Synthesis and application of a bifunctional fluorescent probe for simultaneous detection of homocysteine and viscosity

Abstract

The application discloses a bifunctional fluorescent probe for simultaneously detecting homocysteine and viscosity, and a chemical structural formula of the bifunctional fluorescent probe is as follows: The bifunctional fluorescent probe combines coumarin derivatives and indole derivatives through piperazine, recognizes homocysteine through double binding sites, and detects viscosity through a molecular rotor strategy. When reacting with homocysteine, the probe emits near-infrared fluorescence of 654 nm under an excitation wavelength of 575 nm; when reacting with viscosity, the probe emits near-infrared fluorescence of 726 nm under an excitation wavelength of 650 nm. The probe has good selectivity for detecting homocysteine and viscosity, rapid response and the like, and has a huge application prospect in technical fields such as analytical chemistry, life science and biological medicine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of analytical chemistry, and particularly relates to synthesis and application of a bifunctional fluorescent probe for simultaneously distinguishing homocysteine and viscosity by imaging. The probe combines two fluorophores through piperazine and can rapidly and selectively detect homocysteine and viscosity from various bioactive substances. The red fluorescence channel selectively detects homocysteine, and the near-infrared fluorescence channel selectively detects viscosity. The probe has the advantages of large Stokes shift, high detection sensitivity and visual detection. BACKGROUND

[0002] Homocysteine ​​(Hcy) is a sulfur-containing amino acid and a key intermediate in methionine metabolism. It is cytotoxic. Normally, the total Hcy concentration in human serum is 5–15 µM (Diabetes and Vascular Disease Research 2007, 4, 143-149). Excessive Hcy levels can generate large amounts of reactive oxygen species (ROS), which directly damage the vascular endothelium, promote the formation of oxidized low-density lipoprotein (OXLDL), and platelet aggregation, thereby contributing to the development of atherosclerosis and becoming a risk factor for cardiovascular and cerebrovascular diseases (New England Journal of Medicine 2002, 346, 476-483). Hcy is also a neurotoxic sulfur-containing amino acid that can induce DNA strand breaks, amyloid protein aggregation, oxidative stress, and apoptosis (Cell Death & Disease 2016, 7, e2513-e2513). Clinical studies have shown its accumulation in various neurological diseases. Elevated Hcy concentration is closely associated with various diseases and is the most accurate indicator of health. However, its pathogenic mechanisms are not fully understood (International Journal of Molecular Sciences 2016, 17, 1733). This may be due to a lack of understanding of the substances that Hcy associates with. Viscosity, a key parameter of the cellular microenvironment, is crucial for the transport of nutrients and metabolites, intracellular and intercellular signaling, interactions between biomacromolecules, and other physiological processes (Biochem. Pharmacol., 1995, 49, 1589). When cells undergo pathological changes and apoptosis, the intracellular microenvironment undergoes changes, leading to intermolecular coupling of biomacromolecules and increased intracellular viscosity (J. Am. Chem. Soc., 2019, 141, 18301; Chem. Commun., 2021, 57, 3508). Abnormal changes in intracellular viscosity are closely linked to cancer, atherosclerosis, diabetes, Alzheimer's disease, and other diseases.

[0003] Currently, various methods have been developed to detect Hcy and viscosity, such as liquid chromatography-mass spectrometry (LCMS), high-performance liquid chromatography, gas chromatography-mass spectrometry (GCMS), and fluorescence spectroscopy. Among these detection methods, fluorescence probe analysis is universally favored due to its ability to respond quickly and have high sensitivity, as well as spatial resolution and satisfactory biocompatibility (Angew. Chem. Int. Ed. 2017, 56, 16611-16615; Anal. Chem. 2016, 76, 166-181). Many fluorescence probes for detecting Hcy / viscosity have been reported, and most of them detect only one of them individually (Angew. Chem. Int. Ed. 2018, 57 , 4991; Analyst, 2022, 147, 2470). And homocysteine and cysteine are similar in structure, and it is difficult to distinguish them, so it is particularly difficult to detect homocysteine and viscosity at the same time without the interference of other substances, which is one of the current research difficulties. SUMMARY

[0004] In view of the above, in order to overcome some shortcomings of the prior art, the purpose of the present application is to provide a bifunctional fluorescence probe for simultaneously distinguishing homocysteine and viscosity. The probe can rapidly and selectively detect homocysteine and viscosity from various bioactive substances under specific detection conditions.

[0005] The purpose of the present application is also to provide a synthesis and application method of the above-mentioned bifunctional fluorescence probe, which has simple preparation method, high sensitivity, low detection limit and low cost.

[0006] The specific technical scheme adopted by the present application to solve the problem is a bifunctional fluorescence probe for simultaneously distinguishing homocysteine and viscosity, and a device for quantitatively analyzing homocysteine and viscosity in the environment and simultaneously distinguishing and imaging homocysteine and viscosity in living cells. The chemical structure of the probe is as follows:

[0007] .

[0008] A synthesis of a bifunctional fluorescence probe for simultaneously distinguishing homocysteine and viscosity, characterized in that the preparation method of the bifunctional fluorescence probe comprises the following steps:

[0009] Step 1. Synthesis of 5-5-(4-(tert-butoxycarbonyl)piperazine-l-carbonyl)-2-((lE,3E)-4-(4-(dimethylamino)phenyl)buta-l,3-dien-l-yl)-l-ethyl-3,3-dimethyl-3H-indol-l-ium 4-(tert-butoxycarbonyl)piperazine-l-carbonyl)-2-((lE,3E)-4-(4-(dimethylamino)phenyl)buta-l,3-dien-l-yl)-l-ethyl-3,3-dimethyl-3H-indol-l-ium iodide

[0010] To 5-carboxy-2-((lE,3E)-4-(4-(dimethylamino)phenyl)buta-l,3-dien-l-yl)-l-ethyl-3,3-dimethyl-3H-indol-l-ium iodide in dry dichloromethane was added 4-dimethylaminopyridine (DMAP) and stirred for 5 min, followed by the addition of l-BOC-piperazine and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI). The reaction was stirred at room temperature overnight. After the reaction was complete, the product was purified by column chromatography to give 5-5-(4-(tert-butoxycarbonyl)piperazine-l-carbonyl)-2-((lE,3E)-4-(4-(dimethylamino)phenyl)buta-l,3-dien-l-yl)-l-ethyl-3,3-dimethyl-3H-indol-l-ium 4-(tert-butoxycarbonyl)piperazine-l-carbonyl)-2-((lE,3E)-4-(4-(dimethylamino)phenyl)buta-l,3-dien-l-yl)-l-ethyl-3,3-dimethyl-3H-indol-l-ium iodide;

[0011] Step 2. Synthesis of 2-((lE,3E)-4-(4-(dimethylamino)phenyl)buta-l,3-dien-l-yl)-l-ethyl-3,3-dimethyl-5-(piperazin-l-yl-l-carbonyl)-3H-indol-l-ium 2,2,2-trifluoroacetate iodide

[0012] 5-(4-(tert-Butoxycarbonyl)piperazine-1-carbonyl)-2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-hydrogen iodide 4-(tert-Butoxycarbonyl)piperazine-1-carbonyl)-2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-hydrogen iodide Methyl-3H-indole-1-hydroiodide was added to anhydrous dichloromethane, and then trifluoroacetic acid was added, and the mixture was stirred at room temperature overnight. After the reaction was complete, the reaction system was spin-dried and purified by column chromatography to obtain 2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-5-(piperazin-1-yl-1-carbonyl)-3H-indol-1-yl 2,2,2-trifluoroacetate iodide;

[0013] Step 3. Synthesis ( E )-2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazole-6-carboxylic acid

[0014] 4-(Butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde and 2-(cyanomethyl)benzo[d]thiazole-6-carboxylic acid were added to anhydrous ethanol and stirred at 50°C for 24 hours. After the reaction was complete, the reaction system was dried and purified by column chromatography to obtain ( E )-2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazole-6-carboxylic acid;

[0015] Step 4. Synthesis of the bifunctional fluorescent probe

[0016] Will( E )-2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazole-6-carboxylic acid was added to anhydrous dichloromethane, and then 4-dimethylaminopyridine (DMAP) was added. The reaction was carried out at room temperature for 5 minutes. Subsequently, 2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-5-(piperazin-1-yl-1-carbonyl)-3H-indol-1-yl 2,2,2-trifluoroacetate iodide was added and stirred for 5 minutes. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added and stirred at room temperature overnight. After the reaction was completed, the reaction system was dried by rotary evaporation and purified by column chromatography to obtain the bifunctional fluorescent probe.

[0017] The use method of the bifunctional fluorescent probe for simultaneously distinguishing homocysteine and viscosity according to the application: without special instructions, the bifunctional probe is usually dissolved in dimethyl sulfoxide (DMSO) at room temperature, and is used for analysis and detection in an environment with a volume ratio of organic phase to water phase of 5:5, the organic phase is dimethyl sulfoxide (DMSO), and the water phase is a phosphate buffer solution (PBS) with pH = 7.4.

[0018] The specific features of the bifunctional fluorescent probe for simultaneously distinguishing homocysteine and viscosity according to the application are as follows: the bifunctional fluorescent probe is dissolved in dimethyl sulfoxide (DMSO), and after the bifunctional probe is dissolved in an organic phase and a water phase (5:5, v / v) solution and reacts with homocysteine for 30 minutes, red fluorescence with a wavelength of 654 nm is emitted under excitation with a wavelength of 575 nm; under the action of glycerol-methanol (V:V = 5:5), near-infrared fluorescence with a wavelength of 720 nm is emitted under excitation with a wavelength of 650 nm. Therefore, specific analytes can be detected by using specific excitation and fluorescence emission signals, and when both of the two substances exist, the two substances can also be well distinguished by using different excitation and fluorescence emission signals. The bifunctional fluorescent probe realizes simultaneous distinguishing detection of homocysteine and viscosity under different detection conditions, has no obvious response to other active oxygen, active sulfur, common amino acids, metal ions and active nitrogen, and the detection limits of homocysteine and viscosity are as low as 249.6 μM and 0.14 cP, respectively. Therefore, the bifunctional fluorescent probe disclosed by the application can realize high-sensitivity distinguishing and quantitative detection of the two substances. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the bifunctional fluorescent probe according to the application.

[0020] Figure 2 The ultraviolet and fluorescence spectra of the bifunctional fluorescent probe according to the application in response to homocysteine and viscosity.

[0021] Figure 3 The fluorescence quantitative analysis diagram of the bifunctional fluorescent probe according to the application in response to homocysteine and viscosity.

[0022] Figure 4 The bifunctional fluorescent probe according to the application simultaneously realizes double-channel imaging of endogenous homocysteine and viscosity in cells. EMBODIMENT

[0023] The bifunctional fluorescent probe according to the application is further described in combination with the following drawings.

[0024] The synthesis route of the bifunctional fluorescent probe according to the application is shown in the following figure:

[0025]

[0026] Example 1. Synthesis of 5-5-(4-(tert-butoxycarbonyl)piperazine-l-carbonyl)-2-((lE,3E)-4-(4-(dimethylamino)phenyl)buta-l,3-dien-l-yl)-l-ethyl-3,3-dimethyl-3H-indol-l-ium 4-(tert-butoxycarbonyl)piperazine-l-carbonyl)-2-((lE,3E)-4-(4-(dimethylamino)phenyl)buta-l,3-dien-l-yl)-l-ethyl-3,3-dimethyl-3H-indol-l-ium iodide

[0027] To 300.0 mg (580.92 µmol) of 5-carboxy-2-((lE,3E)-4-(4-(dimethylamino)phenyl)buta-l,3-dien-l-yl)-l-ethyl-3,3-dimethyl-3H-indol-l-ium iodide was added 8 mL of anhydrous dichloromethane, followed by 10 mg of dimethylaminopyridine (DMAP), stirred for 5 min, then 82.7 mg (580.92 µmol) of 1-BOC-piperazine and 222.7 mg (1.16 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) were added, and the reaction was stirred at room temperature overnight. After the reaction was completed, column chromatography was used for separation and purification to obtain 5-5-(4-(tert-butoxycarbonyl)piperazine-l-carbonyl)-2-((lE,3E)-4-(4-(dimethylamino)phenyl)buta-l,3-dien-l-yl)-l-ethyl-3,3-dimethyl-3H-indol-l-ium 4-(tert-butoxycarbonyl)piperazine-l-carbonyl)-2-((lE,3E)-4-(4-(dimethylamino)phenyl)buta-l,3-dien-l-yl)-l-ethyl-3,3-dimethyl-3H-indol-l-ium iodide 230.0 mg, yield 57.83%.

[0028] Example 2. Synthesis of 2-((lE,3E)-4-(4-(dimethylamino)phenyl)buta-l,3-dien-l-yl)-l-ethyl-3,3-dimethyl-5-(piperazin-l-yl-l-carbonyl)-3H-indol-l-ium 2,2,2-trifluoroacetate iodide

[0029] To 200.0 mg (292.11 µmol) 5-5-(4-(tert-butoxycarbonyl)piperazine-1-carbonyl)-2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium 4-(tert-butoxycarbonyl)piperazine-1-carbonyl)-2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium iodide was added 8 mL of anhydrous dichloromethane, then 1 mL of trifluoroacetic acid was added, and it was stirred at room temperature overnight. After the reaction was completed, the reaction system was rotary evaporated, and column chromatography was used for separation and purification to obtain 2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-5-(piperazin-1-yl-1-carbonyl)-3H-indol-1-yl 2,2,2-trifluoroacetate iodide 198.1 mg, with a yield of 97.03%.

[0030] Example 3. Synthesis of E ) -2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazole-6-carboxylic acid

[0031] To 100.0 mg (264.24 µmol) 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-formaldehyde and 57.7 mg (264.24 µmol) 2-(cyanomethyl)benzo[d]thiazole-6-carboxylic acid were added 6 mL of anhydrous ethanol, and the reaction was stirred at 50°C for 24 hours. After the reaction was completed, the reaction system was rotary evaporated, and column chromatography was used for separation and purification to obtain E ) -2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazole-6-carboxylic acid 120.0 mg, with a yield of 78.48%.

[0032] Example 4. Synthesis of the bifunctional fluorescent probe

[0033] To 90.0 mg (155.53 µmol) ( E)-2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazole-6-carboxylic acid was added to 6 mL of anhydrous dichloromethane, and then 10 mg of 4-dimethylaminopyridine (DMAP) was added. The mixture was reacted at room temperature for 5 min. Then, 110.0 mg (155.53 μmol) of 2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-5-(piperazin-1-yl-1-carbonyl)-3H-indol-1-yl 2,2,2-trifluoroacetate iodide was added and stirred for 5 min. Then, 56.3 mg (311.06 μmol) of 2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-5-(piperazin-1-yl-1-carbonyl)-3H-indol-1-yl 2,2,2-trifluoroacetate iodide was added. The reaction mixture was stirred at room temperature overnight. After the reaction was complete, the reaction system was dried and purified by column chromatography to obtain 90.0 mg of the bifunctional fluorescent probe with a yield of 50.53%.

[0034] Example 5. Dual-functional fluorescent probe for distinguishing and detecting homocysteine ​​and viscosity in vitro

[0035] The spectral properties of the bifunctional fluorescent probe described in this invention were tested by dissolving the bifunctional probe in dimethyl sulfoxide (DMSO) to prepare a 1 mM probe solution with a 10 mM homocysteine ​​concentration and a viscosity of 55 cP. The specific testing method involved taking 20 μL of the 1 mM probe solution, adding 20 μL of a 10 mM analyte solution, and finally adding 980 μL of analytical-grade DMSO and 980 μL of PBS. For all tests, the organic phase to aqueous phase volume ratio was maintained at 5:5 (the total volume of each test sample was 2 mL). For example, to measure the fluorescence intensity of homocysteine ​​at a concentration of 100 μM, the sample preparation is as follows: 20 μL of a 1 mM probe solution, 20 μL of a 10 mM homocysteine ​​aqueous solution, 980 μL of analytical-grade DMSO, and 980 μL of PBS buffer are added to a 2 mL sample tube. After shaking at room temperature for 30 minutes, the fluorescence emission intensity can be measured at an excitation wavelength of 575 nm. Other test procedures are similar to those described above. This dual-function probe enables the differentiated detection of two bioactive substances, homocysteine ​​and viscosity, using different excitation wavelengths and fluorescence emission signals. It exhibits high sensitivity, with detection limits as low as 249.6 μM and 0.14 cP, respectively, making it ideal for imaging and quantitative analysis of endogenous homocysteine ​​and viscosity in living cells.

[0036] Example 6. Dual-channel fluorescence imaging analysis of endogenous homocysteine ​​and viscosity in HepG2 (liver cancer cell) cells

[0037] HepG2 cells were passaged into confocal dish cell culture medium, after being cultured under standard growth conditions for 24 hours, an appropriate amount of probe (5 μM) was added to continue to be cultured under standard growth conditions for 30 minutes, and then photographed under a confocal fluorescence microscope, and imaged by using a near-infrared channel of 590-670 nm and a near-infrared fluorescence channel of 700-750 nm, respectively. The homocysteine and viscosity endogenous in HepG2 cells, the fluorescent probe of the present application can emit near-infrared fluorescence of different wavelengths in cells, which indicates that the probe can detect homocysteine and viscosity in cells, and successfully realizes the dual-channel fluorescence imaging analysis of homocysteine and viscosity endogenous in cells.

[0038] The present application provides a bifunctional fluorescent probe for simultaneously distinguishing homocysteine and viscosity, which connects coumarin derivatives and indole derivatives through piperazine, and recognizes homocysteine through double binding sites design, and detects viscosity through a molecular rotor strategy. When reacting with homocysteine, it emits near-infrared fluorescence of 654 nm under an excitation wavelength of 575 nm; when reacting with viscosity, it emits near-infrared fluorescence of 726 nm under an excitation wavelength of 650 nm, and the fluorescence phenomenon is obvious. Moreover, the product after reaction has good water solubility, fast response speed, large Stokes shift and other advantages. It has great practical application value in the fields of biochemistry, analysis and detection. Although the content of the present application has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present application. After reading the above content, various modifications and substitutions of the present application will be obvious to those skilled in the art. Therefore, the fluorescent probes similar to the technical features described herein all fall within the protection scope of the present patent.

Claims

1. A dual-function fluorescent probe for simultaneous detection of homocysteine ​​and viscosity, characterized in that: The chemical structure of the dual-functional fluorescent probe is shown in (1):

2. The synthesis of the dual-function fluorescent probe according to claim 1, wherein The synthesis method of the dual-function fluorescent probe comprises the following steps: Step 1. Synthesis of 5-(4-(tert-Butoxycarbonyl)piperazine-1-carbonyl)-2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium iodide 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-ium iodide was added to anhydrous dichloromethane, and then 4-dimethylaminopyridine (DMAP) was added, and the mixture was stirred for 5 minutes. Subsequently, 1-BOC-piperazine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) were added, and the mixture was stirred at room temperature overnight. After the reaction was complete, 5-(4-(tert-butoxycarbonyl)piperazine-1-carbonyl)-2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-ium iodide was obtained by separation and purification by column chromatography; Step 2. Synthesis of 2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-5-(piperazin-1-yl-1-carbonyl)-3H-indol-1-yl 2,2,2-trifluoroacetate iodide. 2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-5-(piperazin-1-yl-1-carbonyl)-3H-indol-1-yl 2,2,2-trifluoroacetate iodide was obtained; Step 3. Synthesis of (E)-2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazole-6-carboxylic acid 4-(Butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde and 2-(cyanomethyl)benzo[d]thiazole-6-carboxylic acid were added to anhydrous ethanol, and the mixture was stirred at 50° C. for 24 hours. After the reaction was complete, the reaction system was spin-dried, and the reaction mixture was separated and purified by column chromatography to obtain (E)-2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-yl)-1-cyanovinyl)benzo[d]thiazole-6-carboxylic acid; Step 4. Synthesis of the bifunctional fluorescent probe (E)-2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazole-6-carboxylic acid is added to anhydrous dichloromethane, and then 4-dimethylaminopyridine (DMAP) is added, and the reaction is carried out at room temperature for 5 minutes. Subsequently, 2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-5-(piperazin-1-yl-1-carbonyl)-3H-indol-1-yl 2,2,2-trifluoroacetate iodide is added and stirred for 5 minutes. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added, and the mixture is stirred at room temperature overnight. After the reaction is complete, the reaction system is dried by rotary evaporation, and the bifunctional fluorescent probe is purified and separated by column chromatography.

3. The method for synthesizing a bifunctional probe according to claim 2, wherein: The molar ratio of (E)-2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazole-6-carboxylic acid and 2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-5-(piperazin-1-yl-1-carbonyl)-3H-indol-1-yl 2,2,2-trifluoroacetate iodide in step 4 is 1:

1.

4. Use of the dual-function fluorescent probe according to claim 1 for preparing a device, characterized in that: The fabricated device is capable of quantitatively analyzing homocysteine ​​and viscosity in the environment, as well as simultaneously differentiating and imaging homocysteine ​​and viscosity in cells, tissues, and living bodies.

Images