Fluorescent probe and application thereof in distinguishing dead / living cells

Abstract

The invention discloses a fluorescent probe and application of the fluorescent probe in distinguishing dead / living cells. The chemical name of the fluorescent probe is (E)-1-methyl-4-(2-(1-methyl-1H-pyrrole-2) vinyl) pyridine-1-iodised salt. Distinguishing of cell death and viability is closely related to disease diagnosis and drug research and development. Therefore, the fluorescent compound provided by the invention can provide a more accurate and effective tool for research and application in the fields of chemical industry, biological medicine and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a fluorescent probe and an application thereof in distinguishing dead / living cells. Background Art

[0002] Distinguishing between living and dead cells is of great significance in the fields of biology, medicine and scientific research. Living cells have complex structures and functions and can perform life activities such as metabolism, information transmission, cell division, etc. Dead cells have lost these functions. Distinguishing between living and dead cells can help us better understand the life process, physiological state and function of living cells, so as to deeply study the nature and mechanism of life. In addition, in the field of medicine, distinguishing between living and dead cells is crucial for the diagnosis and treatment of diseases. For example, in cancer research, distinguishing between cancer cells and normal cells, as well as the ratio of living and dead cells in cancer cells, helps to evaluate the progression of the disease and the effect of treatment. In pathological examinations, distinguishing between living and dead cells helps to accurately judge the pathological conditions of tissues or organs. Therefore, distinguishing between living and dead cells is of great significance for in-depth understanding of life processes, diagnosis of diseases, optimization of experimental conditions, drug development and biotechnology applications. By accurately distinguishing between living and dead cells, cell resources can be better utilized to promote the development of biology, medicine and scientific research.

[0003] Fluorescent probes have attracted widespread attention due to their ability to observe cells in situ and in real time. However, there are few fluorescent probes that can distinguish between dead and alive cells. It is particularly important to develop a probe that can distinguish between dead and alive cells. This will provide a more accurate and effective tool for research and application in the fields of chemical industry, biomedicine, etc. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a fluorescent probe and its application in distinguishing dead / living cells.

[0005] Technical solution: The fluorescent probe provided by the present invention has the chemical name: (E)-1-methyl-4-(2-(1-methyl-1H-pyrrole-2)vinyl)pyridine-1-iodine salt, and the structural formula is:

[0006]

[0007] The present invention also provides a method for preparing the fluorescent probe, the preparation route being:

[0008]

[0009] Furthermore, compound 1 and compound 2 are reacted by Knoevenagel reaction to synthesize the fluorescent compound, which specifically includes the following steps: dissolving compounds 1 and 2 in methanol, stirring, and adding piperidine; refluxing at 90° C., cooling to room temperature, and washing with petroleum ether; using a CH2Cl2 / CH3OH mixture as an eluent, separating and purifying by column chromatography to obtain the fluorescent compound.

[0010] Furthermore, the volume ratio of the CH2Cl2 / CH3OH mixture is 12:1 to 7:1.

[0011] The present invention also provides the use of the fluorescent probe in preparing a reagent for distinguishing dead / live cells.

[0012] Furthermore, the concentration of the fluorescent probe is 5-15 μM.

[0013] Furthermore, the solvent in the reagent includes one of DMF, DMSO, ethanol (EtOH), 1,4-dioxane, Gly and H2O.

[0014] Furthermore, the excitation wavelength is 350-640 nm, and the fluorescence collection wavelength is 450-600 nm.

[0015] Furthermore, cells with high fluorescence intensity are living cells, and cells with low fluorescence intensity are dead cells.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the fluorescent probe of the present invention can distinguish between live and dead cells. Since distinguishing between live and dead cells is closely related to disease diagnosis and drug development, the fluorescent compound of the present invention can provide a more accurate and effective tool for research and application in the fields of chemical industry, biomedicine, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The absorption and fluorescence spectra of PP-1 (10 μM) in different solvents.

[0018] Figure 2 The absorption and fluorescence spectra of PP-1 (10 μM) in solutions with different pH values.

[0019] Figure 3 Confocal fluorescence images of normal and paraformaldehyde-fixed HeLa cells stained with PP-1 (15 μM, 30 min). DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0021] The absorption spectrum test instrument used in the examples is a Hitachi U-2910 spectrophotometer; the fluorescence spectrum instrument is a Hitachi F-2700 spectrophotometer, and the cell imaging instrument is a Lecia confocal microscope.

[0022] Example 1 Synthesis of fluorescent compounds

[0023] (E)-1-methyl-4-(2-(1-methyl-1H-pyrrole-2)vinyl)pyridine-1-iodine salt (i.e. compound PP-1)

[0024] The reaction route is as follows:

[0025]

[0026] Compound 1 (1 mmol) and compound 2 (1 mmol) were dissolved in 25 mL of methanol, stirred in a flask for 1 h, and 6 drops of piperidine were added. After stirring, the mixture was refluxed at 90 ° C for 10 h, cooled to room temperature, and washed with petroleum ether. A CH2Cl2 / CH3OH mixture (12:1-7:1, v / v) was used as the eluent for column chromatography separation and purification to obtain a yellow solid, which was compound PP-1 (yield: 60%).

[0027] 1 H NMR (400MHz, DMSO-d6) δ (ppm): 8.69 (d, J = 8.0Hz, 2H), 8.11 (d, J = 8.0Hz, 2H), 7.91 (d, J = 16.0H z,1H),7.05-7.10(m,2H),6.88-6.89(m,1H),6.22(q,J=1.3Hz,1H),4.18(s,3H),3.81(s,3H). 13 C NMR (400MHz, DMSO-d6), δ (ppm): 153.75, 144.72, 130.94, 129.94, 129.14, 122.59, 117.91, 112.82, 110.38, 46.84, 34.55.

[0028] Example 2 Photophysical property test experiment

[0029] Different solvents (DMF, DMSO, ethanol (EtOH), 1,4-dioxane (1,4-Dioxane), Gly and H2O) were used to prepare test solutions containing 10 μM PP-1. The absorption spectra of the above solutions were measured by UV-visible spectrophotometer, and the fluorescence emission spectra were measured by fluorescence spectrometer. The results are shown in Figure 1 .

[0030] It can be seen from the figure that the fluorescent compound PP-1 has an absorption peak at 430nm, and the absorption peak range is 350-640nm (see Figure 1 A). It has a fluorescence peak in the range of 450-600nm (see Figure 1 B). The above results show that the fluorescent compound can be excited by light of 350-640nm and its emission spectrum ranges from 450-600nm.

[0031] Example 3 Fluorescence test experiment of fluorescent compound PP-1 in solutions with different pH values

[0032] (1) Take the organic fluorescent compound PP-1 prepared in Example 1 and prepare a probe stock solution with a concentration of 1 mM using DMSO;

[0033] (2) preparing PBS solutions with different pH values ​​(pH = 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0);

[0034] (3) adding the probe mother solution prepared in step (1) to the solutions of different pH values ​​prepared in step (2) to prepare test solutions, so that the final concentration of the fluorescent probe PP-1 in each solution is 10 μM;

[0035] (4) The above solution was tested for its absorption spectrum and fluorescence emission spectrum using a UV-visible spectrophotometer and a fluorescence spectrometer to obtain corresponding curves. PP-1 has absorption in the range of 350-510nm, and the fluorescence emission spectrum range is 420-650nm.

[0036] The absorption spectrum of the above solution was measured by UV spectrophotometer. Figure 2 A. Use a fluorescence spectrometer to test its fluorescence emission spectrum and obtain the corresponding curve, see Figure 2 B.

[0037] As can be seen from the figure, in PBS solutions with different pH values, its absorption peak is around 430nm, and the fluorescence intensity does not change with the change of pH value. This shows that the probe is not affected by pH, which facilitates more accurate cell imaging.

[0038] Example 4 Probe PP-1 distinguishes between dead and alive cells

[0039] 1. Culture HeLa cells adherently in high-glucose H-DMEM medium containing 10% fetal bovine serum in a saturated humidity incubator at 37°C and 5% CO2. Replace the medium every 2-3 days and perform subculture.

[0040] 2. When the cells grow to the logarithmic phase, carry out grafting culture:

[0041] ① Soak the cover glass in anhydrous ethanol for 30 minutes, dry it with an alcohol lamp, and place it in a disposable 35mm culture dish for later use;

[0042] ② Wash the HeLa cells in the 100mL cell bottle three times with PBS, digest with 1mL 0.25% trypsin for 5 minutes, carefully pour out the trypsin, add fresh culture medium, blow evenly and count the cells. Control the cell density by adding culture medium so that the final cell concentration is 1×10 per mL. 5 Then, the cells were inoculated into the culture dish containing the cover glass and placed in a 5% CO2 incubator to allow the cells to grow close to the culture dish. After the HeLa cells grew on the plate and covered the cover glass, they were used for cell experiments.

[0043] 3. Prepare a probe stock solution with a concentration of 1mM using DMSO, and prepare a PP-1 solution with a concentration of 15μM before use. Incubate the cultured active HeLa cells in a culture solution containing 5μM PP-1 for 15min as a control group and observe with a laser confocal microscope. The experimental group was fixed with paraformaldehyde for 1 hour, then washed with PBS, and then incubated in a culture solution with 15μM PP-1 for 15min. Record the coloring site in the cell, fluorescence distribution, brightness changes, etc. The results are shown in Figure 3 .

[0044] in, Figure 3 A is a confocal microscopy image of normal (living cells) and paraformaldehyde-fixed (cells allowed to die) HeLa cells stained with the probe PP-1 (15 μM, 30 min). Figure 3 B is the relative fluorescence intensity of active HeLa cells and fixed cells. The excitation wavelength of PP-1 in the green light channel is 488nm, and the fluorescence collection wavelength is 500-600nm. Compared with normal cells, the fluorescence intensity of the probe is reduced in dead cells. This confirms that the probe can observe the life and death of cells.

[0045] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A fluorescent probe, It is characterized in that The chemical name of the fluorescent probe is: (E)-1-methyl-4-(2-(1-methyl-1H-pyrrole-2)vinyl)pyridine-1-iodine salt, and the structural formula is:

2. A method for preparing the fluorescent probe according to claim 1, It is characterized in that The preparation route is:

3. The method for preparing the fluorescent probe according to claim 2, It is characterized in that The specific steps include: Compounds 1 and 2 were dissolved in methanol, stirred, and piperidine was added; refluxed at 90°C, cooled to room temperature, and washed with petroleum ether; 2 Cl 2 / CH 3 OH mixture is used as eluent, and column chromatography is performed for separation and purification to obtain the product.

4. The method for preparing the fluorescent probe according to claim 3, It is characterized in that The CH 2 Cl 2 / CH 3 The volume ratio of the OH mixture is 12:1 to 7:

1.

5. Use of the fluorescent probe according to claim 1 in preparing a reagent for distinguishing dead / live cells.

6. The use according to claim 5, It is characterized in that The concentration of the fluorescent probe is 5-15 μM.

7. The use according to claim 5, It is characterized in that The solvents in the reagents include DMF, DMSO, ethanol (EtOH), 1,4-dioxane Gly and H 2 One of O.

8. The use according to claim 5, It is characterized in that The excitation wavelength is 350-640nm, and the fluorescence collection wavelength is 450-600nm.

9. The use according to claim 5, It is characterized in that Cells with high fluorescence intensity are living cells, and cells with low fluorescence intensity are dead cells.

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