A fluorescent probe for myeloperoxidase detection, and a preparation method and application thereof

Abstract

The application provides a fluorescent probe for myeloperoxidase detection, and a preparation method and application thereof. The fluorescent probe can detect MPO in living cells, greatly shortens the detection time, has low production cost, high specificity, and can be fixed on the protein of the cell after response, thereby avoiding non-specific fluorescent signal transmission and reducing false positives in the detection process. Meanwhile, the fluorescent probe can realize the function of distinguishing myeloid leukemia cells from lymphoid leukemia cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent probes, and particularly relates to a fluorescent probe for detecting myeloperoxidase, a preparation method and application thereof. BACKGROUND

[0002] Myeloperoxidase (MPO), also known as peroxidase, mainly exists in the benzene blue granules of myeloid cells, and is a specific marker of myeloid cells, which can be used to identify the type of leukemia in clinic. MPO is closely related to the mechanism of many diseases and plays an important role in the oxidative stress process of phagocytes. It can catalyze the intracellular chloride and hydrogen peroxide to produce hypochlorous acid, thereby killing microorganisms phagocytosed by cells.

[0003] At present, the methods for detecting MPO mainly include the following: 1. Continuous monitoring method: under acidic conditions, tetramethylbenzidine, o-methoxyphenol or 3,3'-dimethoxybenzidine is used as a substrate to determine the activity of MPO in blood or tissue. The method is simple to operate and has low cost, but is easily interfered by other peroxidases or heme proteins, and has poor specificity. 2. Enzyme-linked immunosorbent assay (ELISA): MPO specific antibody is prepared by using the specificity of antigen-antibody reaction, and the content of MPO in blood is detected by immunological method. ELISA detection improves the specificity, but has high cost, requires preparation of high titer antibody, and the detection process takes a long time. 3. Flow cytometry detection: after the whole blood sample is treated, fluorescent-labeled anti-MPO antibody or fluorescent probe is finally added, the MPO in the cells is stained and incubated by using antigen-antibody reaction, and finally flow cytometry analysis is carried out. This method has high specificity and sensitivity, and the detection time is short, and the intracellular MPO can be detected.

[0004] Clinically, peroxidase (POX) staining is used to aid in the diagnosis of acute leukemia types and to differentiate acute myeloid leukemia (mainly granulocytic or monocytic) from acute lymphoblastic leukemia. Common methods include the diaminobenzidine (DAB) method, the tetramethylbenzidine method, the WG-KI oxidation method, and the traditional Washburn method. ICSH recommends three methods: DAB, peroxidase-amino-methylcarbazole staining, and diaminobenzidine dihydrochloride staining. The Washburn method works by using peroxidase to decompose hydrogen peroxide in the reagent, releasing oxygen and oxidizing colorless benzidine to blue benzidine. This blue benzidine then combines with sodium nitroferricyanide to form brownish-black or brownish-yellow particles that precipitate within the cells, facilitating clinical observation. The DAB method works by using peroxidase to transfer hydrogen atoms from the colorless DAB to hydrogen peroxide, catalyzing the formation of a colored probe that deposits as a brownish-yellow particle at the POX site in the cytoplasm. However, benzidine (including that used in the DAB method) is toxic and carcinogenic, making it unsuitable for laboratory personnel. The principle of the oxidative WG-KI method is that peroxidase decomposes peroxides to produce nascent oxygen, which reacts with KI (potassium iodide) to produce iodine. The iodine then combines with the active ingredient in the chromogenic reagent to form brownish-red or blue-black particles. Although the reagents for the oxidative WG-KI method are safe, it is difficult to control the appropriate staining time. Furthermore, the probe is water-soluble and easily washed away and faded, which can lead to false negatives. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a fluorescent probe for myeloperoxidase detection, its preparation method and application.

[0006] To achieve the above objectives, a first aspect of the present invention is to provide a fluorescent probe for myeloperoxidase detection, the fluorescent probe having the following structural formula:

[0007] R 1 R 2 Each is independently selected from H, phenyl, substituted phenyl, C 1-8 Substituted or unsubstituted alkyl, C 1-8 Substituted or unsubstituted alkenyl groups, R 3 Halogen atoms;

[0008] The substituted phenyl group is composed of halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Carboxyl group and C 1-6 The ester group may be substituted in any way; the substituted alkyl and substituted alkenyl groups are derived from halogens or C. 1-6 Alkoxy groups can be substituted in any way.

[0009] Preferred, R 1 For methyl, R2 R is hydrogen, R 3 R is fluorine.

[0010] The second aspect of the present application is to provide a preparation method of a fluorescent probe for myeloperoxidase detection, comprising the following steps:

[0011] (1) reacting the compound shown in formula I with p-fluoro nitrobenzene in a N, N-dimethylformamide solution containing cesium carbonate to synthesize the compound shown in formula II;

[0012] (2) reacting the compound shown in formula II with sodium borohydride in a reaction solvent, which is a mixed solvent of methanol and dichloromethane;

[0013] (3) reacting the compound generated in step (2) with a halogen-containing compound to obtain the compound shown in formula III;

[0014] (4) reacting the compound shown in formula III with stannous chloride in a solution.

[0015] Formula I: ; Formula II: ; Formula III: .

[0016] Preferably, in step (1), the molar ratio of the compound shown in formula I to p-fluoro nitrobenzene is 1:4-6, the reaction system temperature is 80-120℃, and the reaction time is 4-6h.

[0017] Preferably, in step (2), the molar ratio of the compound shown in formula II to sodium borohydride is 4-5:1, the volume ratio of methanol to dichloromethane is 1:2-4, the reaction temperature is 10-40℃, and the reaction time is 15-45min.

[0018] Preferably, in step (3), R 3 is fluorine, the halogen-containing compound is diethylamine trifluoride, the compound generated in step (2) is reacted with diethylamine trifluoride according to a molar ratio of 1:0.8-1.2, the reaction solvent is dichloromethane, the reaction system temperature is -40-0℃, and the reaction time is 1-3h.

[0019] Preferably, in step (4), the molar ratio of the compound shown in formula III to stannous chloride is 1:12-18, the solution is methanol: concentrated hydrochloric acid: water = 3:1.5-2.5:2.5-3.5, the reaction temperature is 10-40℃, and the reaction time is 10-14h.

[0020] The third aspect of the present application is to provide the application of the fluorescent probe prepared by the preparation method as described above in the detection of myeloperoxidase.

[0021] A fourth aspect of the present invention is to provide the application of the fluorescent probe prepared by the above preparation method in the detection of myeloid leukemia cells, wherein the myeloid leukemia cells are incubated with the fluorescent probe in the dark, and then H2O2 is added for further incubation. After the incubation is completed, an image is captured by a laser confocal microscope, and the myeloid leukemia cells emit green fluorescence.

[0022] A fifth aspect of the present invention is to provide a reagent or kit for detecting myeloperoxidase, comprising a fluorescent probe prepared by the preparation method described above.

[0023] The beneficial effects of this invention are as follows: This fluorescent probe can detect MPO in living cells, greatly shortening the detection time. At the same time, the probe has low production cost, high specificity, and can be fixed on the protein of the cell after response, avoiding non-specific fluorescent signal transmission and reducing false positives in the detection process. In addition, this fluorescent probe can distinguish between myeloid leukemia cells and lymphoid leukemia cells. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0025] Figure 1 This is the reaction formula for the preparation of the fluorescent probe in Example 1.

[0026] Figure 2 Part A presents the fluorescence spectra of fluorescent probe molecules (10 μM), hydrogen peroxide (100 μM), and different concentrations of MPO (10 mU / mL, 15 mU / mL, 20 mU / mL, 25 mU / mL, 30 mU / mL, 35 mU / mL, 40 mU / mL) in PBS solution (10 mM, pH 7.4) containing 100 μL fetal bovine serum (FBS) using a fluorescence spectrophotometer. Part B plots the linear relationship between the amount added and the fluorescence intensity value, with the fluorescence intensity value plotted on the ordinate and the MPO concentration on the abscissa, using the fluorescence intensity value at the position of the maximum emission peak of the fluorescence spectrum in Part A.

[0027] Figure 3To test the fluorescence spectra of a 10 μM fluorescent probe molecule against 50 μM hypochlorite ions, hydroxyl radicals, singlet oxygen, 300 μM peroxynitrite, 500 μM nitric oxide, and hydrogen peroxide in a PBS solution (10 mM, pH 7.4) containing 100 μL fetal bovine serum (FBS), a fluorescence spectrophotometer was used. The fluorescence intensity at the position of the maximum emission peak was plotted on the ordinate, and each reactive oxygen species was plotted on the abscissa.

[0028] Figure 4 The fluorescence intensity at the maximum emission peak was measured before and after the reaction with 50 μM sodium hypochlorite in 3 mL PBS (containing 100 μL LFBS) buffer solution with pH 2.0 to 12.0, with a concentration of 10 μM probe molecules.

[0029] Figure 5 Part A shows the fluorescence spectra of the fluorescent probe molecule (10 μM) against 50 μM hypochlorite ions at different response times, measured using a fluorescence spectrophotometer in a PBS solution (10 mM, pH 7.4) containing 100 μL fetal bovine serum (FBS). Part B shows the fluorescence intensity at the maximum emission wavelength of the fluorescence spectrum in Part A plotted on the ordinate and the response time on the abscissa to obtain the curve of fluorescence intensity at the maximum emission wavelength versus response time.

[0030] Figure 6 To measure the UV absorption spectra of a fluorescent probe molecule (10 μM) against 50 μM hypochlorite ions at different response times in PBS solution (10 mM, pH 7.4) containing 100 μL fetal bovine serum (FBS), a UV-Vis spectrophotometer was used.

[0031] Figure 7 The probe molecules were used to stain lymphoid leukemia cells (NALM-6) and myeloid leukemia cells (HL-60) and then analyzed by flow cytometry. The concentration of the probe molecules was 10 μM and the concentration of H2O2 was 200 μM.

[0032] Figure 8 Laser confocal microscopy was used to image myeloid leukemia cells (HL-60) after staining with probe molecules. The probe molecule concentration was 10 μM, and the H2O2 concentration was 200 μM. The probe molecule detection channel was the FITC channel, the cell nucleus was stained with DAPI, TD was the white light channel, and Merge was the mixed channel.

[0033] Figure 9Image of NALM-6 lymphoblastic leukemia cells after staining with probe molecules using laser confocal microscopy. The probe molecule concentration was 10 μM, and the H2O2 concentration was 200 μM. The probe molecule detection channel was the FITC channel, the cell nucleus was stained with DAPI, TD was the white light channel, and Merge was the mixed channel.

[0034] Figure 10 The effect of culturing HL-60 cells with different concentrations of probe molecules for 12 h and 24 h on cell viability. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0036] Example 1

[0037] I. The preparation method of the fluorescent probe in this embodiment is as follows: Figure 1 And as shown in the following steps:

[0038] (1) 187 mg of compound 1 was dissolved in 5 mL of anhydrous N,N-dimethylformamide, and 325 mg of cesium carbonate and 350 mg of p-fluoronitrobenzene were added sequentially. The mixture was heated at 100 °C for 5 hours. After the reaction was completed, the solution was poured into 50 mL of water and extracted three times with 90 mL of ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, and the solvent was evaporated to dryness. The solution was separated by column chromatography to obtain yellow compound 2. The structure of compound 2 was characterized by NMR as follows:

[0039] 1H NMR (400 MHz, DMSO) δ 10.68 (s, 1H), 8.25 (t, J = 8.7 Hz, 2H), 8.08 (d, J = 7.6 Hz, 1H), 7.87 (t, J = 7.4 Hz, 1H), 7.79 (t, J = 7.4 Hz, 1H), 7.40 (dd, J = 21.5, 11.8 Hz, 1H), 7.29 – 7.17 (m, 3H), 7.15 (d, J = 2.3 Hz, 1H), 6.97 (d, J = 8.8 Hz, 1H), 6.86 – 6.75 (m, 2H), 3.93 – 3.79 (m, 3H).

[0040] (2) 50 mg of compound 2 was dissolved in 7.5 mL of anhydrous dichloromethane, 2.5 mL of methanol was added, and 1.3 mg of sodium borohydride was added under ice bath conditions. The mixture was slowly heated to room temperature and reacted for 30 minutes. The reaction was then quenched by adding saturated ammonium chloride solution. The product was extracted three times with 90 mL of ethyl acetate, dried over anhydrous sodium sulfate, and the solvent was evaporated. The product was then redissolved in 5 mL of anhydrous dichloromethane and cooled to -20 °C. 15 mg of diethylaminotrifluoride (DAST) was added and the mixture was reacted for 2 hours. After the reaction was completed, the solvent was evaporated, and the yellow compound 3 was obtained by column chromatography. The structure of compound 3 was characterized by NMR as follows:

[0041] 1H NMR (400 MHz, DMSO) δ 8.27 (d, J = 9.2 Hz, 2H), 8.06 (d, J = 7.6Hz, 1H), 7.85 (t, J = 7.1 Hz, 1H), 7.77 (t, J = 7.3 Hz, 1H), 7.37 (t, J =10.6 Hz, 1H), 7.24 (d, J = 9.2 Hz, 2H), 7.13 (d, J = 2.3 Hz, 1H), 7.06 – 6.89(m, 2H), 6.78 (m, 2H), 5.82 (s, 1H), 5.69 (d, J = 10.4 Hz, 1H), 3.85 (d, J =9.5 Hz, 3H).

[0042] (3) 25 mg of compound 3 was dissolved in 10 mL of a mixed solution of methanol:concentrated hydrochloric acid:water = 3:2:3, and 47.5 mg of stannous chloride was added. The mixture was stirred at 25 °C for 12 hours. After the reaction was completed, 20 mL of water was added to dilute the solution, and the pH was adjusted to 7 with 1 mol / L sodium hydroxide solution. The solution was then extracted three times with 90 mL of ethyl acetate, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness. The yellow compound I-FMPO was obtained by column chromatography. The structure of compound I-FMPO was characterized by NMR as follows:

[0043] 1H NMR (400 MHz, DMSO) δ 8.02 (d, J = 7.5 Hz, 1H), 7.80 (t, J = 7.0Hz, 1H), 7.73 (t, J = 7.1 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H), 7.07 (t, J =10.6 Hz, 1H), 6.84 – 6.73 (m, 4H), 6.69 (d, J = 8.8 Hz, 1H), 6.58 (d, J = 8.8Hz, 2H), 6.47 (d, J = 9.0 Hz, 1H), 5.89 (s, 1H), 5.77 (d, J = 3.7 Hz, 1H),5.06 (s, 2H).3.86 (s, 3H).

[0044] II. Investigate the response intensity of the fluorescent probe in this embodiment to different concentrations of target MPO in PBS (10mM, pH 7.4) buffer solution.

[0045] The probe molecule I-FMPO was prepared as a stock solution (concentration 3 mM). Then, in a total test system of 3 mL PBS and 100 μL FBS, the test fluorescent probe molecule (10 μM), hydrogen peroxide (100 μM), and different concentrations of MPO (10 mU / mL, 15 mU / mL, 20 mU / mL, 25 mU / mL, 30 mU / mL, 35 mU / mL, 40 mU / mL) were added. The fluorescence spectrum was scanned using a fluorescence spectrophotometer to obtain the results shown below. Figure 2 The fluorescence spectrum of part A is plotted. The fluorescence intensity at the position of the maximum emission peak is plotted on the ordinate, and the MPO concentration on the abscissa, to show the linear relationship between the amount added and the fluorescence intensity. Figure 2 (Part B). It was found that as the concentration increases, there is a strong linear relationship between concentration and absorption intensity (R0). 2 =0.9837), indicating that the fluorescent probe I-FMPO tested in this embodiment has a good response to MPO in 100 μM hydrogen peroxide in PBS (10 mM, pH 7.4) buffer solution.

[0046] III. Experiments to investigate the selectivity of the fluorescent probe in this embodiment for various reactive oxygen species.

[0047] The probe molecule I-FMPO was prepared as a stock solution (concentration 3 mM). Then, in a total test system of 3 mL PBS and 100 μL FBS, the probe molecule (10 μM) and different reactive oxygen species (50 μM hypochlorite ion, hydroxyl radical, singlet oxygen, 300 μM peroxynitrite, 500 μM nitric oxide, and hydrogen peroxide) were added. The fluorescence spectra of each group were scanned using a fluorescence spectrophotometer, and plotted as shown below. Figure 3 The bar graph showing the comparison of fluorescence intensity values ​​indicates that the fluorescent probe I-FMPO tested in this embodiment has good selectivity for hypochlorous acid, and other reactive oxygen species do not cause interference.

[0048] IV. pH titration experiment to examine the reaction of the probe molecules with sodium hypochlorite in this embodiment.

[0049] The probe molecule was at a concentration of 10 μM in 3 mL PBS containing 100 μL LBS. The change in fluorescence intensity was measured before and after the reaction with 50 μM sodium hypochlorite at pH 2.0 to 12.0, as pH changed. Figure 4 This indicates that the probe of the present invention has a good response in the pH range of 6-7.

[0050] V. Kinetic tests to investigate the reaction between the fluorescent probe and hypochlorous acid in this embodiment.

[0051] The invented fluorescent probe molecule I-FMPO was added to the test solution of 3 mL PBS (pH 7.4) and 100 μL LFBS to make its concentration in the solution 10 μM. Then, 50 equivalents of sodium hypochlorite solution were added, and the changes in fluorescence intensity and ultraviolet absorption after the reaction were observed over time. Figure 5 Part A and Figure 5 Part B of the results all indicate that the probe reacts rapidly with sodium hypochlorite and can generate a strong fluorescence signal in a short time. Figure 6 This indicates that the ultraviolet absorption at around 450 nm changed after the probe reacted with sodium hypochlorite.

[0052] VI. To investigate whether the fluorescent probe in this embodiment can distinguish between myeloid leukemia cells and lymphoid leukemia cells by detecting MPO.

[0053] NALM-6 lymphocytic leukemia cells (low MPO expression) and HL-60 myeloid leukemia cells (high MPO expression) were selected. I-FMPO (10 μM) was added to each cell, and the cells were incubated at 37°C in the dark for 30 minutes. Then, H2O2 (200 μM) was added, and incubation continued for another 30 minutes. After incubation, fluorescence signals were detected using a BD-C6 flow cytometer. Figure 7As shown, HL-60 cells produced a fluorescent signal after the addition of the probe of this invention and H2O2, indicating that the fluorescent probe of this embodiment can detect the presence of MPO.

[0054] VII. To investigate whether the fluorescent probe in this embodiment can distinguish between myeloid leukemia cells and lymphoid leukemia cells.

[0055] To better illustrate the ability of this fluorescent probe to distinguish between myeloid leukemia cells and lymphoid leukemia cells, laser confocal microscopy was used to observe the imaging of the probe molecules within the cells. NALM-6 lymphoid leukemia cells and HL-60 myeloid leukemia cells were selected, and the probe molecule I-FMPO (10 μM) was added to each. After incubation at 37°C in the dark for 30 minutes, H2O2 (200 μM) was added, and incubation continued for another 30 minutes. Images were then captured using a laser confocal microscope. Figure 8 (HL-60) and Figure 9 As shown in (NALM-6), only the HL-60 group emitted green fluorescence after the addition of the probe and hydrogen peroxide, indicating that the probe in this embodiment can distinguish between cells with high and low MPO expression.

[0056] 8. Investigate the effect of the fluorescent probe of this invention on cytotoxicity.

[0057] We used the CCK-8 reagent to detect whether the probe had a toxic effect on cells. Cell suspension (100 μL / well, 5000 HL-60 cells per well) was seeded into two 96-well plates, and different concentrations of the probe (5-30 μM) were added. After incubating the plates for 12 and 24 hours, 10 μL of CCK-8 reagent was added, and the plates were incubated at 37°C for 2 hours. The absorbance at 450 nm was then measured using a microplate reader. Figure 10 This indicates that the working concentration of this probe at 10 μM is not toxic to cells and does not affect cell proliferation and growth.

[0058] Example 2

[0059] The difference between the preparation method of the fluorescent probe in this embodiment and that in Example 1 is only that: in step (2), the product is extracted three times with ethyl acetate, dried with anhydrous sodium sulfate, the solvent is evaporated, phosphorus oxychloride is added to redissolve the product, and the temperature is raised to 60°C. Phosphorus oxychloride (POCl3) is added and the reaction is carried out for 2 hours to obtain R. 3 It is a fluorescent probe for Cl.

[0060] Example 3

[0061] The difference between the preparation method of the fluorescent probe in this embodiment and that in Example 1 is only that: in step (2), the product is extracted three times with ethyl acetate, dried with anhydrous sodium sulfate, the solvent is evaporated, dichloromethane is added to redissolve the product and the temperature is controlled at 25°C, phosphorus tribromide (PBr3) is added and reacted for 12 hours to obtain R. 3 It is a fluorescent probe for Br, wherein the product and phosphorus tribromide react in a molar ratio of 1:1.

[0062] Example 4

[0063] The difference between the preparation method of the fluorescent probe in this embodiment and that in Example 1 is only that: in step (2), the product is extracted three times with ethyl acetate, dried with anhydrous sodium sulfate, the solvent is evaporated, dichloromethane is added to redissolve the product and the temperature is controlled at 25°C, phosphorus tribromide (PBr3) is added and reacted for 4 hours to obtain R. 3 The fluorescent probe is I, wherein the product reacts with triphenylphosphine, imidazole, and iodine in a molar ratio of 1:2:2:2.

[0064] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. Use of a fluorescent probe for the detection of myeloid leukemia cells for non-diagnostic and therapeutic purposes, characterized in that: The myeloid leukemia cells are incubated with the fluorescent probe in dark, and then H2O2 is added for further incubation, and then the images are taken by laser confocal microscope, and the myeloid leukemia cells emit green fluorescence, The fluorescent probe has the following structural formula: ， R 1 is methyl, R 2 is hydrogen, R 3 is fluorine.

2. Use of the fluorescent probe according to claim 1 for the detection of myeloid leukemia cells for non-disease diagnostic and therapeutic purposes, characterized in that, The preparation method of the fluorescent probe comprises the following steps: (1) the compound shown in formula I is reacted with p-fluoroniobenzene in a N,N-dimethylformamide solution containing cesium carbonate to synthesize the compound shown in formula II; (2) the compound shown in formula II is reacted with sodium borohydride in a reaction solvent, and the reaction solvent is a mixed solvent of methanol and dichloromethane; (3) the compound generated in step (2) is reacted with a halogen-containing compound to obtain the compound shown in formula III; (4) the compound shown in formula III is reacted with stannous chloride in a solution; Formula I: ; Formula II: ; Formula III: .

3. Use of the fluorescent probe according to claim 2 for the detection of myeloid leukemia cells for non-disease diagnostic and therapeutic purposes, characterized by: In step (1), the molar ratio of the compound shown in formula I to p-fluoroniobenzene is 1:4-6, the reaction system temperature is 80-120 DEG C, and the reaction time is 4-6 h.

4. The use of the fluorescent probe according to claim 2 for the detection of myeloid leukemia cells for non-disease diagnostic and therapeutic purposes, characterized by: In step (2), the molar ratio of the compound shown in formula II to sodium borohydride is 4-5:1, the volume ratio of methanol to dichloromethane is 1:2-4, the reaction temperature is 10-40 DEG C, and the reaction time is 15-45 min.

5. Use of the fluorescent probe according to claim 2 for the detection of myeloid leukemia cells for non-disease diagnostic and therapeutic purposes, characterized by: In step (3), the halogen-containing compound is diethylamine trifluoride, the compound generated in step (2) is reacted with diethylamine trifluoride according to a molar ratio of 1:0.8-1.2, the reaction solvent is dichloromethane, the reaction system temperature is -40-0 DEG C, and the reaction time is 1-3 h.

6. The use of the fluorescent probe according to claim 2 for the detection of myeloid leukemia cells for non-disease diagnostic and therapeutic purposes, characterized by: In step (4), the molar ratio of the compound shown in formula III to stannous chloride is 1:12-18, the solution is methanol: concentrated hydrochloric acid: water = 3:1.5-2.5:2.5-3.5, the reaction temperature is 10-40 DEG C, and the reaction time is 10-14 h.

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