other reactive oxygen species, but only by ClO - It has good response ability, and the fluorescence intensity after response is much higher than that of other detection substances.

[0047] Example 5: Temperature change after reaction of CMI-ClO and ClO-

[0048] During the experiment, it was found that CMI-ClO and ClO - After the reaction, an exothermic reaction occurred and the temperature rose. Figure 4 (a) Infrared thermal imaging camera showing the dropwise addition of ClO to CMI-ClO (100 μM). - The temperature change of (10 μM) within 180 s can reach 43°C in 20 s, and the temperature increases by 10°C within 30 s. Figure 4 (b) At the same time, as ClO - As the concentration increases, the temperature peaks and variations also increase. Figure 4 (c) is an infrared thermal image, which clearly shows that with the increase of ClO - The dropping temperature increases.

[0049] Example 6: CMI-ClO and ClO - After the reaction, various active oxygen species are generated

[0050] In order to compare CMI-ClO and ClO - The ability to release ROS after the reaction was evaluated by analyzing the fluorescence emission and ESR. Figure 5 (a) (b) (c) show that CMI-ClO and ClO - After the reaction, there are obvious hydroxyl free radicals (OH·), superoxide anions (O2 •− ) and singlet oxygen ( 1 O2) fluorescence signal. Figure 5 (d) (e) (f) As the temperature increases, CMI-ClO and ClO - After the reaction, the ESR signals of hydroxyl radicals, superoxide anions, and singlet oxygen increased, indicating that thermal energy provided energy to generate reactive oxygen species. These results show that CMI-ClO and ClO - After the response, an exothermic reaction occurs to produce various reactive oxygen species.

[0051] Example 7: Imaging the mitochondrial targeting ability of CMI-ClO

[0052] In order to explore the ability of CMI-ClO to image mitochondria in cells, we used Mito-Tracker Red to perform co-localization studies with CMI-ClO in cells. Figure 6 As shown, the green channel (λ em =560±20 nm; λ ex = 475 nm) and the red channel of Mito-Tracker Red (λ em = 600 ± 20 nm; λ ex = 579 nm) overlapped well, and the Pearson colocalization coefficient of CMI-ClO and Mito-Tracker Red was calculated to be 0.82. These results indicate that CMI-ClO has the ability to track mitochondria.

[0053] Example 8: Exogenous ClO in cells with CMI-ClO - Concentration monitoring

[0054] To investigate the effect of CMI-ClO on monitoring exogenous ClO in cells - Variation of the capacity of HeLa cells using different concentrations of ClO - Processing ( Figure 7 ). With the exogenous ClO - As the concentration increases, the cell fluorescence intensity increases. The two-photon fluorescence intensity is higher than the single-photon fluorescence intensity, the brightness is more obvious, and it has a higher resolution. Experiments have shown that CMI-ClO can detect exogenous ClO - Changes

[0055] Example 9: Endogenous ClO in cells with CMI-ClO - Concentration monitoring

[0056] In order to investigate the role of CMI-ClO in monitoring cellular ClO - Changes in the ability to produce endogenous ClO using LPS-induced cellular oxidative stress - .like Figure 8 As shown in the figure, the fluorescence intensity of CMI-ClO increased after LPS pretreatment compared with the control group. NAC and ABH are antioxidants, and the fluorescence intensity after pretreatment with NAC and ABH was significantly reduced compared with cells treated with LPS alone. This indicates that the antioxidants NAC and ABH can scavenge LPS-induced ClO - Fluorescence imaging shows that CMI-ClO can detect endogenous ClO - .

[0057] Example 10: CMI-ClO and ClO- Detection of reactive oxygen species generated in cells after the reaction

[0058] In order to explore the relationship between CMI-ClO and ClO - The ability to produce reactive oxygen species in cells after the reaction. DCFH-DA (Ex / Em: 495 nm / 525 nm), HPF (Ex / Em: 492 nm / 515 nm), DHE (Ex / Em: 510 nm / 610 nm) and SOSG (Ex / Em: 504 nm / 525 nm) were used to detect the CMI-ClO and ClO in cells at different temperatures (LT: 0℃; RT: 25℃; HT: 37℃). - Fluorescence imaging showed that compared with the addition of CMI-ClO alone, CMI-ClO and ClO - Generate bright fluorescence signals. As the temperature increases, the fluorescence signal of the cells increases significantly, which is consistent with the properties of the optical test part. Fluorescence imaging shows that CMI-ClO and ClO - After the reaction, various active oxygen species (OH·, O2 •− , 1 O2), has certain biological application value.

Claims

1. A mitochondrial-targeted two-photon fluorescent probe, abbreviated as CMI-ClO, characterized in that Its structure is as follows: 。 2. The method for preparing the two-photon fluorescent probe according to claim 1, characterized in that The steps include: Step 1: Dissolve potassium hydroxide in acetone and stir at room temperature, then add 3-iodocarbazole to continue the reaction; drip acetone containing ethyl bromide into the above solution and react for 8-12 hours; after the reaction is completed, separate and purify to obtain intermediate 1; Step 2: under ice-water bath conditions, add phosphorus oxychloride dropwise to the DMF solution, and then place the reactants at room temperature for reaction; add intermediate 1 dropwise to the system, stir and heat to reflux for reaction; after the reaction is completed, separate and purify to obtain intermediate 2; Step 3: Add palladium acetate, tri-o-tolylphosphine, potassium carbonate and intermediate 2 into a round-bottom flask, then add solvents N-methyl-2-pyrrolidone and 4-vinylpyridine, and heat to react at 130° C. for 2 days under nitrogen protection to obtain a deep red suspension solution; and obtain intermediate 3 after separation and purification; Step 4: Dissolve the intermediate 3 and dicyanoisophorone in anhydrous ethanol, add piperidine dropwise, and heat to reflux for 12 hours; after the reaction, separate and purify to obtain the intermediate 4; Step 5: Dissolve the intermediate 4 in anhydrous tetrahydrofuran, add iodomethane, stir and react at room temperature for 12 hours, and finally recrystallize from ethyl acetate to obtain solid CMI-ClO; The synthetic route is as follows: 。 3. The two-photon fluorescent probe according to claim 1 is used to prepare ClO - Application in detection reagents.

4. The use according to claim 3, characterized in that: The detection reagent is in I 560nm The fluorescence ratio at ClO - The concentration is linearly related.

5. The use according to claim 3, characterized in that: The detection reagent targets mitochondria.

6. The use according to claim 3, characterized in that: The detection reagent is effective for ClO - The detection limit was 0.32 μM.

7. Use of the two-photon fluorescent probe according to claim 1 in the preparation of thermal therapy drug preparations and / or photodynamic therapy drug preparations.

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