Viscosity response type fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides a viscosity response type fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of biochemical detection and analysis. The chemical structural formula of the fluorescent probe is shown in the specification, X represents halogen, and n is an integer between 0 and 10. The method can be applied to specific viscosity detection, targeted positioning of cell membranes and distinguishing of normal and tumor cells by utilizing the viscosity difference of the cell membranes. The probe synthesized by the invention can realize accurate detection of the viscosity change of the cell membrane.

Description

Technical Field

[0001] This invention belongs to the field of biochemical detection and analysis technology, and relates to a fluorescent probe for measuring cell membrane viscosity, specifically a viscosity-responsive fluorescent probe, its preparation method, and its application. Background Technology

[0002] The cell membrane, as a vital organelle, plays an indispensable role in maintaining cellular homeostasis. Composed of different types of phospholipids, cholesterol, and proteins, the cell membrane possesses complex biophysical properties (polarity, viscosity, and electrostatic properties, etc.), among which viscosity plays a crucial role in biological processes such as membrane fluidity, transmembrane transport, and biological signal transduction. Numerous studies have shown that abnormal changes in cell membrane viscosity are closely related to the pathogenesis and progression of diseases such as Alzheimer's disease, diabetes, local inflammation, and cancer. Therefore, developing a tool for accurately detecting changes in cell membrane viscosity is of significant value in clinical research and early disease diagnosis.

[0003] Small molecule fluorescent probes possess advantages such as high specificity, high sensitivity, and excellent biocompatibility, and have become effective tools in clinical research and disease diagnosis. While some viscosity fluorescent probes have been developed, those specifically targeting cell membranes are extremely rare. Currently available commercial cell membrane fluorescent probes such as Did, Dir, and CellMaskGreen are all lipid-soluble molecules. Probes that do not bind to the cell membrane during staining will generate background signals, impairing the accuracy of viscosity detection. Therefore, these probes can only be used for fluorescent labeling of cell membranes and are insufficient for precise detection of changes in cell membrane viscosity. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a viscosity-responsive fluorescent probe, its preparation method, and its application, thereby solving the technical problem that existing cell membrane fluorescent probes are all lipid-soluble molecules, and probes that do not bind to the cell membrane during staining will generate background signals, making it difficult to accurately detect changes in cell membrane viscosity.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A viscosity-responsive fluorescent probe has the following chemical structure: (Formula V) Formula V.

[0006] In the formula: X represents a halogen, selected from F, Cl, Br and I; preferably Br.

[0007] n is an integer between 0 and 10; preferably 0, 1, 4 or 7; most preferably 1.

[0008] This invention also protects a method for preparing the viscosity-responsive fluorescent probe as described above, the synthetic route of which is shown below: .

[0009] The method first uses 2-(4-pyridyl)acetonitrile and alkylaminobenzaldehyde as reactants to carry out a Knoevenagel condensation reaction to obtain heteroaryl-arylacrylonitrile; then, using heteroaryl-arylacrylonitrile and 3-bromopropyltrimethylammonium as reactants, a nucleophilic substitution reaction is carried out to obtain a viscosity-responsive fluorescent probe.

[0010] Specifically, the method includes the following steps: Step 1, Synthesis of the intermediate: 1 mmol of the compound shown in Formula II and 2 mmol of the compound shown in Formula I are dissolved in an organic solvent and heated under nitrogen protection. After the reaction is complete, the reaction system is cooled, filtered, and the yellow solid is collected, which is the intermediate. Specifically, in Step 1, the organic solvent is at least one of toluene, acetonitrile, and acetic anhydride, preferably acetonitrile; the reaction temperature is 60–180°C, preferably 90°C; the reaction time is 2–8 h, preferably 6 h.

[0011] Step two, probe synthesis: 1 mmol of the intermediate synthesized in step one and 1 mmol of the compound shown in formula IV are dissolved in an organic solvent and reacted under nitrogen protection. After the reaction, the solvent is removed by rotary evaporation, and the crude product is purified by silica gel column chromatography to obtain a dark red solid, which is the viscosity-responsive fluorescent probe. Specifically, in step two, the organic solvent is anhydrous ethanol; the reaction temperature is 90℃; and the reaction time is 2–6 h, preferably 4 h.

[0012] This invention also protects the application of the viscosity-responsive fluorescent probe described above for specific viscosity detection. This application includes: firstly, dissolving the viscosity-responsive fluorescent probe in dimethyl sulfoxide to prepare a 20 mM probe stock solution; then diluting the probe stock solution in a methanol-glycerol mixed solution, with the concentration of the viscosity-responsive fluorescent probe being 20 μM; and measuring the fluorescence signal intensity of the viscosity-responsive fluorescent probe at 592 nm using 470 nm as the excitation wavelength.

[0013] This invention also protects the application of the viscosity-responsive fluorescent probe described above in specific viscosity detection under different interfering substances. This application includes: firstly, dissolving the viscosity-responsive fluorescent probe in dimethyl sulfoxide to prepare a 20 mM probe stock solution; then diluting the probe stock solution in a 10 mM phosphate buffer solution at pH 7.4; the concentration of the viscosity-responsive fluorescent probe used is 20 μM; then adding Na... + K + Fe 3+ Cu 2+ Mg2+ Histidine, alanine, leucine, glycine, threonine, glutathione, cysteine, hydrogen peroxide, and hypochlorous acid were used as excitation wavelengths. Finally, the fluorescence signal intensity of the viscosity-responsive fluorescent probe at 592 nm was measured using 470 nm as the excitation wavelength.

[0014] This invention also protects the application of the viscosity-responsive fluorescent probe described above for targeted localization of cell membranes. This application includes: adding the viscosity-responsive fluorescent probe to cultured cells at a final concentration of 10 μM; incubating for 30 minutes; then removing the cell culture medium, washing several times with PBS, adding fresh cell culture medium, and then collecting fluorescence signals near a wavelength of 592 nm using a 488 nm laser. Specifically, the cells are human breast cancer cells MCF-7.

[0015] This invention also protects the application of the viscosity-responsive fluorescent probe described above for targeted localization on the cell membrane at different temperatures. The application includes: adding the viscosity-responsive fluorescent probe to cultured cells at a final concentration of 5 μM; incubating at 4°C, 25°C, and 37°C for 30 minutes each; then removing the cell culture medium, washing several times with PBS, adding fresh cell culture medium, and then collecting fluorescence signals near a wavelength of 592 nm using a 488 nm laser. Specifically, the cells are human breast cancer cells MCF-7.

[0016] This invention also protects the application of the viscosity-responsive fluorescent probe described above in distinguishing normal and tumor cells based on differences in cell membrane viscosity. This application includes: adding the viscosity-responsive fluorescent probe to cultured cells at a final concentration of 5 μM; incubating at 37°C in the dark for 30 minutes; removing the cell culture medium; washing several times with PBS; adding fresh cell culture medium; and then collecting fluorescence signals near a wavelength of 592 nm using a 488 nm laser. Specifically, the cells used are normal human breast epithelial cells MCF-10A, human breast cancer cells MBA-MD-231, human breast cancer cells MCF-7, normal human pancreatic ductal epithelial cells HPDE6-C7, human pancreatic cancer cells QGP1, human gastric mucosal epithelial resuscitation cells GES1, human gastric cancer cells MKN-45, and human gastric cancer cells NCI-N87.

[0017] Compared with the prior art, the present invention has the following technical effects: (I) The probe synthesized in this invention is an amphiphilic molecule with a rotatable double bond structure. The rotation of the double bond in the free probe molecule leads to fluorescence quenching. When the lipophilic end (dimethylamine moiety) is inserted into the cell membrane, the double bond rotation is restricted, resulting in fluorescence emission. Furthermore, the fluorescence signal increases with increasing cell membrane viscosity. Simultaneously, thanks to the introduction of the quaternary ammonium salt structure, the probe exhibits excellent water solubility and does not produce non-specific fluorescence due to self-aggregation in aqueous solution. During cell imaging, only probes bound to the cell membrane produce fluorescence emission signals; unbound probes do not cause signal interference, thus achieving wash-free imaging. Therefore, the probe of this invention can achieve precise detection of changes in cell membrane viscosity.

[0018] (II) The probe synthesis route of the present invention is simple, and probe synthesis can be achieved through only two steps.

[0019] (III) Compared with normal cells, tumor cells generally have a lower membrane potential. The two positive charges carried by the probe molecules of this invention will promote the binding of the probe to the tumor cell membrane, thereby achieving the distinction between tumor cells and normal cells. Attached Figure Description

[0020] Figure 1 The fluorescence response spectra of the fluorescent probe Pyr shown in Equation V-1 under different viscosity conditions are shown.

[0021] Figure 2 The fluorescence response of the fluorescent probe Pyr, as shown in Equation V-1, to different interfering substances at 592 nm is given.

[0022] Figure 3 This is a colocalization fluorescence imaging image of the fluorescent probe Pyr (as shown in Formula V-1) and the commercial cell membrane dye Did in human breast cancer cells MCF-7.

[0023] Figure 4 The images show fluorescence imaging of human breast cancer cells MCF-7 after incubation with the fluorescent probe Pyr shown in Formula V-1 at different temperatures.

[0024] Figure 5 The fluorescence imaging results of the fluorescent probe Pyr shown in Formula V-1 in normal human mammary epithelial cells MCF-10A, human breast cancer cells MBA-MD-231 and human breast cancer cells MCF-7 are shown. Figure 5 In the image: (a) is a fluorescence imaging image. (b) is the quantization result of (a).

[0025] Figure 6 The fluorescence imaging results of the fluorescent probe Pyr shown in Formula V-1 in normal human pancreatic ductal epithelial cells HPDE6-C7 and human pancreatic cancer cells QGP1 are shown. Figure 6 In the image: (a) is a fluorescence imaging image. (b) is the quantization result of (a).

[0026] Figure 7 The fluorescence imaging results of the fluorescent probe Pyr shown in Formula V-1 in human gastric mucosal epithelial resuscitation cells GES1, human gastric cancer cells MKN-45, and human gastric cancer cells NCI-N87 are shown. Figure 7 In the image: (a) is a fluorescence imaging image. (b) is the quantization result of (a).

[0027] The specific content of the present invention will be further explained in detail below with reference to the embodiments. Detailed Implementation

[0028] It should be noted that, unless otherwise specified, all raw materials used in this invention are those known in the art.

[0029] The following are specific embodiments of the present invention. It should be noted that the present invention is not limited to the following specific embodiments. All equivalent modifications made based on the technical solutions of this application fall within the protection scope of the present invention.

[0030] Example 1: This embodiment provides a method for preparing a viscosity-responsive fluorescent probe, and its synthetic route is shown below: .

[0031] The method specifically includes the following steps: Step 1, Synthesis of the intermediate: 1 mmol of 2-(4-pyridyl)acetonitrile (the compound shown in Formula II) and 2 mmol of 4-dimethylaminobenzaldehyde (the compound shown in Formula I-1) were dissolved in acetonitrile and reacted under nitrogen protection at a temperature of 90 °C for 6 h. After the reaction was completed, the reaction system was cooled and filtered to collect the yellow solid (i.e., the intermediate 3-(4-dimethylaminophenyl)-2-(pyridin-4-yl)acrylonitrile, as shown in Formula III-1).

[0032] Step 2, Probe Synthesis: 1 mmol of the intermediate synthesized in Step 1 and 1 mmol of 3-bromopropyltrimethylammonium (the compound shown in Formula IV-1) were dissolved in anhydrous ethanol and reacted under nitrogen protection at a temperature of 90°C for 4 hours. After the reaction was completed, the solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography to obtain a dark red solid (Pyr).

[0033] Example 2: This embodiment provides a viscosity-responsive fluorescent probe prepared using the method of Example 1 or 2, named Pyr, whose chemical structure is shown in Formula V-1 below: Equation V-1.

[0034] The proton NMR spectrum, carbon NMR spectrum, and high-resolution mass spectrometry data of the fluorescent probe Pyr shown in Equation V-1 are as follows: ¹H NMR (400 MHz, DMSO) δ 8.86 (d, J = 7.0 Hz, 2H), 8.44 (s, 1H), 8.17 (d, J = 7.0 Hz, 2H), 7.98 (d, J = 9.2 Hz, 2H), 6.81 (d, J = 9.2 Hz, 2H), 4.47 (t, J = 7.3 Hz, 2H), 3.02 (s, 6H), 2.98 (s, 9H), 2.38 (t, J = 7.5 Hz, 2H), 2.37 – 2.26 (m, 2H).

[0035] 13C NMR (100 MHz, DMSO) δ 157.12, 153.56, 150.37, 146.03, 146.16, 129.72, 129.60, 124.81, 124.73, 124.58, 118.85, 111.73, 117, 63, 99.51, 65.39, 56.64, 54.81, 41.36, 19.22.

[0036] High-resolution mass spectrometry (HR-ESI-MS): m / z calcd for [M]2+([C22H30N4]2+): 175.12297, found: 175.12289.

[0037] Example 3: This embodiment describes the application of the viscosity-responsive fluorescent probe from Example 2 for specific viscosity detection. The application includes the following steps: the fluorescent probe Pyr is dissolved in dimethyl sulfoxide (DMSO) to prepare a 20 mM standard solution, which is then aliquoted and stored at 0–4°C for later use. Fluorescence spectroscopy is performed in a 1 cm × 1 cm quartz cuvette. The probe stock solution is uniformly dispersed in methanol-glycerol mixed solutions of different volume ratios, resulting in a final probe concentration of 20 μM. Then, using a fluorescence spectrophotometer with an excitation wavelength of 470 nm, the fluorescence signal of the probe in solutions of different viscosities is measured.

[0038] In this embodiment, the final test result is as follows: Figure 1 As shown, the results indicate that the fluorescence signal intensity of the probe near 592 nm increases with increasing ambient viscosity; when the ambient viscosity increases from 1 cp to 1499 cp, the fluorescence signal of the probe at 592 nm is enhanced by 7556 times.

[0039] Example 4: This embodiment demonstrates the application of the viscosity-responsive fluorescent probe from Example 2 in specific viscosity detection under different interfering substances. The application includes the following steps: fluorescence spectroscopy was performed in a 1 cm × 1 cm quartz cuvette; the probe stock solution was uniformly dispersed in a phosphate buffer solution (10 mM, pH 7.4), resulting in a final probe concentration of 20 μM; then, using 470 nm as the excitation wavelength, the fluorescence signal intensity of the probe at 592 nm was measured on a fluorescence spectrophotometer; subsequently, different interfering substances (Na+, Na ... + K + Fe 3+ Cu 2+ Mg 2+ The fluorescence signal intensity of the probe at 592 nm was measured using histidine, alanine, leucine, glycine, threonine, glutathione, cysteine, hydrogen peroxide, and hypochlorous acid (glycerol was used as a control) at an excitation wavelength of 470 nm.

[0040] In this embodiment, the final test result is as follows: Figure 2 As shown, the results indicate that, by comparing the fluorescence signal intensity of the probe before and after the addition of interfering substances, the addition of interfering substances such as salt ions, amino acids, and common redox substances has a negligible effect on the fluorescence signal of the probe at 592 nm, indicating that the probe can specifically detect viscosity changes.

[0041] Example 5: This embodiment describes the application of the viscosity-responsive fluorescent probe from Example 2 for targeted localization of the cell membrane. The application includes the following steps: Step 1, Cell Culture: In this example, the cells used are human breast cancer cells MCF-7. The culture medium is DMEM or 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cells are cultured in a sterile incubator at 37°C and 5% carbon dioxide. After the cells reach confluence, they are digested with trypsin, divided into flasks, and passaged.

[0042] Step 2, Cellular Fluorescence Co-localization Assay of Fluorescent Probe Pyr: This experiment used cell membrane red fluorescence detection reagent (Did) and the fluorescent probe Pyr shown in Formula V-1 to co-stain cells for probe co-localization testing. Cells were seeded in confocal culture dishes, and after full adhesion, 5 μM of the prepared Did dye and 10 μM of the fluorescent probe shown in Formula V-1 were added for co-incubation. After 30 minutes, the culture medium was removed, the cells were washed three times with PBS, fresh culture medium was added, and confocal imaging was performed. A 644 nm laser was used for Did dye, with a collection wavelength range of 663 nm; a 488 nm laser was used for Pyr, with a collection wavelength range of 592 nm.

[0043] In this embodiment, the final test result is as follows: Figure 3 As shown, the fluorescent probe Pyr exhibits a high degree of overlap with the fluorescence signal of commercially available cell membrane fluorescent dyes. The calculated Pearson coefficient is 0.8, indicating that the fluorescent probe Pyr can be targeted and localized to the cell membrane.

[0044] Example 6: This embodiment describes the application of the viscosity-responsive fluorescent probe from Example 2 for targeted localization on the cell membrane at different temperatures. The application includes the following steps: Step 1, Cell Culture: In this example, the cells used are human breast cancer cells MCF-7. The culture medium is DMEM or 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cells are cultured in a sterile incubator at 37°C and 5% carbon dioxide. After the cells reach confluence, they are digested with trypsin, divided into flasks, and passaged.

[0045] Step 2: Fluorescence imaging experiment of the fluorescent probe Pyr at different temperatures: MCF-7 cells were seeded in confocal culture dishes. After full adhesion, 5 μM Pyr was added and co-incubated with the cells. Incubation was performed at 4℃, 25℃, and 37℃ for 30 minutes in the dark. The culture medium was then removed, the cells were washed three times with PBS, and fresh culture medium was added for confocal imaging. A 488 nm laser was used to collect fluorescence signals around 592 nm.

[0046] In this embodiment, the final test result is as follows: Figure 4 As shown, the results indicate that the fluorescence signal of the fluorescent probe Pyr on the cell membrane decreases with increasing temperature under different temperature conditions.

[0047] Example 7: This embodiment describes the application of the viscosity-responsive fluorescent probe from Example 2 in distinguishing between normal and tumor cells based on differences in cell membrane viscosity. The application includes the following steps: Step 1, Cell Culture: In this example, the cells used were normal human mammary epithelial cells MCF-10A, human breast cancer cells MBA-MD-231, human breast cancer cells MCF-7, normal human pancreatic ductal epithelial cells HPDE6-C7, human pancreatic cancer cells QGP1, human gastric mucosal epithelial resuscitation cells GES1, human gastric cancer cells MKN-45, and human gastric cancer cells NCI-N87. The culture medium was DMEM or 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were cultured in a sterile incubator at 37°C and 5% carbon dioxide. After the cells reached confluence, they were digested with trypsin, divided into flasks, and passaged.

[0048] Step 2: Fluorescence imaging experiment of fluorescent probes in different cells: Different cells were seeded separately in confocal culture dishes. After full adhesion, 5 μM Pyr was added and co-incubated with the cells at 37°C in the dark for 30 minutes. The culture medium was then removed, the cells were washed three times with PBS, and fresh culture medium was added for confocal imaging. A 488 nm laser was used to collect fluorescence signals around 592 nm. The results are as follows: Figures 5 to 7 As shown, the imaging results of the three cell lines all indicate that the fluorescence imaging signal of normal cells is significantly weaker than that of tumor cells.

Claims

1. A viscosity-responsive fluorescent probe, having a chemical structure as shown in the following formula: wherein X represents halogen; and n is an integer between 0 and 10. Formula V; The method comprises: firstly, using 2-(4-pyridyl)acetonitrile and alkylaminobenzaldehyde as raw materials, performing Knoevenagel condensation reaction to obtain a heteroaryl-aryl acrylonitrile; and then, using the heteroaryl-aryl acrylonitrile and 3-bromopropyl trimethylammonium as raw materials, performing nucleophilic substitution reaction to obtain the viscosity-responsive fluorescent probe.

2. A method for preparing the viscosity-responsive fluorescent probe according to claim 1, characterized by, The molar ratio of 2-(4-pyridyl)acetonitrile to alkylaminobenzaldehyde is 1:2; and the molar ratio of the heteroaryl-aryl acrylonitrile to 3-bromopropyl trimethylammonium is 1:

1.

3. The method for preparing the viscosity-responsive fluorescent probe as described in claim 2, characterized in that, The Knoevenagel condensation reaction is performed at a temperature of 60-180℃ for 2-8 h; and the nucleophilic substitution reaction is performed at a temperature of 90℃ for 2-6 h.

4. The method for preparing the viscosity-responsive fluorescent probe as described in claim 2, characterized in that, 5.The viscosity-responsive fluorescent probe of claim 1 for specific viscosity detection. The application comprises: diluting a mother solution of the viscosity-responsive fluorescent probe to 20 μM, and then measuring the fluorescence signal intensity at 592 nm with 470 nm as the excitation wavelength.

6. The use according to claim 5, wherein the compound is ###0002### 7.The viscosity-responsive fluorescent probe of claim 1 for targeted localization of cell membranes. The application comprises: adding the viscosity-responsive fluorescent probe to cultured cells, with a final concentration of 5-10 μM; incubating for 30 minutes, removing the cell culture medium, washing several times with PBS, and then adding new cell culture medium; and then using a laser with a wavelength of 488 nm to collect the fluorescence signal near 592 nm.

8. Use according to claim 7, wherein the compound is ###0002### 9.The viscosity-responsive fluorescent probe of claim 1 for distinguishing normal cells from tumor cells by using the viscosity difference of cell membranes. The application comprises: adding the viscosity-responsive fluorescent probe to cultured cells, with a final concentration of 5 μM; incubating for 30 minutes at 37℃ in the dark, removing the cell culture medium, washing several times with PBS, and then adding new cell culture medium; and then using a laser with a wavelength of 488 nm to collect the fluorescence signal near 592 nm; and the fluorescence imaging signal of normal cells is weaker than that of tumor cells.

10. Use according to claim 9, wherein ​

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