Application of fluorescent probe in simultaneous imaging of lysosome and nucleolus and nucleic acid detection
By preparing (E)-2-(4-(ethylamino)-2-hydroxyvinyl)-3-(2-hydroxyethyl)benzothiazol-3-iodine salt fluorescent probes, the problem of the inability to simultaneously image lysosomes and nucleolus in the prior art is solved, precise cell structure observation and nucleic acid detection are achieved, and the development of research and diagnostic tools for related diseases has been promoted.
Patent Information
- Application Number
- CN202510452114.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-07-11
AI Technical Summary
The lack of fluorescent probes that can simultaneously image lysosomes and nucleolus in the prior art limits the accuracy of research and diagnostic tools for related diseases.
A fluorescent probe was developed with a specific structure of (E)-2-(4-(ethylamino)-2-hydroxyvinyl)-3-(2-hydroxyethyl)benzothiazole-3-iodite salt. It was prepared by a specific synthetic route and was able to emit 550-675nm fluorescence at an excitation wavelength of 400-600nm, which was used to simultaneously image lysosomes and nucleolus and combine nucleic acid detection.
Accurate observations of simultaneous imaging of lysosomes and nucleolus are achieved, providing more accurate cell and tissue structure analysis tools to support disease diagnosis and drug development.
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Figure CN120289381A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the application of a fluorescent probe in simultaneously imaging lysosomes and nucleoli and nucleic acid detection, belonging to the technical field of fluorescent probes. Background Art
[0002] Lysosomes are the "digestive factories" of cells. They contain a variety of acidic hydrolases and are responsible for the degradation and recycling of intracellular substances, playing a crucial role in organisms. Lysosomes are involved in autophagy and signal transduction and have functions such as maintaining cell morphology, which are essential for the normal physiological activities of cells. Abnormal activities of lysosomes are related to various diseases, including certain genetic diseases and cancers.
[0003] The nucleolus is the most prominent structure in the interphase nucleus of eukaryotic cells. The nucleolus usually appears as a single or multiple homogeneous spherical bodies located in the nucleus. There are various ribosomal proteins and rRNAs in the nucleolus, and they are assembled in the nucleolus to form ribosomal subunits. Ribosomes are important sites for protein synthesis. The size, shape, and number of nucleoli vary with the type of organism, cell type, and cell metabolic state. Abnormal nucleolar function may lead to uncontrolled cell proliferation and thus trigger tumorigenesis. Nucleolar dysfunction may cause abnormal RNA structure or function, resulting in incorrect protein synthesis. Therefore, the nucleolus is of great significance in the fields of biology and medicine and plays an important role in aspects such as cell cycle regulation, RNA processing, ribosome synthesis and assembly, signal transduction, and growth factor-mediated biosynthesis.
[0004] Fluorescent probes have received extensive attention due to their advantage of being able to observe cells in situ in real time. However, there are few fluorescent probes that can simultaneously image nucleoli and lysosomes. Such probes are of great significance for the research of diseases related to lysosomes and nucleoli. It is particularly important to develop a probe that can simultaneously image mitochondria and nucleoli. This will provide a more accurate and effective tool for research and application in fields such as the chemical industry and biomedicine. Summary of the Invention
[0005] Object of the Invention: The object of the present invention is to provide the application of a fluorescent probe in simultaneously imaging lysosomes and nucleoli and nucleic acid detection.
[0006] Technical Solution: The present invention provides the application of a fluorescent probe in simultaneously imaging lysosomes and nucleoli and nucleic acid detection, and the structural formula of the fluorescent probe is:
[0007]
[0008] Furthermore, the preparation route of the fluorescent probe is:
[0009]
[0010] Furthermore, the preparation method of the fluorescent probe specifically includes the following steps: Dissolve compound 1 and 2-iodoethanol in ethanol, stir, reflux at 100 °C, cool and filter, wash with anhydrous EtOH, dry to obtain compound 2; Dissolve compound 2 and 3 in ethanol, stir, add piperidine, reflux at 85 °C, cool to room temperature and wash with petroleum ether; Use a CH2Cl2 / CH3OH mixture as the eluent for column chromatography purification to obtain the product.
[0011] Furthermore, the volume ratio of the CH2Cl2 / CH3OH mixture is 10:1 to 6:1.
[0012] The present invention also provides a reagent for simultaneously imaging lysosomes and nucleoli / nucleic acid detection reagent, and the structural formula of the fluorescent probe in the reagent is:
[0013]
[0014] Furthermore, the concentration of the fluorescent probe is 2 to 10 μM.
[0015] Furthermore, the excitation wavelength of the fluorescent probe in the reagent for simultaneously imaging lysosomes and nucleoli is 400 - 600 nm, and the fluorescence collection wavelength is 550 - 675 nm.
[0016] Furthermore, the solvent in the reagent for simultaneously imaging lysosomes and nucleoli includes one of DMF, DMSO, ethanol, Gly, H2O, and acetonitrile.
[0017] Furthermore, the reagent for simultaneously imaging lysosomes and nucleoli also includes a lysosome-tracking deep red fluorescent probe LTDR.
[0018] Furthermore, the nucleic acid concentration range detected by the nucleic acid detection reagent is 0 to 4 mM.
[0019] Advantages: Compared with the prior art, the present invention has the following remarkable advantages: The present invention discloses that (E)-2-(4-(ethylamino)-2-hydroxyvinyl)-3-(2-hydroxyethyl)benzothiazole-3-iodide can be used as a fluorescent probe to simultaneously image lysosomes and nucleoli, and as a fluorescent probe for detecting nucleic acids. The probe of the present invention is a red fluorescent probe, which helps to image the microstructure of cells and tissues. Since lysosomes and nucleoli are closely related to the diagnosis of various diseases (such as cancer) and drug development. Therefore, the fluorescent compound of the present invention can provide a more accurate and effective tool for research and application in fields such as the chemical industry and biomedicine. Description of the Drawings
[0020] Figure 1Absorption spectra (A), fluorescence spectra (B) of TN (10 μM) in different solvents, and absorption and fluorescence spectra (C) in acetonitrile solution.
[0021] Figure 2 Fluorescence spectra (A) of TN (10 μM) in RNA solutions with different concentrations, relationship diagram (B) between concentration and the strongest fluorescence, and RNA fitting curve (C).
[0022] Figure 3 Confocal fluorescence image of normal HeLa cells stained with TN (5 μM, 30 min).
[0023] Figure 4 Confocal fluorescence image of active HeLa cells co-stained with TN (5 μM, 30 min) and LTDR (2 μM, 30 min). Detailed implementation manners
[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings.
[0025] The test instrument for absorption spectra used in the examples is Hitachi U-2910 spectrophotometer; the fluorescence spectrum instrument is Hitachi F-2700 spectrophotometer, and the cell imaging instrument is lecia confocal microscope.
[0026] Example 1 Synthesis of fluorescent compound
[0027] (E)-2-(4-(dimethylamino)-2-hydroxyvinyl)-3-(2-hydroxyethyl) benzothiazole-3-iodide (i.e., compound TN)
[0028] The reaction route is as follows:
[0029]
[0030] 1) Synthesis of benzothiazole iodide (compound 2)
[0031] Compound 1 (2-methylbenzothiazole, 1.49 g, 10 mmol) and 2-iodoethanol (1.72 mL, 10 mmol) were dissolved in 20 mL of anhydrous ethanol. Under room temperature conditions, it was stirred in a flask for 1 hour. Then it was refluxed at 100 °C for 8 h, cooled and filtered, and washed 3 times with anhydrous EtOH. After drying, a white solid was obtained, namely compound 2 (mass: 3.23 g, yield: 90%).
[0032] 2) Synthesis of compound TN
[0033] Dissolve Compound 2 (1 mmol) and Compound 3 (1 mmol) in 20 mL of ethanol, stir in a flask for 1 h, and add 5 drops of piperidine. After stirring, reflux at 85 °C for 8 h. After cooling to room temperature, wash with petroleum ether. Use a CH2Cl2 / CH3OH mixture (10:1 - 6:1, v / v) as the eluent for column chromatography purification to obtain a yellow solid, which is Compound TN (mass: 0.25 g, yield: 62%).
[0034] Example 2 Photophysical Property Test Experiment
[0035] 1. Prepare test solutions containing 10 μM TN with different solvents (DMF, DMSO, ethanol (EtOH), Gly, H2O, and acetonitrile (MeCN)). Measure the absorption spectra of the above solutions using a UV-visible spectrophotometer and the fluorescence emission spectra using a fluorescence spectrometer. The results are shown in Figure 1 .
[0036] It can be seen from the figure that the fluorescent compound TN has an absorption peak at 540 nm, and the absorption peak range is 400 - 600 nm (see Figure 1 A). It has a fluorescence peak in the range of 550 - 675 nm (see Figure 1 B). The above results indicate that this fluorescent compound can be excited by light with a wavelength of 400 - 600 nm, and the range of its emission spectrum is 550 - 675 nm. In addition, in the acetonitrile solvent, its Stokes shift is approximately 60 nm( Figure 1 C), indicating that this probe can avoid the influence of the excitation wavelength.
[0037] 2. Dissolve the probe TN in tris buffer to prepare a test solution of 10 μM TN. Add different concentrations of RNA, measure the fluorescence emission spectra using a fluorescence spectrometer, and analyze the fluorescence intensity measured at 600 nm using the Scatchard equation to calculate the RNA binding constant of the probe. The results are shown in Figure 2 .
[0038] The calculation formula is as follows (Formula 1):
[0039] r / Cf = kn - kr(1)
[0040] Where r represents the ratio of the concentration of the bound probe to the RNA concentration (in terms of phosphate), Cf represents the concentration of the free probe, k represents the binding constant, and n represents the number of probe binding sites per phosphate.
[0041] The concentration of the probe bound to RNA is calculated according to formula (2)
[0042] C b = C t ((F - F0) / (Fmax -F0))(2)
[0043] Where C t is the concentration of the total compound, F is the fluorescence intensity of the probe measured at a certain RNA concentration, F0 is the fluorescence intensity when the RNA concentration is 0, and F max is the fluorescence intensity of the fully bound probe.
[0044] From Figure 2 A, it can be seen that as the RNA concentration increases, the fluorescence intensity of the probe gradually increases. When the RNA concentration reaches 4 mM, the fluorescence intensity of the probe reaches saturation ( Figure 2 B). Since RNA mainly exists in the nucleolus, the probe may stain the nucleolus. By fitting, the fitting curve of RNA is obtained ( Figure 2 C), and the binding constant of the probe is calculated to be 1.2×10 7 M -1 .
[0045] Example 3 Cell Imaging Experiment of Probe TN
[0046] 1. Culture HeLa cells adherently in high-glucose H-DMEM culture medium containing 10% fetal bovine serum in an incubator with saturated humidity at 37°C and 5% CO2. Replace the culture medium every 2 - 3 days and perform subculture.
[0047] 2. When the cells grow to the logarithmic phase, perform slide culture:
[0048] ① Immerse the cover glass in absolute ethanol for 30 min, dry it with an alcohol lamp, and then place it in a disposable 35 mm culture dish for later use;
[0049] ② Wash the HeLa cells grown in a 100 mL cell bottle three times with PBS, digest them with 1 mL of 0.25% trypsin for 5 minutes, carefully pour out the trypsin, add fresh culture medium, pipette evenly, and count the cells. Control the cell density by the addition amount of the culture medium to make the final cell concentration 1×10 5 cells per milliliter, and then inoculate them into the above-mentioned culture dish containing the cover glass, and place them in a 5% CO2 incubator for culture to make the cells adhere tightly to the culture dish. After the HeLa cells grow on the coverslip and cover the coverslip, they are used for cell experiments.
[0050] 3. Prepare a stock solution of the probe with a concentration of 1 mM using DMSO. Incubate the cultured active HeLa cells in the culture medium containing 5 μM TN for 30 min, then wash them with PBS and image them with a laser confocal microscope. Record the colored parts, fluorescence distribution, and brightness changes in the cells, and the results are shown in Figure 3 .
[0051] Among them, Figure 3Confocal microscopy image of normal HeLa cells treated with probe TN (5 μM, 30 min). The excitation wavelength of TN is 561 nm, and the fluorescence collection wavelength is 570 - 700 nm. In addition, it can be seen from the overlapping pictures that the probe can illuminate the nucleolus and dot-like lysosomes in the nucleus, indicating that the probe can image both the nucleolus and lysosomes simultaneously.
[0052] Example 4: Cellular co-localization experiment of probe TN
[0053] The steps of culturing and seeding HeLa cells are the same as those in Example 3. Prepare a stock solution of the probe with a concentration of 1 mM using DMSO. Incubate the cultured active HeLa cells in a culture medium containing 5 μM TN for 30 min, then stain the cells with 2 μM LTDR (lysosome-tracking deep red fluorescent probe) for 30 min, wash the cells twice with PBS, and then image them using a confocal microscope.
[0054] Figure 4 Confocal picture of active HeLa cells co-stained with TN and LTDR. Among them, the excitation wavelength of TN is 561 nm, and the fluorescence collection wavelength is 570 - 700 nm; the excitation wavelength of LTDR in the red light channel is 635 nm, and the fluorescence collection wavelength is 640 - 740 nm. It can be seen from the figure that the cytoplasmic part stained by TN overlaps significantly with the part stained by the commercial lysosome fluorescent probe, and its co-localization coefficient is 0.82, indicating that the probe can image lysosomes.
[0055] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as a limitation of the present invention itself. Various changes can be made in its form and details without departing from the spirit and scope of the present invention defined by the appended claims.
Claims
1. Application of a fluorescent probe in simultaneous imaging of lysosomes and nucleoli and nucleic acid detection, characterized in that, The structural formula of the fluorescent probe is as follows:
2. The application according to claim 1, wherein The preparation route of the fluorescent probe is as follows:
3. The application according to claim 1, characterized in that, The preparation method of the fluorescent probe specifically includes the following steps: Dissolve compound 1 and 2-iodoethanol in ethanol, stir, reflux at 100 °C, cool and filter, wash with anhydrous EtOH, dry to obtain compound 2; Dissolve compound 2 and 3 in ethanol, stir, add piperidine, reflux at 85 °C, cool to room temperature and wash with petroleum ether; Use a CH2Cl2 / CH3OH mixture as the eluent for column chromatography purification to obtain the product.
4. The application according to claim 3, wherein The volume ratio of the CH2Cl2 / CH3OH mixture is 10:1 to 6:
1.
5. A reagent for simultaneously imaging lysosomes and nucleoli / nucleic acid detection reagent, characterized in that, The structural formula of the fluorescent probe in the reagent is as follows:
6. The reagent according to claim 5, wherein The concentration of the fluorescent probe is 2 - 10 μM.
7. The reagent according to claim 5, characterized in that, The excitation wavelength of the fluorescent probe in the reagent for simultaneous imaging of lysosomes and nucleoli is 400 - 600 nm, and the fluorescence collection wavelength is 550 - 675 nm.
8. The reagent according to claim 5, wherein The solvent in the reagent for simultaneous imaging of lysosomes and nucleoli includes one of DMF, DMSO, ethanol, Gly, H2O, and acetonitrile.
9. The reagent according to claim 5, wherein The reagent for simultaneous imaging of lysosomes and nucleoli further includes the lysosome-tracking deep red fluorescent probe LTDR.
10. The reagent according to claim 5, wherein The nucleic acid concentration range detected by the nucleic acid detection reagent is 0 - 4 mM.