Fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate as well as preparation method, use method and application of fluorescent probe

Abstract

The invention provides a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate as well as a preparation method, a use method and application of the fluorescent probe, and relates to the field of analytical chemistry. The invention discloses a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate. The structural formula of the fluorescent probe is shown in the specification. The fluorescent probe provided by the invention is good in selectivity, high in sensitivity and strong in anti-interference performance, and can be used for directly and simultaneously quantifying Cys, Hcy, GSH and ATP in an environment and simultaneously distinguishing Cys, Hcy, GSH and ATP in biological systems such as fluorescence imaging tumor cells.

Description

Technical Field

[0001] This application relates to the field of analytical chemistry, and in particular to a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate, as well as its preparation method, usage method and application. Background Technology

[0002] Cysteine ​​(Cys), homocysteine ​​(Hcy), and reduced glutathione (GSH) are common biothiols in organisms, playing important roles in various physiological and pathological processes. Cys, a precursor to GSH, is involved in many aspects of protein synthesis, detoxification, and metabolism. Hcy has been identified as an independent risk factor for many diseases. Even small increases in Hcy (above 15 μM) can lead to severe hyperhomocysteinemia. As the most abundant intracellular thiol, GSH plays a central role in combating oxidative stress and maintaining redox homeostasis. Abnormal levels of these three biothiols are associated with various diseases, such as cancer, HIV / AIDS, cardiovascular disease, liver damage, and neurodegenerative diseases (Anal. Chim. Acta., 2019, 1074, 123–130). Adenosine triphosphate (ATP) is considered the "currency unit" of energy transfer within living cells; it transports chemical energy for metabolism. Fundamental life activities involving ATP production or consumption include protein transport, regulation of ion channels, and activation of signaling cascades. Cellular, tissue, and organ systems maintain a constant cellular ATP concentration during extensive use; this is known as ATP homeostasis. For example, a lack of ATP levels may indicate disease states such as ischemia, cancer, Parkinson's disease, and hypoglycemia (Chem. Commun., 2012, 48, 3206–3208). The roles, levels, localization, distribution, and biological functions of the four substances Cys, Hcy, GSH, and ATP in tumor cells are highly complex. Therefore, the simultaneous detection of Cys, Hcy, GSH, and ATP is of great significance in the pathological analysis and early diagnosis of tumors.

[0003] In recent years, fluorescent probes have attracted widespread attention due to their high selectivity, sensitivity, ease of operation, and ability to monitor and visualize biomolecules in living cells. To date, some fluorescent probes have been reported for the selective detection of Cys / Hcy / GSH / ATP, and others for the simultaneous detection of two of these three. However, no fluorescent probe has yet achieved the simultaneous differentiation and detection of Cys, Hcy, GSH, and ATP, failing to meet the application requirements for the simultaneous differentiation, detection, and imaging of endogenous Cys, Hcy, GSH, and ATP in biological systems such as tumor cells. Summary of the Invention

[0004] The purpose of this application is to provide a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate, as well as its preparation method, usage method, and application, in order to solve the above-mentioned problems.

[0005] To achieve the above objectives, this application adopts the following technical solution: A fluorescent probe that can simultaneously distinguish and detect the tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate has the following structural formula: .

[0006] This application also provides a method for preparing a fluorescent probe that can simultaneously distinguish and detect the tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate, comprising: 4-((4-(butyrothio)-3-formyl-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid and 2-(benzo[d]thiazo-2-yl)acetonitrile were added to anhydrous ethanol to carry out the first reaction. After the reaction was completed, the reaction solution was concentrated and purified by column chromatography to obtain a red solid product. Under a protective atmosphere, (E)-4-((3-(2-(benzo[d]thiazo-2-yl)-2-cyano)-4-(butyro)-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid and 4-dimethylaminopyridine were added to anhydrous dichloromethane for a second reaction. Then, 2-(2-((2-aminoethyl)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthine]-3-one and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added for a third reaction. After the reaction was complete, water and dichloromethane were added to the reaction solution, the mixture was separated, the organic layer was concentrated, and purified by column chromatography to obtain the fluorescent probe that simultaneously distinguishes and detects tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate.

[0007] Preferably, the temperature of the first reaction is 50-90℃ and the time is 12-24h.

[0008] Preferably, the temperature of the second reaction is room temperature, and the reaction time is 10-30 min.

[0009] Preferably, the temperature of the third reaction is room temperature, and the reaction time is 4-12 hours.

[0010] Preferably, the first reaction, the second reaction, and the third reaction are all carried out under stirring.

[0011] Preferably, the protective atmosphere includes argon.

[0012] This application also provides a method for using the fluorescent probe described above for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate, including: The fluorescent probe that can simultaneously distinguish and detect tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate is dissolved in an organic phase and an aqueous phase mixture, and then detected under specific wavelength conditions after the reaction. Among them, after reacting with cysteine, it emits blue fluorescence at an excitation wavelength of 455 nm at 375 nm; after reacting with homocysteine, it emits red fluorescence at an excitation wavelength of 555 nm at 493 nm; after reacting with glutathione, it emits green fluorescence at an excitation wavelength of 529 nm at 455 nm; and after reacting with adenosine triphosphate, it emits pink fluorescence at an excitation wavelength of 587 nm at 520 nm.

[0013] Preferably, when detecting cysteine, homocysteine, and glutathione, the organic phase is dimethyl sulfoxide, the aqueous phase is a phosphate buffer solution with pH = 7.4, and the aqueous solution of the analyte is used. When detecting adenosine triphosphate (ATP), the organic phase is methanol, the aqueous phase is a phosphate buffer solution with pH 7.4, and the analyte is an aqueous solution.

[0014] This application also provides an application of the fluorescent probe described above for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate, for the quantitative detection of tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate.

[0015] Compared with the prior art, the beneficial effects of this application include: This application provides a fluorescent probe for the simultaneous detection of tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate (ATP). It possesses multiple active reaction sites and, based on different reaction mechanisms, can simultaneously distinguish and detect Cys (λex / λem = 375 / 455 nm), Hcy (λex / λem = 493 / 555 nm), GSH (λex / λem = 455 / 529 nm), and ATP (λex / λem = 520 / 587 nm). It exhibits good selectivity, high sensitivity, and strong anti-interference properties, and can be used for the direct and simultaneous quantification of Cys, Hcy, GSH, and ATP in the environment, as well as for the simultaneous differentiation of Cys, Hcy, GSH, and ATP in biological systems such as tumor cells during fluorescence imaging.

[0016] This application provides a simple method for preparing a fluorescent probe that can simultaneously distinguish and detect tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation on the scope of this application.

[0018] Figure 1 The hydrogen spectrum of compound 2-1 obtained in Example 1; Figure 2 The carbon spectrum of compound 2-1 obtained in Example 2; Figure 3 The hydrogen spectrum of compound 2-2 obtained in the example; Figure 4 The carbon spectrum of compound 2-2 obtained in the example; Figure 5 The hydrogen spectroscopy spectra of compounds 2-3 obtained in Examples 2-3 are shown below. Figure 6 The carbon spectra of compounds 2-3 obtained in Examples 2-3 are shown below. Figure 7 The hydrogen spectroscopy spectra of compounds 2-4 obtained in Examples 2-4 are shown below. Figure 8 The carbon spectra of compounds 2-4 obtained in the examples are shown below; Figure 9 The 1H NMR spectrum of (E)-4-((3-(2-(benzo[d]thiazo-2-yl)-2-cyano)-4-(butyrothio)-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid obtained in the examples; Figure 10 The carbon spectrum of (E)-4-((3-(2-(benzo[d]thiazo-2-yl)-2-cyano)-4-(butyrothio)-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid obtained in the examples; Figure 11 This shows the change in fluorescence emission intensity of the fluorescent probe with Cys concentration in the quantitative detection of Cys. Figure 12 This shows the change in fluorescence emission intensity of the fluorescent probe with Hcy concentration in the quantitative detection of Hcy. Figure 13 This shows the change in fluorescence emission intensity of the fluorescent probe with the concentration of GSH in the quantitative detection of GSH. Figure 14 This shows the change in fluorescence emission intensity of a fluorescent probe with ATP concentration in the quantitative detection of ATP. Figure 15 Images showing how fluorescent probes can simultaneously distinguish and image endogenous Cys, Hcy, GSH, and ATP in HeLa tumor cells. Detailed Implementation

[0019] The implementation schemes of this application will be described in detail below with reference to specific embodiments. However, those skilled in the art will understand that the following embodiments are only for illustrating this application and should not be regarded as limiting the scope of this application. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used without specified manufacturers are all conventional products that can be purchased commercially. Example 1

[0020] This embodiment provides a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate (ATP). Its preparation method is as follows: 1. Synthesis of (E)-4-((3-(2-(benzo[d]thiazo-2-yl)-2-cyano)-4-(butyrothio)-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid: First, 4-((4-(butthio)-3-formyl-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid was synthesized, as shown in the following equation:

[0021]

[0022] The preparation method is as follows: Synthesis of compound 2-1: 3.01 g (21.93 mmol) of compound 1 (prepared in reference

[127] ) was dissolved in 50 mL of anhydrous acetonitrile, and 5.87 g (26.31 mmol) of tert-butyl 4-bromobutyrate, 3.04 g (23.02 mmol) of diammonium hydrogen phosphate and 1.82 g (10.96 mmol) of potassium iodide were added. The reaction mixture was stirred overnight at 95 ºC. After the reaction was completed, the mixture was concentrated and purified by column chromatography to obtain a yellow oily compound 2-1 (3.34 g, 54.52%).

[0023] The proton NMR spectrum of the obtained compound 2-1 is as follows: Figure 1 As shown, the carbon spectrum is as follows Figure 2 As shown.

[0024] Synthesis of compound 2-2: 3.34 g (11.96 mmol) of compound 2-1 was dissolved in 30 mL of anhydrous toluene solution, and then 5.53 g (11.96 mmol) of bis(2,4,6-trichlorophenyl)malonate was added. The reaction mixture was refluxed at 110 °C and stirred overnight. After the reaction was complete, the mixture was cooled to room temperature, concentrated, and purified by column chromatography to give compound 2-2 (2.34 g, 56.34%) as a yellow solid.

[0025] The proton NMR spectrum of the obtained compound 2-2 is shown below. Figure 3As shown, the carbon spectrum is as follows Figure 4 As shown.

[0026] Synthesis of Compound 2-3: Under argon protection and an ice bath, anhydrous DMF solution (3 mL) was slowly added to POCl3 solution (2 mL) via a constant-pressure dropping funnel. The mixture was stirred at room temperature for 30 minutes to obtain an orange-red precursor solution. 2.34 g (6.74 mmol) of compound 2-2 was dissolved in 10 mL of dry DMF and then added dropwise to the above mixture. The mixture was stirred overnight at 80 °C under argon protection. After the reaction was complete, it was quenched with 200 mL of ice water. The pH was adjusted to 6-7 using 20% ​​sodium hydroxide solution. The mixture was extracted three times successively with EA and saturated brine. After drying and concentration, the extract was purified by column chromatography to obtain a yellow solid compound 2-3 (1.56 g, 68.57%).

[0027] The proton NMR spectra of compounds 2-3 are as follows: Figure 5 As shown, the carbon spectrum is as follows Figure 6 As shown.

[0028] Synthesis of compounds 2-4: 1.56 g (4.62 mmol) of compound 2-3 was dissolved in 20 mL of anhydrous dichloromethane. After stirring the reaction mixture for 2 minutes at room temperature, 701.1 mg (6.93 mmol) of triethylamine and 499.8 mg (5.54 mmol) of n-butanethiol were added, and the reaction was carried out at room temperature in the dark for 8 hours. After the reaction was completed by TLC monitoring, the solvent was removed by concentration under reduced pressure. The crude product was purified by column chromatography to give a brownish-red oily compound 2-4 (1.01 g, 55.86%).

[0029] The proton NMR spectra of compounds 2-4 are as follows: Figure 7 As shown, the carbon spectrum is as follows Figure 8 As shown.

[0030] Sufficient amounts of 4-((4-(butthio)-3-formyl-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid were prepared multiple times for subsequent experiments.

[0031] (1.04 g, 2.66 mmol) 4-((4-(butyrothio)-3-formyl-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid and (462.83 mg, 2.66 mmol) 2-(benzo[d]thiazo-2-yl)acetonitrile were added to 10 mL of anhydrous ethanol and stirred at 50 °C for 24 hours. After the reaction was complete, the reaction solution was concentrated and purified by column chromatography to give 1.02 g of red solid product, with a yield of 73.3%. The 1H NMR spectrum of the obtained substance is shown below. Figure 9 As shown, the carbon spectrum is as follows Figure 10 As shown.

[0032] 4-((4-(butyrothio)-3-formyl-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid (200.0 mg, 510.9 μmol) and 2-(benzo[d]thiazo-2-yl)acetonitrile (89.0 mg, 510.9 μmol) were added to 10 mL of anhydrous ethanol and stirred at 50 °C for 24 hours. After the reaction was completed, the reaction solution was concentrated and purified by column chromatography to give 205.0 mg of red solid product, with a yield of 73.3%.

[0033] 2. Synthesis of probe (E)-4-((3-(2-(benzo[d]thiazo-2-yl)-2-cyano)-4-(butyrothio)-2-oxo-2H-chromen-7-yl)(ethyl)amino)-N-(2-((2',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthine]-2-yl)ethyl)amino]ethyl)butyramide: Under argon protection, (E)-4-((3-(2-(benzo[d]thiazo-2-yl)-2-cyano)-4-(butyrothio)-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid (96.0 mg, 175.3 μmol) and 4-dimethylaminopyridine (2.1 mg, 17.53 μmol) were added to 10 mL of anhydrous dichloromethane and stirred at room temperature for 20 minutes. Then, 2-(2-((2-aminoethyl)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthine]-3-one (Reference: Preparation: Turn-on fluorogenic and chromogenic detection of Fe) 3+ and Cr 3+ in completely water medium with polyacrylamide covalently bonding to rhodamine Busing diethylenetriamine as a linker. RSC Adv. 2014, 4(86), 46332-46339) (92.5 mg, 175.3 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (purchased from Aladdin Reagent, CAS No. 1892-57-5) (50.4 mg, 262.9 μmol) were added to the above reaction solution in one step, and the reaction was continued to be stirred under argon protection for 6 hours. After the reaction was completed by TLC monitoring, an appropriate amount of dichloromethane and water were added, the mixture was separated, the organic layer was washed with water 3 times, concentrated to obtain crude product, and purified by column chromatography to obtain 100.0 mg of red solid, with a yield of 53.9%.

[0034] The reaction equation is as follows:

[0035] The above fluorescent probes can be used to simultaneously distinguish and detect Cys, Hcy, GSH, and ATP. Spectroscopic properties of Cys, Hcy, GSH, and ATP detected by fluorescent probes: The probe was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM probe solution. 1 mM aqueous solutions of Cys, Hcy, GSH, and ATP were prepared separately. The specific testing method for Cys, Hcy, GSH, and ATP was as follows: 20 μL of the 1 mM probe solution, 980 μL / 580 μL of analytical grade DMSO / MeOH, the required amount of 1 mM Cys / Hcy / GSH / ATP aqueous solution, and the required amount of PBS buffer solution were placed in a 2 mL sample tube. The final volume ratio of organic phase to aqueous phase was maintained at 7:3 / 5:5 (total volume of each test sample was 2 mL). After shaking and mixing at room temperature for 20 / 30 minutes, the fluorescence emission spectra of Cys, Hcy, GSH, and ATP were measured using excitation wavelengths of 375 nm, 493 nm, 455 nm, and 520 nm, respectively. Other testing procedures were similar to the above steps. The probe has low detection limits for Cys, Hcy, GSH and ATP and high sensitivity.

[0036] Figure 11 The graph shows the change in fluorescence emission intensity of the fluorescent probe of the present invention with the concentration of Cys in quantitative detection. The horizontal axis represents wavelength and the vertical axis represents fluorescence intensity. Figure 12 The graph shows the change in fluorescence emission intensity with Hcy concentration in the quantitative detection of Hcy using the fluorescent probe of the present invention. The horizontal axis represents wavelength, and the vertical axis represents fluorescence intensity. Figure 13 The graph shows the change in fluorescence emission intensity with GSH concentration in the quantitative detection of GSH using the fluorescent probe of the present invention. The horizontal axis represents wavelength, and the vertical axis represents fluorescence intensity. Figure 14The graph shows the change in fluorescence emission intensity with ATP concentration in the quantitative detection of ATP using the fluorescent probe of the present invention. The horizontal axis represents wavelength, and the vertical axis represents fluorescence intensity.

[0037] Application of simultaneously differentiating endogenous Cys, Hcy, GSH, and ATP in HeLa tumor cells during imaging: HeLa cells were passaged into confocal imaging cell culture dishes and cultured under standard growth conditions for 24 hours. Then, a suitable amount of probe (5 μM) was added, and the cells were cultured under standard growth conditions for another 30 minutes. Imaging was then performed under a confocal fluorescence microscope using the blue, red, green, and red fluorescence channels. Figure 15 As can be seen, the fluorescent probe of this invention successfully achieves simultaneous fluorescence imaging analysis of endogenous Cys, Hcy, GSH and ATP in cells, which has great application value in the fields of biochemistry, analytical detection and environmental science.

[0038] The method for synthesizing the fluorescent probe and its application in simultaneously distinguishing and detecting tumor markers Cys, Hcy, GSH and ATP provided in this application has the advantages of high efficiency, simplicity, low cost and high accuracy, and can realize the simultaneous distinguishing detection and imaging analysis of Cys, Hcy, GSH and ATP in environmental and biological samples.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A fluorescent probe for simultaneously distinguishing and detecting the tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate, characterized in that, Its structural formula is: .

2. A method for preparing a fluorescent probe that can simultaneously distinguish and detect the tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate, characterized in that, include: 4-((4-(butyrothio)-3-formyl-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid and 2-(benzo[d]thiazo-2-yl)acetonitrile were added to anhydrous ethanol to carry out the first reaction. After the reaction was completed, the reaction solution was concentrated and purified by column chromatography to obtain a red solid product. Under a protective atmosphere, (E)-4-((3-(2-(benzo[d]thiazo-2-yl)-2-cyano)-4-(butyro)-2-oxo-2H-chromen-7-yl)(ethyl)amino)butyric acid and 4-dimethylaminopyridine were added to anhydrous dichloromethane for a second reaction. Then, 2-(2-((2-aminoethyl)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthine]-3-one and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added for a third reaction. After the reaction was complete, water and dichloromethane were added to the reaction solution, the mixture was separated, the organic layer was concentrated, and purified by column chromatography to obtain the fluorescent probe that simultaneously distinguishes and detects tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate.

3. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate according to claim 2, characterized in that, The temperature of the first reaction is 50-90℃, and the time is 12-24h.

4. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate according to claim 2, characterized in that, The second reaction was carried out at room temperature for 10-30 minutes.

5. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate according to claim 2, characterized in that, The third reaction is carried out at room temperature for 4-12 hours.

6. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate according to claim 2, characterized in that, The first reaction, the second reaction, and the third reaction are all carried out under stirring.

7. The method for preparing a fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate according to any one of claims 2-6, characterized in that, The protective atmosphere includes argon.

8. A method of using the fluorescent probe of claim 1 for simultaneously distinguishing and detecting the tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate, characterized in that, include: The fluorescent probe that can simultaneously distinguish and detect tumor markers cysteine, homocysteine, glutathione and adenosine triphosphate is dissolved in an organic phase and an aqueous phase mixture, and then detected under specific wavelength conditions after the reaction. Among them, after reacting with cysteine, it emits blue fluorescence at an excitation wavelength of 455 nm at 375 nm; after reacting with homocysteine, it emits red fluorescence at an excitation wavelength of 555 nm at 493 nm; after reacting with glutathione, it emits green fluorescence at an excitation wavelength of 529 nm at 455 nm; and after reacting with adenosine triphosphate, it emits pink fluorescence at an excitation wavelength of 587 nm at 520 nm.

9. The method of using the fluorescent probe for simultaneously distinguishing and detecting tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate according to claim 8, characterized in that, When detecting cysteine, homocysteine, and glutathione, the organic phase is dimethyl sulfoxide, the aqueous phase is a phosphate buffer solution with pH = 7.4, and the aqueous solution of the analyte is used. When detecting adenosine triphosphate (ATP), the organic phase is methanol, the aqueous phase is a phosphate buffer solution with pH 7.4, and the analyte is an aqueous solution.

10. The application of the fluorescent probe of claim 1 for simultaneously distinguishing and detecting the tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate, characterized in that, It is used for the quantitative detection of tumor markers cysteine, homocysteine, glutathione, and adenosine triphosphate.

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