Application of fluorescent probe in long-range observation of cell membrane

Abstract

The invention discloses an application of a fluorescent probe in long-range observation of a cell membrane. The chemical name of the pyrrole fluorescent probe is (E)-4-(2-(1-methyl-1H-pyrrole-2) vinyl)-1-octadecyl-1-iodised salt. The pyrrole fluorescent probe provided by the invention can be used for tracking and observing the dynamic change of a cell membrane for a long time, and has the characteristics of weaker internalization, no influence of pH value, sensitivity to viscosity and the like. Due to the fact that cell membranes are closely related to cell growth, division, metabolism, disease diagnosis (tumors) and the like, the fluorescent compound can be used in the fields of preparation of tumor diagnosis reagents, fluorescence imaging reagents or radioactive imaging reagents, cell growth and division diagnosis reagents or long-range cell membrane observation reagents and the like.

Description

Technical Field

[0001] The invention relates to application of a fluorescent probe in long-range observation of cell membranes, belonging to the technical field of fluorescent probes. Background Art

[0002] As the outer structure of the cell, the cell membrane plays a role in protecting the cell and maintaining the stability of the cell. Its permeability and fluidity are crucial to the life activities of the cell, such as growth, division and metabolism. The cell membrane plays an obvious role in maintaining the stability of the cell, transporting substances, cell recognition and signal transmission, and cell division and differentiation. Therefore, the study of the cell membrane is of great significance for a deep understanding of the structure and function of the cell membrane, revealing the mechanism of cell life activities, and promoting the development of the biomedical field.

[0003] At present, scanning electron microscopes, transmission electron microscopes, etc. are widely used to observe changes in cell membranes. However, these methods will destroy the cell structure and cannot observe the dynamic changes of the cell membrane of living cells in situ and in real time. Fluorescent probes have attracted widespread attention because of their simple operation and the ability to observe the dynamic changes of cells in situ, non-destructively, and in real time. Current cell membrane probes often have the characteristic of internalization, which is not conducive to long-term observation of cell membranes. Therefore, it is necessary to develop cell membrane fluorescent probes that are not easy to internalize. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a fluorescent probe suitable for long-range tracking of dynamic changes of cell membranes, as well as a preparation method and application thereof.

[0005] Technical solution: The present invention provides a pyrrole fluorescent probe, whose chemical name is: (E)-4-(2-(1-methyl-1H-pyrrole-2)vinyl)-1-octadecyl-1-iodine salt, and whose structural formula is:

[0006]

[0007] The fluorescent compound consists of two parts: pyridinium salt and pyrrole.

[0008] The present invention also provides a method for preparing the fluorescent probe, the preparation route being:

[0009]

[0010] Compound 1 and compound 2 are reacted by Knoevenagel reaction to synthesize the fluorescent compound.

[0011] Furthermore, the method 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; and separating and purifying by column chromatography using a CH2Cl2 / CH3OH mixture as an eluent to obtain the product.

[0012] Furthermore, the reflux time is 10 hours.

[0013] Furthermore, the volume ratio of the CH2Cl2 / CH3OH mixture is 10:1 to 9:1.

[0014] The present invention also provides the use of the fluorescent probe in preparing tumor diagnostic reagents, fluorescent imaging reagents or radioactive imaging reagents.

[0015] The present invention also provides the use of the fluorescent probe in preparing a diagnostic reagent for cell growth and division.

[0016] The present invention also provides the use of the fluorescent probe in preparing a reagent for long-range observation of cell membranes.

[0017] Furthermore, the concentration of the fluorescent probe is 1-15 μM.

[0018] Furthermore, the concentration of the fluorescent probe is 5 μM.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: The present invention discloses a pyrrole fluorescent probe, which can be used to track and observe the dynamic changes of cell membranes for a long time. The probe has the characteristics of weak internalization, no influence of pH value, and sensitivity to viscosity. Since the cell membrane is closely related to cell growth, division, metabolism and disease diagnosis (tumor), the fluorescent compound of the present invention can be used in the fields of preparing tumor diagnostic reagents, fluorescent imaging reagents or radioactive imaging reagents, diagnostic reagents for cell growth and division, or long-term observation of cell membrane reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Absorption spectra (A) and fluorescence spectra (B) of PP-18 (10 μM) in different solvents.

[0021] Figure 2 Absorption spectra (A) and fluorescence spectra (B) of PP-18 (10 μM) in solutions with different pH values.

[0022] Figure 3 Confocal fluorescence images of live or fixed HeLa cells stained with PP-18 (5 μM, 15 min).

[0023] Figure 4These are confocal fluorescence images of HeLa cells at different time points after staining with PP-18 (5 μM, 15 min). DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0025] The absorption spectrum test instrument used in the following 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.

[0026] Example 1 Synthesis of fluorescent compounds

[0027]

[0028] Compound 1 (1 mmol) and compound 2 (1 mmol) were dissolved in 15 mL of methanol, stirred in a flask for 1 h, and 5 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 (10:1-9:1, v / v) was used as the eluent for column chromatography separation and purification to obtain a yellow solid, namely (E)-4-(2-(1-methyl-1H-pyrrole-2)vinyl)-1-octadecyl-1-iodide, hereinafter referred to as compound PP-18 (yield: 60%).

[0029] 1 H NMR (400MHz, DMSO-d6) δ (ppm): 8.78 (d, J = 4.0Hz, 2H), 8.12 (d, J = 8.0Hz, 2H), 7.93 (d, J = 16.0Hz, 1H), 7.09 (t, J = 12.0Hz, 2H), 6.88 ( d,J=4.0Hz,1H),6.22(t,J=8.0Hz,1H),4.41(t,J=6.0Hz,2H),3.81(s,3H),1.85-1.91(m,2H),1.23(s,30H),0.86(t,J=6.0Hz,3H). 13 C NMR (400MHz, DMSO-d6), δ (ppm): 154.16, 144.83, 130.55, 130.39, 129.30, 123. 06,116.66,113.94,110.77,60.55,35.42,31.93,31.51,26.12,22.77,14.14.

[0030] Example 2 Photophysical property test experiment

[0031] DMF, DMSO, ethanol (EtOH), 1,4-dioxane (1,4-Dioxane), Gly, H2O and MeCN were used as solvents to prepare test solutions containing 10 μM PP-18 prepared in Example 1. The absorption spectra of the probe PP-18 in DMF, DMSO and ethanol were first tested to determine the excitation wavelength ( Figure 1 A). Then, the fluorescence spectra of the probe PP-18 in all the above solvents (1,4-dioxane, DMF, DMSO, EtOH, Gly, H2O and MeCN) were measured using 450 nm light excitation ( Figure 1 B).

[0032] It can be seen from the figure that the fluorescent compound PP-18 has an absorption peak at 440nm, and the absorption peak range is 350-650nm (see Figure 1 A). It has a fluorescence peak in the range of 460-620nm (see Figure 1 B). The above results show that the fluorescent compound can be excited by light of 350-650nm, and its emission spectrum ranges from 460-620nm. In addition, it can be seen that the fluorescence intensity of the fluorescent compound is stronger in glycerol (Gly), and lower in low viscosity solvents, indicating that the probe is sensitive to viscosity, which is conducive to high-fidelity imaging of organelles.

[0033] Example 3 Fluorescence test experiment of fluorescent compound PP-18 in solutions with different pH values

[0034] (1) Take the organic fluorescent compound PP-18 prepared in Example 1 and prepare a probe stock solution with a concentration of 1 mM using DMSO;

[0035] (2) preparing PBS solutions with different pH values ​​(pH = 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0);

[0036] (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-18 in each solution is 10 μM;

[0037] (4) The above solution was tested for its absorption spectrum using a UV-visible spectrophotometer and a fluorescence spectrometer ( Figure 2 A) and fluorescence emission spectra ( Figure 2 B), and the corresponding curve is obtained. PP-18 has absorption in the range of 355-510nm, and the fluorescence emission spectrum range is 420-650nm.

[0038] As can be seen from the figure, the probe PP-18 is not affected by pH value, indicating that the probe can accurately observe the dynamic changes of organelles.

[0039] Example 4 Cell Imaging Experiment of Probe PP-18

[0040] 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.

[0041] 2. When HeLa cells grow to the logarithmic phase, perform grafting culture:

[0042] ① Soak the coverslip in anhydrous ethanol for 30 minutes, dry it with an alcohol lamp, and place it in a disposable 35mm culture dish for later use.

[0043] 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.

[0044] 3. Prepare a 1mM probe PP-18 stock solution with DMSO, and prepare a 5μM PP-18 solution before use. Incubate the cultured active HeLa cells in the culture solution containing 5μM PP-18 for 15 minutes and observe with a laser confocal microscope.

[0045] Staining of fixed cells: HeLa cells were first fixed with 5% paraformaldehyde for 45 min, then incubated in culture medium containing 5μM PP-18 for 15 min, and imaged using a laser confocal microscope to record the stained sites in the cells, fluorescence distribution and brightness changes, co-localization information, etc.

[0046] Results Figure 3 , where the excitation wavelength of PP-18 in the green light channel is 405nm, and the fluorescence collection wavelength is 500-600nm. From the figure, it can be seen that the probe stains the cell membrane, indicating that the probe targets the cell membrane.

[0047] Example 5 Internalization experiment of probe PP-18

[0048] 1. HeLa cells were cultured in a high-glucose culture medium containing 10% fetal bovine serum at 37°C in a saturated humidity incubator with 5% CO2. The culture medium was replaced every 2-3 days and subculture was performed.

[0049] 2. When HeLa cells grow to the logarithmic phase, perform grafting culture:

[0050] ① Soak the coverslip in anhydrous ethanol for 30 minutes, dry it with an alcohol lamp, and place it in a disposable 35mm culture dish for later use.

[0051] ② Wash the cells in the 100mL cell bottle three times with PBS, digest them with 1mL 0.25% trypsin for 5 minutes, pour out the trypsin carefully, add fresh culture medium, blow and mix 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, inoculate the culture dish containing the cover glass and place it in a 5% CO2 incubator to allow the cells to grow close to the culture dish. After the HeLa cells grow on the plate and cover the cover glass, they are used for cell experiments.

[0052] 3. Prepare a probe stock solution with a concentration of 1mM using DMSO. Incubate the cultured active HeLa cells in a culture solution containing 5μM PP-18 for 15min and observe with a laser confocal microscope. Take photos at different time points to record the coloring sites in the cells, fluorescence distribution and brightness changes, etc. The results are shown in Figure 4 .

[0053] Among them, the excitation wavelength of PP-18 in the green light channel is 405nm, and the fluorescence collection wavelength is 500-600nm. As time goes by, the fluorescence intensity of the probe hardly changes. This confirms that the internalization of the probe is weak, and the cell membrane can still be seen at 2.5h, which helps to track the dynamic changes of the cell membrane over a long distance.

[0054] 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 pyrrole fluorescent probe, characterized in that: Its chemical name is: (E)-4-(2-(1-methyl-1H-pyrrole-2)vinyl)-1-octadecyl-1-iodide, and its structural formula is:

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

3. The method for preparing a fluorescent probe according to claim 2, characterized in that: The specific steps include: Dissolve compounds 1 and 2 in methanol, stir, add piperidine; reflux at 90°C, cool to room temperature and wash with petroleum ether; separate and purify by column chromatography using a CH2Cl2 / CH3OH mixture as eluent to obtain the product.

4. The method for preparing a fluorescent probe according to claim 3, characterized in that: The reflux time is 10 hours.

5. The method for preparing a fluorescent probe according to claim 3, characterized in that: The volume ratio of the CH2Cl2 / CH3OH mixture is 10:1 to 9:

1.

6. Use of the fluorescent probe according to claim 1 in the preparation of tumor diagnostic reagents, fluorescent imaging reagents or radioactive imaging reagents.

7. Use of the fluorescent probe according to claim 1 in preparing a diagnostic reagent for cell growth and division.

8. Use of the fluorescent probe according to claim 1 in preparing a reagent for long-range observation of cell membranes.

9. The use according to claims 6, 7 and 8, characterized in that: The concentration of the fluorescent probe is 1-15 μM.

10. The use according to claim 9, characterized in that: The concentration of the fluorescent probe was 5 μM.

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