Fluorescent probe for detecting viscosity of cell membrane as well as preparation method and application of fluorescent probe
By designing fluorescent probes with negatively charged sulfonates to enhance cell membrane lipophilicity and near-infrared emission, the problems of specificity and accuracy in cell membrane viscosity detection were solved, enabling real-time monitoring and imaging of cell membrane viscosity.
Patent Information
- Application Number
- CN202511613536.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-06
- Publication Date
- 2026-02-24
AI Technical Summary
Current technologies lack fluorescent probes that can specifically target cell membranes and detect viscosity fluctuations, making it difficult to achieve real-time monitoring of the cell membrane microenvironment.
A fluorescent probe was designed that introduces a negatively charged sulfonate to prevent the probe from entering the cell, enhances its lipophilicity to the cell membrane, and expands near-infrared emission through a conjugated structure. It also utilizes the carbon-carbon single bond rotation restriction to emit fluorescence in a high-viscosity environment, thereby enabling the detection of cell membrane viscosity.
This fluorescent probe has excellent cell membrane anchoring ability, can accurately detect changes in cell membrane viscosity, reduce the influence of background fluorescence, improve analytical accuracy, and is suitable for cell membrane and in vivo viscosity fluorescence imaging.
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Figure CN121554446A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of small molecule probe technology, and more specifically to a fluorescent probe for detecting cell membrane viscosity, its preparation method, and its application. Background Technology
[0002] The cell membrane is the first line of defense for maintaining cellular homeostasis. Besides playing a crucial role in certain cellular activities such as survival, growth, division, and differentiation, the cell membrane also participates in processes like material exchange, energy transfer, and signal transduction with the external environment. Abnormalities in certain indicators within the cell membrane microenvironment can directly affect normal cellular function, leading to the development of various diseases and even cell carcinogenesis.
[0003] Viscosity is a crucial physiological parameter in biology, participating in numerous cellular processes. In particular, viscosity within the cell membrane microenvironment directly impacts cell membrane fluidity, protein-protein interactions, and signal transduction. Furthermore, abnormal viscosity is associated with certain diseases, including diabetes, atherosclerosis, Parkinson's disease, and Alzheimer's disease. Therefore, real-time monitoring of cell membrane viscosity is of paramount importance.
[0004] Fluorescence imaging technology possesses inherent advantages such as high sensitivity, non-invasiveness, rapid response, ease of operation, and high spatiotemporal adaptability, giving it significant advantages in the detection of analytes in vivo. To date, various fluorescent probes have been developed for viscosity measurement, among which probes capable of targeting specific organelles can detect viscosity in subcellular structures (such as mitochondria and lysosomes). However, monitoring viscosity fluctuations in the cell membrane microenvironment has remained a challenging task, likely due to the lack of a probe that possesses both specific cell membrane targeting capabilities and viscosity-activating properties.
[0005] Therefore, how to develop a specific cell membrane targeting probe is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0006] In view of this, the purpose of the present invention is to provide a fluorescent probe for detecting cell membrane viscosity, its preparation method and application, so as to overcome the shortcomings of the prior art.
[0007] To achieve the above objectives, the present invention adopts the following technical solution: A fluorescent probe for detecting cell membrane viscosity, with the following structural formula: .
[0008] A method for preparing the above-mentioned fluorescent probe for detecting cell membrane viscosity specifically includes the following steps: (1) Compound 1, 4-diethylaminoketo acid, was reacted with cyclohexanone in concentrated sulfuric acid, poured into crushed ice and stirred, then perchloric acid was added and stirred, allowed to stand, and filtered to obtain compound 2; (2) Compound 2 and 4-diethylaminobenzaldehyde were dissolved in anhydrous ethanol, heated to reflux, the solvent was removed, and the mixture was subjected to chromatography to obtain compound 3; (3) Compound 3, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA) were dissolved in N,N-dimethylformamide (DMF), stirred at room temperature, and then compound 4 3-(tetradecylamino)propane-1-sulfonate was added. The mixture was stirred and stirred to remove the solvent. The mixture was then subjected to chromatography to obtain a fluorescent probe (M1) for detecting cell membrane viscosity. The reaction equation is: .
[0009] Furthermore, in step (1) above, the mass fraction of concentrated sulfuric acid is 60%-80%, and the mass ratio of concentrated sulfuric acid to crushed ice is 1:(10~30); the molar ratio of cyclohexanone, 4-diethylaminoketo acid and concentrated sulfuric acid is 1:(0.25~0.75):(30~100).
[0010] Furthermore, in step (1) above, the mass fraction of perchloric acid is 70%, and the molar ratio of perchloric acid to 4-diethylaminoketo acid is 1:(5~10).
[0011] Furthermore, in step (1) above, the reaction temperature is 60~90℃ and the time is 1~3 h; the stirring time in the crushed ice is 2 min; the stirring time in the perchloric acid is 10~20 min; and the standing time is 1~3 h.
[0012] Furthermore, in step (2) above, the molar ratio of compound 2 to 4-diethylaminobenzaldehyde is 1:(1~2).
[0013] Furthermore, in step (2) above, the heating reflux time is 6~8 h; the chromatography is specifically: using methanol and dichloromethane with a volume ratio of 1:(40~60) as eluents, and using silica gel column chromatography.
[0014] Furthermore, in step (3) above, the molar ratio of compound 3, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine and 3-(tetradecylamino)propane-1-sulfonate is 1:2:2:2.
[0015] Furthermore, in step (3) above, the stirring time at room temperature is 20~40 min; the stirring time is 12~24 h; the chromatography is specifically: using methanol and dichloromethane with a volume ratio of 1:(20~50) as eluents, and using silica gel column chromatography.
[0016] The present invention also claims protection for the application of the above-described fluorescent probe or the fluorescent probe prepared by the above-described method in cell and in vivo viscosity fluorescence imaging.
[0017] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: 1. The negatively charged sulfonate in the fluorescent probe of this invention can effectively prevent the probe from entering the cell, thus giving it good cell membrane anchoring ability; the introduction of diethylaminophenyl not only enhances the lipophilicity of the probe to the cell membrane, but also expands the conjugated structure of the probe, ensuring the near-infrared emission of the probe.
[0018] 2. In low-viscosity phosphate buffer, the fluorescent probe of this invention does not fluoresce because the carbon-carbon single bonds can rotate freely; however, in a high-viscosity environment (such as glycerol), the rotation of the single bonds is restricted, and the probe can emit near-infrared fluorescence at 775 nm, thereby achieving viscosity detection.
[0019] 3. The fluorescent probe of this invention has advantages in detecting cell membrane viscosity. It can firmly adhere to the cell membrane over time, accurately measure the viscosity changes on the cell membrane, and this detection is almost unaffected by background fluorescence, which can greatly reduce the probability of false positive results and improve the accuracy of analysis.
[0020] 4. The invented fluorescent probe has excellent anchoring and imaging capabilities as well as viscosity-triggered near-infrared (NIR) fluorescence response characteristics. It can be used for fluorescence imaging of cell membrane viscosity and viscosity imaging analysis in breast cancer mice. Attached Figure Description
[0021] Figure 1 The image shows the viscosity response of probe M1; where A is the fluorescence emission spectrum of the probe in glycerol-phosphate buffer at different ratios; and B is the linear relationship between the logarithm of the fluorescence intensity at 775 nm and the logarithm of the viscosity. Figure 2 This is a schematic diagram illustrating the selectivity of probe M1; where the vertical axis represents the fluorescence intensity of the probe at 775 nm, and the horizontal axis represents the order of substances from 1 to 1. - (100 μM); 2. Mn 2+ (100 μM); 3. CH3COO - (100 μM); 4. Na + (100 μM); 5. Na2S2O3(100 μM); 6. NH4 + (100 μM); 7. SCN - (100 μM); 8. Cu 2+(100 μM); 9.HCO3 - (100 μM); 10. NO3 - (100 μM); 11. Mg 2+ (100 μM); 12. ClO - (100 μM); 13. Al 3+ (100 μM); 14. CO3 2- (100 μM); 15. NO2 - (100 μM); 16. SO3 2- (100 μM); 17. SO4 2- (100 μM); 18. K + (100 μM); 19. Fe 3+ (100 μM); 20. glutathione (1 mM); 21. H2O2(100 μM); 22. PO4 3- (100 μM); 23. bovine serum albumin (10 μg / mL); 24. glycine (1 mM); 25. leucine (1 mM); 26. cysteine (1 mM); 27. glutamic acid (1 mM); 28.homocysteine (1 mM); 29. 1 O2(100 μM); 30. NO (100 μM); 31. OH . (100 μM); Figure 3 This is a confocal image of probe M1 on the cell membrane over time; in A, the red channel row is the fluorescence image of the probe in the cell, and the bright field row is the bright field image of the cell; B is a statistical graph of cell fluorescence intensity. Figure 4 The images show confocal images of probe M1 in HeLa cells under different viscosity conditions; in A, the red channel row is the fluorescence image of the probe in the cell, and the bright field row is the bright field image of the cell; B is a statistical graph of cell fluorescence intensity. Detailed Implementation
[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0023] Example 1 The preparation method of a fluorescent probe for detecting cell membrane viscosity specifically includes the following steps: (1) 10 mL of 70% concentrated sulfuric acid was cooled to 0 °C. Then, 3.92 g of cyclohexanone (40 mmol) and 6.26 g of compound 1,4-diethylaminoketo acid (20 mmol) were added to the concentrated sulfuric acid. The reaction solution was heated to 90 °C and stirred for 1.5 h. The reaction mixture was then poured into 100 g of ice water and stirred for 2 min. Then, 2 mL of perchloric acid was added dropwise and stirring was continued for 20 min. After standing for 1 h, the precipitate was collected by filtration to obtain compound 2 (red solid, 1 g, yield 70%). The reaction equation is: ; (2) 475 mg of compound 2 (1 mmol) and 177 mg of 4-diethylaminobenzaldehyde (1 mmol) were dissolved in 20 mL of anhydrous ethanol, and the mixture was heated under reflux for 4 h. After the reaction was completed, the solvent was removed under reduced pressure, and then the mixture was purified by silica gel column chromatography using methanol and dichloromethane in a volume ratio of 1:60 to obtain compound 3 (black solid, 350 mg, yield 55%). The reaction equation is: ; (3) 128 mg of compound 3 (0.2 mmol), 152 mg of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (0.4 mmol) and 51 mg of N,N-diisopropylethylamine (0.4 mmol) were dissolved in N,N-dimethylformamide. After stirring at room temperature for 30 min, 123 mg of compound 4 3-(tetradecylamino)propane-1-sulfonate (0.4 mmol) was added and stirring was continued for 12 h. After the reaction was completed, the mixture was extracted with dichloromethane, the solvent was removed under reduced pressure, and then the mixture was eluted with methanol and dichloromethane at a volume ratio of 1:50. The solution was then purified by silica gel column chromatography to obtain fluorescent probe M1 (blue powder, 100 mg, yield 90%). The reaction equation is: .
[0024] Performance testing 1. Characterization of probe M1 probe M1 1 H NMR and13 The C NMR values are as follows: 1 H NMR (400 MHz, DMSO) δ 8.21-8.10 (m, 1H), 7.70-7.61 (m, 4H), 7.58-7.49 (m, 1H), 7.44-7.38 (m, 1H), 7.35-7.27 (m, 1H), 7.26-7.20 (m, 1H), 7.19-7.07 (m, 1H), 6.88-6.81 (d, J =8.8 Hz, 2H), 3.73-3.56 (m, 4H), 3.54-3.42 (m,4H), 3.32-3.08 (m, 4H), 3.07-2.80 (m,4H), 1.88-1.62 (m,4H), 1.30-0.88 (m,41H). 13 C NMR (201 MHz, DMSO) δ 168.17, 164.08, 157.57, 157.06, 154.69,150.25, 140.85, 136.10, 135.50, 135.42, 131.58, 130.44, 129.95, 129.81,129.14, 127.22, 123.16, 122.23, 116.93, 116.03, 112.10, 95.90, 55.40, 48.83,47.97, 45.55, 44.56, 42.95, 31.79, 29.63, 29.62, 29.57, 29.54, 29.53, 29.50,29.34, 29.32, 29.20, 27.61, 26.57, 26.34, 24.70, 22.58, 21.39, 14.43, 13.01.HR-MS: calcd for C 52 H 74 N3O5S + (M+H + ): 852.5349; found 852.5356, and forC 52 H 73 N3O5SNa + (M+Na + ): 874.5169; found 874.5172. 2. Response of probe M1 to viscosity Probe M1 was prepared as a 1 mM stock solution using dimethyl sulfoxide. Glycerol and phosphate buffer (pH 7.4) were mixed in different proportions to adjust sample viscosity. The probe was dissolved in solutions of varying viscosities, maintaining a final probe concentration of 10 µM. Figure 1 As shown in Figure A, the fluorescence intensity of the probe at 775 nm gradually increases with the increase of the glycerol volume ratio in the medium (glycerol from 0% to 99%). Additionally, as... Figure 1 As shown in Figure B, the logarithm of the fluorescence intensity at 775 nm of the probe exhibits a good linear relationship with the logarithm of the viscosity.
[0025] 3. Selectivity of probe M1 In a phosphate buffer solution containing 10 µM probe M1, common ions, reactive oxygen species, and amino acids were added to investigate the probe's viscosity selectivity. Figure 2 As shown, after the addition of the above substances, the fluorescence signal of the probe at 775 nm hardly changed, but it showed obvious fluorescence in glycerol, indicating that the probe has high selectivity for viscosity.
[0026] 4. Imaging capability of probe M1 on the cell membrane Cells were incubated with probe M1 at a concentration of 10 µM, and irradiated with 640 nm excitation light. Fluorescence signals at 700 nm to 800 nm were collected over 10–60 min. Figure 3 As shown in Figure A, the probe remained firmly attached to the cell membrane even after 60 minutes. Figure 3 In the figure, B represents the statistical value of cell fluorescence intensity. Experimental results show that probe M1 has excellent targeting and anchoring ability to the cell membrane, and can serve as a reliable tool for long-term detection of cell membrane-related microenvironment parameters (such as viscosity).
[0027] 5. Probe M1 is used for cell viscosity imaging. HeLa cells were divided into three groups: Group 1 was incubated with probe M1 at a concentration of 10 µM for 15 min (control group); Group 2 was treated with nystatin at a concentration of 25 µM for 15 min, followed by incubation with the probe for 15 min; Group 3 was treated with nystatin at a concentration of 50 µM for 15 min, followed by incubation with the probe for 15 min. Cells were then irradiated with 640 nm excitation light, and fluorescence signals at 700–800 nm were collected. Figure 4 As shown in Figure A, compared with the control group, the cells in the second and third groups showed stronger fluorescence signals. Figure 4 In the figure, B represents the statistical value of cell fluorescence intensity. Experimental results show that probe M1 can be used for imaging analysis of cell viscosity.
[0028] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A fluorescent probe for detecting cell membrane viscosity, characterized in that, The structural formula is .
2. A method for preparing a fluorescent probe for detecting cell membrane viscosity as described in claim 1, characterized in that, Specifically, the following steps are included: (1) Compound 1, 4-diethylaminoketo acid, was reacted with cyclohexanone in concentrated sulfuric acid, poured into crushed ice and stirred, then perchloric acid was added and stirred, allowed to stand, and filtered to obtain compound 2; (2) Compound 2 and 4-diethylaminobenzaldehyde were dissolved in anhydrous ethanol, heated to reflux, the solvent was removed, and the mixture was subjected to chromatography to obtain compound 3; (3) Compound 3, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine were dissolved in N,N-dimethylformamide and stirred at room temperature. Then, compound 4 3-(tetradecylamino)propane-1-sulfonate was added and stirred continuously. The solvent was removed and the mixture was subjected to chromatography to obtain the fluorescent probe for detecting cell membrane viscosity.
3. The method for preparing a fluorescent probe for detecting cell membrane viscosity according to claim 2, characterized in that, In step (1), the mass fraction of the concentrated sulfuric acid is 60%-80%, and the mass ratio of the concentrated sulfuric acid to the crushed ice is 1:(10~30); the molar ratio of the cyclohexanone, 4-diethylaminoketo acid and the concentrated sulfuric acid is 1:(0.25~0.75):(30~100).
4. The method for preparing a fluorescent probe for detecting cell membrane viscosity according to claim 2, characterized in that, In step (1), the mass fraction of perchloric acid is 70%, and the molar ratio of perchloric acid to 4-diethylaminoketo acid is 1:(5~10).
5. A method for preparing a fluorescent probe for detecting cell membrane viscosity according to claim 2, characterized in that, In step (1), the reaction temperature is 60~90℃ and the time is 1~3 h; the stirring time when pouring into crushed ice is 2 min; the stirring time when adding perchloric acid is 10~20 min; and the standing time is 1~3 h.
6. The method for preparing a fluorescent probe for detecting cell membrane viscosity according to claim 2, characterized in that, In step (2), the molar ratio of compound 2 to 4-diethylaminobenzaldehyde is 1:(1~2).
7. A method for preparing a fluorescent probe for detecting cell membrane viscosity according to claim 2, characterized in that, In step (2), the heating reflux time is 6-8 h; the chromatography is specifically: using methanol and dichloromethane with a volume ratio of 1:(40-60) as eluents, and using silica gel column chromatography.
8. A method for preparing a fluorescent probe for detecting cell membrane viscosity according to claim 2, characterized in that, In step (3), the molar ratio of compound 3, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine and 3-(tetradecylamino)propane-1-sulfonate is 1:2:2:
2.
9. A method for preparing a fluorescent probe for detecting cell membrane viscosity according to claim 2, characterized in that, In step (3), the stirring time at room temperature is 20-40 min; the stirring time is 12-24 h; the chromatography is specifically: using methanol and dichloromethane with a volume ratio of 1:(20-50) as eluents, and using silica gel column chromatography.
10. The application of a fluorescent probe as described in claim 1 or a fluorescent probe prepared by any one of claims 2-9 in cell and in vivo viscosity fluorescence imaging.