A citrate-based fluorescent dye for distinguishing between dead and live cells and a method of preparation and use thereof

Abstract

The present application relates to the technical field of fluorescent dyes, in particular to a citric acid-based fluorescent dye for distinguishing dead cells and living cells, a preparation method and application thereof. The citric acid-based fluorescent dye for distinguishing dead cells and living cells comprises at least one of compound 1 and compound 2; the citric acid-based fluorescent dye can specifically enter dead cells, specifically dye the cytoplasm of dead cells, so that the dead cells can be dyed or imaged. The citric acid-based fluorescent dye is difficult to dye living cells, and further can realize rapid and accurate distinction of dead cells and living cells, and can be applied to the field of biological medicine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescent dye technology, and particularly relates to a citric acid-based fluorescent dye for distinguishing between dead cells and live cells, a preparation method thereof and use thereof. BACKGROUND

[0002] Cell death is a key biological process for maintaining normal functions of the human body. On the one hand, abnormal cell death is often closely related to various diseases, such as Alzheimer's disease, cardiovascular disease, and rheumatoid arthritis. On the other hand, the absence of cell death can also lead to other serious health problems such as cancer. Therefore, it is of great significance in the biomedical field to quickly, sensitively and accurately distinguish between live cells and dead cells, which can effectively promote the development of drug development, disease diagnosis and treatment, and immunology research.

[0003] Currently, several typical methods have been developed for distinguishing between dead cells and live cells, such as atomic force microscopy, Fourier transform infrared spectroscopy, electron microscopy, Raman spectroscopy, and nucleic acid sequence-based amplification technology. However, these techniques are often time-consuming, complex, labor-intensive and costly, which severely limits their practical application in distinguishing between live cells and dead cells. In addition, colorimetric methods are also commonly used to evaluate cell activity. Methods such as CCK-8 and MTT assay, which mainly rely on absorbance changes, are one of the most commonly used cell survival assays. However, this method is usually indirect, tedious and labor-intensive, and may not always be very reliable due to factors such as excessive cell confluence or glucose concentration in the medium.

[0004] Compared with the above methods, fluorescent labeling technology is attracting attention due to its significant advantages such as simple operation, rapid response, high sensitivity, and visual detection. To date, a variety of fluorescent dyes for distinguishing between dead cells and live cells have been developed. Commonly used fluorescent probes on the market, such as propidium iodide (PI) and Sytox Green, can selectively penetrate cells with damaged cell membranes, then bind to intracellular DNA and release strong fluorescence, and then stain the cell nucleus to locate dead cells. However, these commercial dyes have certain limitations, they are relatively expensive, have high toxicity, and have poor light stability. Therefore, the development of new fluorescent dyes with economic, low toxicity and rapid imaging is promising, and is expected to achieve rapid and sensitive differentiation between live cells and dead cells.

[0005] Cytoplasm plays a vital role in cell metabolism, synthesis and transport. As the core of cell metabolism, it can synthesize and decompose various organic substances. At the same time, there are various organelles in the cytoplasm, which can synthesize and process the substances required by cells through synergistic operation. In addition, cytoplasm is also involved in the transport and transport of cell substances, and the mechanism of proteins and cytoskeleton can transfer substances from one location to another. Therefore, cytoplasm imaging helps to understand the distribution and dynamic changes of cytoplasm, which has important significance in blood tests (such as observing the cell morphology of various white blood cells, red blood cells, etc.), and can provide answers to many problems related to cytoplasm.

[0006] Although the prior art has successfully synthesized small molecule fluorescent dyes capable of staining cytoplasm, such as BCECF AM, Cyto Tell, etc. However, most of these dyes are mainly used for staining the cytoplasm of living cells, not the cytoplasm of dead cells. Therefore, it is of great significance to develop small molecule fluorescent dyes that can specifically stain the cytoplasm of dead cells. SUMMARY

[0007] Therefore, the technical problem to be solved by the present application is to provide a citric acid-based fluorescent dye for distinguishing between dead cells and living cells, which can specifically stain the cytoplasm of dead cells, thereby achieving the purpose of quickly and accurately distinguishing between dead cells and living cells. The synthesis method of the fluorescent dye provided by the present application is simple and easy to operate, the raw materials are cheap and easy to obtain, and the preparation cost is low. It is a universal dead cell cytoplasmic dye suitable for various types of cells such as normal cells, cancer cells and immune cells, and has a wide application prospect in cell fluorescence imaging.

[0008] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0009] The present application provides a citric acid-based fluorescent dye for distinguishing between dead cells and living cells, which comprises at least one of compound 1 and compound 2; the structural formula of the compound 1 and the compound 2 is as follows:

[0010] .

[0011] The present application provides a preparation method of the compound 1, comprising the following steps:

[0012] S1, heating citric acid and 1,2-diphenyl ethylenediamine to obtain compound TPA-N-COOH;

[0013] S2, adding potassium carbonate to the compound TPA-N-COOH for stirring reaction, then adding methyl iodide for heating reaction to obtain the compound 1.

[0014] The synthetic route is as follows:

[0015] .

[0016] In some embodiments, in the S1 step, the heating reaction condition is 120-140℃ stirring reaction for 4-8 h; for example, the heating temperature can be any one of 120, 125, 130, 135, 140℃ or a range value between any two of them. For example, the heating time can be any one of 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 h or a range value between any two of them.

[0017] In some embodiments, in the S1 step, the molar ratio of citric acid to 1,2-diphenylethylenediamine is 1:1.

[0018] In some embodiments, in the S1 step, after the heating reaction, a washing, extraction, drying and / or purification step is further included. In some embodiments, after the heating reaction, the temperature is lowered to room temperature, and then dissolved with anhydrous methanol, washed with water, extracted with dichloromethane, dried with anhydrous sodium sulfate and / or purified by column chromatography. In some embodiments, in the column chromatography purification step, the chromatography liquid is dichloromethane (DCM): methanol (MeOH)=8:1, V / V.

[0019] In some embodiments, in the S2 step, the reaction condition of compound TPA-N-COOH and potassium carbonate is 30-40℃ stirring reaction for 20-30 min; for example, the heating temperature can be any one of 30, 35, 40℃ or a range value between any two of them. For example, the heating time can be any one of 20, 25, 30 min or a range value between any two of them.

[0020] In some embodiments, in the S2 step, the heating reaction condition is 30-40℃ stirring reaction for 18-24 h; for example, the heating temperature can be any one of 30, 35, 40℃ or a range value between any two of them. For example, the heating time can be any one of 18, 20, 22, 24 h or a range value between any two of them.

[0021] In some embodiments, in the S2 step, the molar ratio of TPA-N-COOH, potassium carbonate, and iodomethane is 1:1.5:1.05; in some embodiments, the reaction solvent is an organic solvent, such as N,N-dimethylformamide.

[0022] In some embodiments, after the heating reaction in the S2 step, a washing, extraction, drying and / or purification step is further included; in some embodiments, after the heating reaction, water washing, dichloromethane extraction, anhydrous sodium sulfate drying and / or column chromatography separation and purification are performed. In some embodiments, in the column chromatography separation and purification step, the chromatography liquid is petroleum ether (PE): ethyl acetate (EA) = 1:1, V / V.

[0023] The present application provides a preparation method of the compound 2, comprising the following steps:

[0024] S1, citric acid is subjected to a heating reaction with 1,2-diphenylethylenediamine to obtain compound TPA-N-COOH;

[0025] S2, compound TPA-N-COOH is subjected to a stirring reaction with potassium carbonate, and then 1,4-dibromobutane is added to perform a heating reaction to obtain compound TPA-N-COO(CH2)4Br;

[0026] S3, compound TPA-N-COO(CH2)4Br is subjected to a heating reflux reaction with 4-methylpyridine to obtain compound 2;

[0027] The synthesis route is as follows:

[0028] .

[0029] In some embodiments, in the S1 step, the heating reaction is performed at 120-140°C for 4-8 h; for example, the heating temperature can be any one of 120, 125, 130, 135, 140°C or a range value between any two of them. For example, the heating time can be any one of 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 h or a range value between any two of them.

[0030] In some embodiments, in the S1 step, the molar ratio of citric acid to 1,2-diphenylethylenediamine is 1:1.

[0031] In some embodiments, after the heating reaction in the S1 step, a washing, extraction, drying and / or purification step is further included; in some embodiments, after the heating reaction, the temperature is lowered to room temperature, and then anhydrous methanol is used for dissolution, water washing, dichloromethane extraction, anhydrous sodium sulfate drying and / or column chromatography separation and purification are performed. In some embodiments, in the column chromatography separation and purification step, the chromatography liquid is dichloromethane (DCM): methanol (MeOH) = 8:1, V / V.

[0032] In some embodiments, in the S2 step, the reaction conditions of compound TPA-N-COOH and potassium carbonate are stirring at 30-40 °C for 20-30 min; the heating temperature can be any one of 30, 35, 40 °C or a range between any two of them. The heating time can be any one of 20, 25, 30 min or a range between any two of them.

[0033] In some embodiments, in the S2 step, the heating reaction conditions are stirring at 30-40 °C for 18-24 h; the heating temperature can be any one of 30, 35, 40 °C or a range between any two of them. The heating time can be any one of 18, 20, 22, 24 h or a range between any two of them.

[0034] In some embodiments, in the S2 step, the molar ratio of TPA-N-COOH, potassium carbonate, and 1,4-dibromobutane is 1:1.5:1; in some embodiments, the reaction solvent is an organic solvent, such as N,N-dimethylformamide.

[0035] In some embodiments, in the S2 step, after the heating reaction, a washing, extraction, drying, and / or purification step is further included; in some embodiments, after the heating reaction, water washing, dichloromethane extraction, anhydrous sodium sulfate drying, and / or column chromatography separation and purification are performed. In some embodiments, in the column chromatography separation and purification step, the chromatography liquid is petroleum ether (PE): ethyl acetate (EA) = 1:1, V / V.

[0036] In some embodiments, in the S3 step, the heating reflux reaction conditions are refluxing at 85 °C for 12-18 h; the heating time can be any one of 12, 14, 16, 18 h or a range between any two of them.

[0037] In some embodiments, the molar ratio of compound TPA-N-COO(CH2)4Br and 4-methylpyridine is 1:1.5; the reaction solvent is an organic solvent, such as acetonitrile.

[0038] In some embodiments, in the S3 step, after the heating reflux reaction, a washing, extraction, drying, and / or purification step is further included; in some embodiments, after the heating reflux reaction, water washing, dichloromethane extraction, anhydrous sodium sulfate drying, and / or column chromatography separation and purification are performed. In some embodiments, in the column chromatography separation and purification step, the chromatography liquid is dichloromethane (DCM): methanol (MeOH) = 10:1, V / V.

[0039] The citric acid-based fluorescent dye for distinguishing dead cells and living cells, the compound 1 prepared by the preparation method of the compound 1, or the compound 2 prepared by the preparation method of the compound 2 has the advantages that the citric acid-based fluorescent dye can specifically enter dead cells, specifically stains the cytoplasm of the dead cells, and thus can be used for staining or imaging the dead cells.

[0040] In some embodiments, the cells include, but are not limited to, normal cells, cancer cells or immune cells; the normal cells include, but are not limited to, embryonic kidney cells or fibroblasts; the immune cells include, but are not limited to, monocyte macrophages; and the cancer cells include, but are not limited to, cervical cancer cells.

[0041] The technical scheme of the present application has the following advantages:

[0042] 1. A citric acid-based fluorescent dye for distinguishing dead cells and living cells, comprising at least one of compound 1 and compound 2; the citric acid-based fluorescent dye can specifically enter dead cells, specifically stains the cytoplasm of the dead cells, and thus can be used for staining or imaging the dead cells.

[0043] Further, the citric acid-based fluorescent dye specifically stains the cytoplasm of the dead cells, has cell universality, and experiments have verified that it can be used for different types of cell lines, and has the advantage of wide universality.

[0044] Further, the citric acid-based fluorescent dye is safe and non-cytotoxic, has good biocompatibility, and can be widely used in biological systems, such as fluorescence imaging of cells.

[0045] Further, the citric acid-based fluorescent dye can specifically stain the cytoplasm of the dead cells at very low concentration and very short staining time, and cannot stain the living cells at long staining time and high concentration, which shows that the citric acid-based fluorescent dye has the advantage of strong specificity.

[0046] Further, the citric acid-based fluorescent dye and the commercial dead cell dye propidium iodide (PI) are added into the cells at the same time, and the citric acid-based fluorescent dye can well stain the dead cells, and has similar ability to stain the dead cells as the commercial dye. Different from PI which enters the cells and binds to DNA, the staining area of the citric acid dye is mainly concentrated in the cytoplasm. Therefore, the citric acid-based fluorescent dye has good application prospects in cytoplasm-related research and detection.

[0047] 2. The present application also provides a preparation method of the citric acid-based fluorescent dye, which has the advantages of simple synthesis, easy operation, cheap and easily obtained raw materials, and low cost. Attached Figure Description

[0048] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0049] Figure 1 The 1H NMR spectrum of the dye TPA-N-COOH in Example 1;

[0050] Figure 2 The 1H NMR spectrum of the dye TPA-N-Me in Example 1;

[0051] Figure 3 The image shows the carbon NMR spectrum of the dye TPA-N-Me in Example 1.

[0052] Figure 4 This is a high-resolution mass spectrum of the dye TPA-N-Me used in Example 1;

[0053] Figure 5 The 1H NMR spectrum of TPA-N-COO(CH2)4Br in Example 1;

[0054] Figure 6 The 1H NMR spectrum of the dye TPA-N-Py in Example 1;

[0055] Figure 7 The carbon NMR spectrum of the dye TPA-N-Py in Example 1;

[0056] Figure 8 This is a high-resolution mass spectrum of the dye TPA-N-Py in Example 1;

[0057] Figure 9 The fluorescence spectra of dyes TPA-N-Me and TPA-N-Py in water in Example 2 are shown; where (a) is TPA-N-Me and (b) is TPA-N-Py.

[0058] Figure 10 The image shows the HeLa cytotoxicity of dyes TPA-N-Me and TPA-N-Py in Example 3; where (a) is TPA-N-Me and (b) is TPA-N-Py.

[0059] Figure 11 The images show fluorescence images of dead and live cells stained with dyes TPA-N-Me and TPA-N-Py in Example 4, respectively.

[0060] Figure 12 Flow cytometry results of dye TPA-N-Me and TPA-N-Py distinguishing dead cells and live cells in Example 5; where (a) is TPA-N-Me, (b) is TPA-N-Py;

[0061] Figure 13 Fluorescence imaging figures of dye TPA-N-Me and TPA-N-Py distinguishing dead cells and live cells at different concentrations in Example 6; where (a) is TPA-N-Me, (b) is TPA-N-Py;

[0062] Figure 14 Fluorescence imaging figures of dye TPA-N-Me and TPA-N-Py distinguishing dead cells and live cells at different staining time in Example 7; where (a) is TPA-N-Me, (b) is TPA-N-Py;

[0063] Figure 15 Fluorescence imaging figures of dye TPA-N-Me and TPA-N-Py distinguishing dead cells and live cells at very low concentrations in Example 8; where (a) is TPA-N-Me, (b) is TPA-N-Py;

[0064] Figure 16 Fluorescence imaging figures of dye TPA-N-Me and TPA-N-Py distinguishing dead cells and live cells of different cell lines in Example 9, the cell lines include mouse breast cancer cells (4T1), human breast cancer cells (MDA-MB-231), human cervical cancer cells (Hela), human embryonic kidney cells (293T), human skin fibroblasts (HSF), mouse monocyte macrophages (RAW264.7), where (a) is TPA-N-Me, (b) is TPA-N-Py;

[0065] Figure 17 Colocalization figures of dye TPA-N-Me and TPA-N-Py respectively with commercial dead cell dye propidium iodide PI staining cells in Example 10, where (a) is TPA-N-Me, (b) is TPA-N-Py. DETAILED DESCRIPTION

[0066] The following examples are provided to better enable those skilled in the art to further understand the application, and are not intended to limit the scope of the application or the protection afforded. Any person skilled in the art who learns of the present application or combines the present application with other prior art features will be able to derive any product that is the same or similar to the present application, which falls within the scope of the present application.

[0067] The specific experimental steps or conditions are not indicated in the examples, which can be carried out according to the conventional experimental steps described in the literature or the operation or conditions. The reagents or instruments used are not indicated by the manufacturer, which are conventional reagent products that can be obtained by purchase.

[0068] Example 1 Citrate-based fluorescent dyes TPA-N-Me and TPA-N-Py for distinguishing dead cells and live cells.

[0069] This example provides a synthesis method of citrate-based fluorescent dyes TPA-N-Me and TPA-N-Py for distinguishing dead cells and live cells, and the chemical structural formulas of TPA-N-Me (compound 1) and TPA-N-Py (compound 2) are as follows (a) and (b):

[0070]

[0071] The synthesis route of TPA-N-Me is as follows:

[0072] .

[0073] The synthesis route of TPA-N-Py is as follows:

[0074] .

[0075] The synthesis of TPA-N-Me includes the following steps:

[0076] (1) Mix citric acid (10 mmol) and 1,2-diphenylethylenediamine (10 mmol), stir at 140°C for 4 h, cool to room temperature after the reaction is completed, add anhydrous methanol to dissolve, and then the reaction solution is washed with water, extracted with dichloromethane, dried with anhydrous sodium sulfate, and then separated and purified by column chromatography (the chromatography liquid is DCM:MeOH=8:1, V / V) to obtain compound TPA-N-COOH.

[0077] The nuclear magnetic hydrogen spectrum is as shown in Figure 1 1 H NMR (600 MHz, DMSO- d 6) δ 8.27 (s, 1H), 7.44-7.32 (m, 8H), 7.23-7.22 (d, 2H), 5.98 (s, 1H), 5.93 (s, 1H), 5.29 (d, 1H),4.81 (d, 1H).

[0078] ​(2) Synthesis of compound TPA-N-Me: purified TPA-N-COOH (10 mmol) and potassium carbonate (15 mmol) were dissolved in N,N-dimethylformamide (25 mL), stirred at 40 °C for 30 min, then iodomethane (10.5 mmol) was added and the reaction was continued for 20 h. After the reaction was completed, the reaction solution was washed with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and column chromatography was used for separation and purification (chromatography liquid was PE:EA=1:1, V / V) to obtain fluorescent dye TPA-N-Me (compound name: 5-oxo-2,3-diphenyl-1,2,3,5-tetrahydroimidazo[1,2-a]pyridine-7-methyl formate).

[0079] The nuclear magnetic hydrogen spectrum thereof is shown in Figure 2 , 1 H NMR (600 MHz, DMSO- d 6) δ 8.5 (s, 1H), 7.44-7.32 (m, 8H), 7.23-7.22 (d, 2H), 6.01 (s, 1H), 5.91 (s, 1H), 5.33-5.32 (d,1H), 4.87-4.86 (d, 1H), 3.83 (s, 3H).

[0080] The nuclear magnetic carbon spectrum thereof is shown in Figure 3 , 13 C NMR (151 MHz, DMSO- d 6) δ 166.14, 159.87,154.40, 143.83, 142.02, 139.77, 129.53, 129.30, 128.81, 128.52, 126.21,126.09, 105.66, 80.45, 68.10, 66.59, 53.12.

[0081] The high resolution mass spectrum thereof is shown in Figure 4 , 21 H 18 N2O3[M+H] + : 347.1396, found: 347.1395.

[0082] Synthesis of TPA-N-Py, including the following steps:

[0083] (1) Synthesis of compound TPA-N-COO(CH2)4Br: TPA-N-COOH (10 mmol) and potassium carbonate (15 mmol) purified in the previous step were dissolved in N,N-dimethylformamide (25 mL), stirred at 40°C for 30 min, and then 1,4-dibromobutane (10 mmol) was added and the reaction was continued for 20 h. After the reaction was completed, the reaction solution was washed with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and column chromatography was performed to separate and purify (the chromatography solution was PE:EA=1:1, V / V) to obtain the compound TPA-N-COO(CH2)4Br.

[0084] The nuclear magnetic hydrogen spectrum thereof is shown in Figure 5 1 H NMR (600 MHz, DMSO- d 6) δ 8.48 (s, 1H), 7.40-7.32 (m, 8H), 7.23-7.22 (d, 2H), 6.02 (s, 1H), 5.91 (s, 1H), 5.32 (d, 1H),4.86 (d, 1H), 4.29-4.27 (t, 2H), 3.62-3.60 (t, 2H), 1.97-1.92 (m, 2H), 1.85-1.80 (m, 2H).

[0085] (2) Synthesis of compound TPA-N-Py: TPA-N-COO(CH2)4Br (10 mmol) and 4-methylpyridine (15 mmol) purified in the previous step were dissolved in acetonitrile (25 mL) and refluxed at 85°C for 12 h. After the reaction was completed, the reaction solution was washed with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and column chromatography was performed to separate and purify (the chromatography solution was DCM:MeOH=10:1, V / V) to obtain the fluorescent dye TPA-N-Py (the compound name is 1-[4-((5-oxo-2,3-diphenyl-1,2,3,5-tetrahydroimidazo[1,2-a]pyridine-7-carbonyl)oxy)butyl]-4-methylpyridinium-1-yl bromide).

[0086] The nuclear magnetic hydrogen spectrum thereof is shown in Figure 6 1 H NMR (600 MHz, DMSO- d ​​6) δ 8.95-8.94 (d, 2H), 8.5 (s, 1H), 8.00-7.99 (d, 2H), 7.44-7.31 (m, 8H), 7.23-7.22 (d, 2H), 6.03 (s, 1H), 5.89 (s, 1H), 5.32 (d, 1H), 4.87 (d, 1H), 4.60-4.57 (t, 2H), 4.29-4.26 (t, 2H), 2.60 (s, 3H), 2.04-2.00 (m, 2H), 1.73-1.68 (m, 2H).

[0087] The carbon magnetic resonance spectrum thereof is shown in Figure 7 13 C NMR (151 MHz, DMSO- d 6) δ 165.62, 159.90, 159.35, 154.34, 144.22, 143.88, 142.02, 139.75, 129.53, 129.30, 128.88, 128.82, 128.55, 126.21, 126.07, 105.69, 80.47, 68.07, 66.53, 64.99, 59.95, 27.82, 25.19, 21.84.

[0088] The high resolution mass spectrum thereof is shown in Figure 8 HRMS (ESI, m / z): calculated C 30 H 30 N3O3Br [M-Br] + : 480.2287, found: 480.2287.

[0089] Example 2 Determination of optical properties

[0090] This example investigates the optical properties of the citric acid-based fluorescent dyes TPA-N-Me and TPA-N-Py.

[0091] Take 1 μL of fluorescent dye TPA-N-Me or TPA-N-Py stock solution with a concentration of 10 mM and add it to 1 mL of aqueous solution to obtain a working solution with a concentration of 10 μM; use a fluorescence spectrometer to excite with 360 nm laser and scan the emission spectrum from 370-700 nm to obtain the fluorescence emission spectrum of the dye.

[0092] As Figure 9 ​As shown from the fluorescence emission spectra, the maximum emission wavelength of dyes TPA-N-Me and TPA-N-Py in aqueous solution is 478 ± 5 nm.

[0093] Example 3

[0094] This example investigates the Hela cell toxicity of citric acid-based fluorescent dyes TPA-N-Me and TPA-N-Py.

[0095] Cell culture: Hela cells were cultured with DMEM high glucose medium containing 10% fetal bovine serum, 1% 100 U / mL penicillin and 100 μg / mL streptomycin, and the culture conditions of the cell incubator were 5% CO2, 37°C.

[0096] Cell toxicity test: cell counting CCK-8 method was used to determine cell toxicity. Appropriate density of Hela cells were inoculated into 96-well plates and cultured overnight. After the cells were completely adherent, the cells were incubated with a series of final concentrations (1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM) of TPA-N-Me or TPA-N-Py dissolved in complete culture medium for 24 h, the control group was the cell sample without dye, and the blank group was the complete culture medium. After incubation, the original culture medium was replaced with complete culture medium containing 10% CCK-8 solution and incubated for another 2 h. Finally, the absorbance at 450 nm wavelength was measured by a microplate reader, and the cell survival rate (cell viability%= (experimental group-blank group) / (control group-blank group) x 100%) was calculated based on the measured absorbance results.

[0097] As shown in Figure 10 , the survival rate of Hela cells was still as high as 80% or more when the concentration of dyes TPA-N-Me and TPA-N-Py was 60 μM. These results show that dyes TPA-N-Me and TPA-N-Py have good biocompatibility and can be used for fluorescence imaging of biological systems such as cells.

[0098] Example 4

[0099] This example investigates the fluorescence imaging experiment of citric acid-based fluorescent dyes TPA-N-Me and TPA-N-Py respectively staining dead cells and live cells.

[0100] The ability of dyes TPA-N-Me and TPA-N-Py to distinguish between dead cells and live cells was investigated using Hela cells, and the steps were as follows:

[0101] Take 1 μL of 10 mM concentration of fluorescent dye TPA-N-Me or TPA-N-Py stock solution to 1 mL of culture medium (DMEM high glucose medium containing 10% fetal bovine serum, 1% 100 U / mL penicillin and 100 μg / mL streptomycin) to obtain a working solution with a concentration of 10 μM. Add 1 mL of working solution to each well containing 4x10 5 Live cells or dead cells (live cells treated with 75% ethanol for 30 min) in a 12-well plate, and continue to culture in the incubator for 90 min. Fluorescence imaging was performed at an excitation wavelength of 405 nm.

[0102] As Figure 11 shown, after incubation of dead cells with TPA-N-Me or TPA-N-Py (10 μM), obvious blue fluorescence was observed in the cytoplasm, while no obvious fluorescence was observed after incubation of live cells with the probes. This indicates that the dye can specifically enter dead cells and stain the cytoplasm of dead cells, but cannot stain live cells, thereby achieving the purpose of distinguishing live cells and dead cells.

[0103] Example 5

[0104] This example investigates the flow cytometry experiment of citrate-based fluorescent dyes TPA-N-Me and TPA-N-Py to distinguish dead cells and live cells.

[0105] The ability of dyes TPA-N-Me and TPA-N-Py to distinguish dead cells and live cells was quantitatively investigated using Hela cells, and the steps were as follows:

[0106] Take 1 μL of 10 mM concentration of fluorescent dye TPA-N-Me or TPA-N-Py stock solution to 1 mL of culture medium (DMEM high glucose medium containing 10% fetal bovine serum, 1% 100 U / mL penicillin and 100 μg / mL streptomycin) to obtain a working solution with a concentration of 10 μM. Add 1 mL of working solution to each well containing 4x10 5 Live cells or dead cells (live cells treated with 75% ethanol for 30 min) in a 12-well plate, and continue to culture in the incubator for 90 min. Fluorescence imaging was performed at an excitation wavelength of 405 nm.

[0107] As Figure 12As shown, after culturing dead cells with TPA-N-Me (10 μM), the percentage of positive cells was 97.8%, significantly higher than that of the live cell group co-incubated with the dye (0.15%) and the untreated control group (0.012%). After culturing dead cells with TPA-N-Py (10 μM), the percentage of positive cells was 91.9%, significantly higher than that of the live cell group co-incubated with the dye (5.83%) and the untreated control group (0.037%). These results indicate that these citrate-based fluorescent dyes, TPA-N-Me and TPA-N-Py, can specifically stain dead cells but have difficulty staining live cells, thus achieving the purpose of distinguishing between dead and live cells.

[0108] Example 6

[0109] This embodiment investigated fluorescence imaging experiments using citrate-based fluorescent dyes TPA-N-Me and TPA-N-Py at different concentrations to distinguish between dead and live cells.

[0110] The experiment used HeLa cells to investigate the ability of the dyes TPA-N-Me and TPA-N-Py at different concentrations to distinguish between dead and live cells. The steps are as follows:

[0111] Working solutions of 10 μM, 30 μM, and 50 μM were prepared using 10 mM stock solutions of the fluorescent dyes TPA-N-Me or TPA-N-Py. These solutions were cultured in DMEM high-glucose medium containing 10% fetal bovine serum, 1% 100 U / mL penicillin, and 100 μg / mL streptomycin. The different concentrations of working solution were added to each well containing 4 × 10⁻⁶ cells / mL. 5 Fill 12-well plates with either live or dead cells (live cells treated with 75% ethanol for 30 min) and incubate for 90 min. Perform fluorescence imaging at an excitation wavelength of 405 nm.

[0112] like Figure 13 As shown, after culturing dead cells with different concentrations of TPA-N-Me or TPA-N-Py, all groups of dead cells exhibited obvious blue fluorescence, while live cells showed no obvious fluorescence after incubation with the dyes. This indicates that these citrate-based fluorescent dyes, TPA-N-Me and TPA-N-Py, can specifically enter dead cells at different concentrations, thus staining dead cells but not live cells, thereby achieving the purpose of distinguishing between dead and live cells.

[0113] Example 7

[0114] This embodiment investigates fluorescence imaging experiments using citrate-based fluorescent dyes TPA-N-Me and TPA-N-Py at different staining times to distinguish between dead and live cells.

[0115] The ability of dyes TPA-N-Me and TPA-N-Py to distinguish between dead and live cells at different staining times was investigated using Hela cells in experiments, and the steps were as follows:

[0116] 1 μL of fluorescent dye TPA-N-Me or TPA-N-Py stock solution with a concentration of 10 mM was added to 1 mL of culture medium (DMEM high-sugar culture medium containing 10% fetal bovine serum, 1% 100 U / mL penicillin, and 100 μg / mL streptomycin) to obtain a working solution with a concentration of 10 μM. The dye working solution was added to each well containing 4×10 5 live cells or dead cells (live cells treated with 75% ethanol for 30 min) in a 12-well plate, and incubation was continued in the incubator for different times (5 min, 10 min, 15 min, 30 min, 60 min, 90 min). Fluorescence imaging was performed at an excitation wavelength of 405 nm.

[0117] As shown in Figure 14 , after incubation of dead cells with TPA-N-Me or TPA-N-Py for different times, there was obvious blue fluorescence in each group of dead cells even at a very short time of 5 min or after long-term incubation (90 min), while there was no obvious fluorescence after incubation of live cells with the dyes. This indicates that the citrate-based fluorescent dyes TPA-N-Me and TPA-N-Py can specifically enter dead cells at different staining times, thereby staining dead cells in a very short time, and will not non-specifically stain live cells at a long time (90 min), thereby achieving the purpose of distinguishing between dead and live cells.

[0118] Example 8

[0119] This example investigates the fluorescence imaging experiment of citrate-based fluorescent dyes TPA-N-Me and TPA-N-Py to distinguish between dead and live cells at very low concentrations.

[0120] The ability of dyes TPA-N-Me and TPA-N-Py to distinguish between dead and live cells at very low concentrations was investigated using Hela cells in experiments, and the steps were as follows:

[0121] Fluorescent dye TPA-N-Me or TPA-N-Py stock solution with a concentration of 10 mM was prepared into working solutions with concentrations of 1 μM, 2 μM, 5 μM, and 10 μM, respectively, using DMEM high-sugar culture medium containing 10% fetal bovine serum, 1% 100 U / mL penicillin, and 100 μg / mL streptomycin. The dye working solutions were added to each well containing 4×10 5Fill 12-well plates with either live or dead cells (live cells treated with 75% ethanol for 30 min) and incubate for 15 min. Perform fluorescence imaging at an excitation wavelength of 405 nm.

[0122] like Figure 15 As shown, after culturing dead cells with low concentrations of TPA-N-Me or TPA-N-Py, even at extremely low concentrations (1 μM), obvious blue fluorescence was observed in all groups of dead cells, while no obvious fluorescence was observed in live cells. This indicates that these citrate-based fluorescent dyes, TPA-N-Me and TPA-N-Py, can stain dead cells even at extremely low concentrations, thereby achieving the purpose of distinguishing between dead and live cells.

[0123] Example 9

[0124] This embodiment examines fluorescence imaging experiments using citrate-based fluorescent dyes TPA-N-Me and TPA-N-Py to distinguish between dead and live cells from different cell lines.

[0125] In this experiment, mouse breast cancer cells (4T1), human breast cancer cells (MDA-MB-231), human cervical cancer cells (HeLa), human embryonic kidney cells (293T), human skin fibroblasts (HSF), and mouse monocytes / macrophages (RAW 264.7) were used to investigate the ability of the dyes TPA-N-Me and TPA-N-Py to distinguish between dead and live cells in different cell lines. The steps are as follows:

[0126] Add 1 μL of 10 mM TPA-N-Me or TPA-N-Py stock solution to 1 mL of DMEM high-glucose medium (containing 10% fetal bovine serum, 1% 100 U / mL penicillin, and 100 μg / mL streptomycin) to obtain a 10 μM working solution. Add the dye working solution to different cell lines, either live or dead (live cells treated with 75% ethanol for 30 min) (each well contains 4 × 10⁶ cells). 5 The cells were placed in 12-well plates and cultured for 15 min in an incubator. Fluorescence imaging was performed at an excitation wavelength of 405 nm.

[0127] like Figure 16 As shown, dead cells from different cell lines exhibited significant blue fluorescence after being cultured with TPA-N-Me or TPA-N-Py, while no significant fluorescence was observed in live cells. This indicates that these citrate-based fluorescent dyes, TPA-N-Me or TPA-N-Py, can stain dead cells from different cell lines, thereby distinguishing between dead and live cells, and have a wide range of applications.

[0128] Example 10

[0129] This example investigates the co-localization experiment of citric acid-based fluorescent dyes TPA-N-Me and TPA-N-Py with commercialized dead cell dye (propidium iodide, PI) for cell staining.

[0130] The experiment uses Hela cells to compare the effect of dyes TPA-N-Me and TPA-N-Py with commercialized dead cell dye (propidium iodide, PI) for staining dead cells.

[0131] Take 1 μL of 10 mM fluorescent dye TPA-N-Me or TPA-N-Py stock solution and add it to 1 mL of culture medium, and then add PI dye (1 μL of 10 mM) and mix well to obtain a working solution containing both PI dye and citric acid dye (TPA-N-Me or TPA-N-Py). Add the dye working solution to each well of a 12-well plate containing 4 x 10 5 dead cells (live cells treated with 75% ethanol for 30 min) and continue to culture in the incubator for 15 min. Fluorescence imaging is performed at excitation wavelengths of 405 nm and 525 nm.

[0132] As shown in Figure 17 , after co-incubation of dead cells with mixed dyes, obvious blue fluorescence and red fluorescence can be observed simultaneously, where the blue fluorescence is mainly concentrated in the cytoplasm, and the red fluorescence is mainly concentrated in the nucleus. This shows that the citric acid-based fluorescent dyes TPA-N-Me and TPA-N-Py can well stain dead cells, have similar ability to stain dead cells as commercialized dyes, and their staining area is mainly concentrated in the cytoplasm, which can be applied to many experimental studies related to the cytoplasm.

[0133] Obviously, the above examples are only examples for clarity, and do not limit the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and impossible to exhaust all embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A citrate-based fluorescent dye for distinguishing between dead and live cells, characterized in that, The citric acid-based fluorescent dye is compound 2; the structural formula of the compound 2 is as follows: 。 2. A process for the preparation of compound 2 as claimed in claim 1, wherein, The method comprises the following steps: S1, citric acid is subjected to a heating reaction with 1,2-diphenylethylenediamine to obtain compound TPA-N-COOH; S2, compound TPA-N-COOH is subjected to a stirring reaction with potassium carbonate, and then 1,4-dibromobutane is added to perform a heating reaction to obtain compound TPA-N-COO(CH2)4Br; S3, compound TPA-N-COO(CH2)4Br is subjected to a heating reflux reaction with 4-methylpyridine to obtain compound 2; The synthesis route is as follows: 。 3. The process for the preparation of compound 2 according to claim 2, characterized in that, In the S1 step, the heating reaction is performed at 120-140 DEG C for 4-8 h under stirring; In the S1 step, the molar ratio of citric acid to 1,2-diphenylethylenediamine is 1:1; In the S1 step, after the heating reaction, a washing, extraction, drying and / or purification step is further included; in the purification step, column chromatography separation and purification are adopted, and the chromatography liquid is dichloromethane:methanol = 8:1, V / V.

4. Process for the preparation of compound 2 according to claim 2 or 3, characterized in that, In the S2 step, the reaction conditions of compound TPA-N-COOH with potassium carbonate are stirring at 30-40 DEG C for 20-30 min; In the S2 step, the heating reaction is performed at 30-40 DEG C for 18-24 h under stirring; In the S2 step, the molar ratio of TPA-N-COOH, potassium carbonate and 1,4-dibromobutane is 1:1.5:1; In the S2 step, after the heating reaction, a washing, extraction, drying and / or purification step is further included; in the purification step, column chromatography separation and purification are adopted, and the chromatography liquid is petroleum ether:ethyl acetate = 1:1, V / V.

5. The process for the preparation of compound 2 according to claim 2 or 3, characterized in that, In the S3 step, the heating reflux reaction is performed at 85 DEG C for 12-18 h under reflux; In the S3 step, the molar ratio of compound TPA-N-COO(CH2)4Br to 4-methylpyridine is 1:1.5; In the S3 step, after the heating reflux reaction, a washing, extraction, drying and / or purification step is further included; in the purification step, column chromatography separation and purification are adopted, and the chromatography liquid is dichloromethane:methanol = 10:1, V / V.

6. The citric acid-based fluorescent dye for distinguishing dead cells and living cells according to claim 1, the compound 2 prepared by the preparation method of the compound 2 or compound 1 according to any one of claims 2-5, or the application of the compound 2 or compound 1 in distinguishing dead cells and living cells, wherein the application is for a non-disease diagnosis purpose. The structural formula of the compound 1 is as follows: 。 7. Use according to claim 6, characterized in that, The cells are selected from normal cells, cancer cells or immune cells.

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