Fluorescent probe for detecting viscosity change and preparation and detection methods thereof

Abstract

The invention discloses a fluorescent probe for detecting viscosity change as well as a preparation method and a detection method thereof. The specific chemical structural formula of the fluorescent probe is shown in the specification. The fluorescent probe disclosed by the invention is simple to prepare, low in cost, high in detection sensitivity, good in selectivity and good in biocompatibility; the fluorescent probe can be used for detecting the change of mitochondrial viscosity in apoptotic and inflammatory cells; the detection method provided by the invention is simple to operate, the adopted solvent is low in price, and post-treatment is convenient.

Description

Technical Field

[0001] The present invention relates to the field of chemical analysis and detection technology, and in particular to a fluorescent probe for detecting viscosity changes and a preparation and detection method thereof, and more specifically to a fluorescent probe for detecting mitochondrial viscosity changes in apoptotic and inflammatory cells and a preparation and detection method thereof. Background Art

[0002] Mitochondria, as one of the most important organelles in cells, provide energy for cellular life and participate in a variety of cellular metabolic processes, including central metabolism, ATP production, and apoptosis. Mitochondrial damage and a range of cellular dysfunctions can lead to abnormal changes in mitochondrial viscosity. For example, during mitophagy, cells clear dysfunctional mitochondria and recycle their nutrients, a process closely associated with changes in mitochondrial viscosity. Therefore, visualizing changes in mitochondrial viscosity can provide a better understanding of the pathogenic mechanisms of physiological processes and may become an effective method for diagnosing mitochondrial diseases.

[0003] Due to the complexity of living systems, the detection of mitochondrial viscosity remains a significant challenge. Traditional viscosity detection methods often suffer from complex pre-processing, low sensitivity, and the inability to monitor in real time, making them difficult to use in living cells or for in vivo imaging. In contrast, fluorescent probes are widely used in biological research and disease monitoring due to their ease of operation, high sensitivity, and real-time monitoring. However, their shorter emission wavelengths make it difficult to effectively overcome the scattering and absorption effects of tissues, thus affecting signal transmission and detection accuracy. Furthermore, the smaller Stokes shift causes the emission spectrum of the fluorescent probe to overlap with background fluorescence, increasing background fluorescence interference and further reducing the probe's signal-to-noise ratio. This problem is particularly significant in cell or tissue viscosity detection, reducing the sensitivity and accuracy of fluorescent probes in practical applications. Therefore, the development of fluorescent probes with longer emission wavelengths and larger Stokes shifts can effectively reduce background interference and enhance penetration into deep tissues, providing important reference and application value for the current development of viscosity-sensitive fluorescent probes. Summary of the Invention

[0004] To address the above issues, the present invention provides a fluorescent probe for detecting viscosity changes, as well as a preparation and detection method thereof. Through molecular design, a fluorescent probe structure with a large Stokes shift and excellent biocompatibility is obtained. This fluorescent probe can detect changes in mitochondrial viscosity in apoptotic and inflammatory cells, giving it the advantages of specific selectivity, high sensitivity, and good biocompatibility.

[0005] According to one object of the present invention, the present invention provides a fluorescent probe for detecting viscosity changes, the specific chemical structure of which is as follows: .

[0006] According to the second object of the present invention, the present invention provides a method for preparing the above-mentioned fluorescent probe for detecting viscosity changes, comprising the following steps: S1. Dissolve dimethylaminocinnamaldehyde and quinaldine iodoethane in anhydrous ethanol, add piperidine, and reflux under cooling at 78°C for 12 h. After the reaction is complete, remove the solvent under reduced pressure and purify by column chromatography to obtain SKL as a black solid. S2. Dissolve quinaldine iodoethane and 4-(dimethylamino)benzaldehyde in anhydrous ethanol, add piperidine, and reflux under cooling at 78°C for 12 hours. After the reaction is complete, remove the solvent under reduced pressure and purify by column chromatography to obtain a dark green solid DKL. S3. Dissolve 4-methylpyridinium iodide and dimethylaminocinnamaldehyde in anhydrous ethanol, add piperidine, and reflux at 78°C for 12 hours. After the reaction is complete, remove the solvent under reduced pressure. Purify by column chromatography to obtain a purple solid SBD. S4. Dissolve 4-methylpyridinium iodide and 4-(dimethylamino)benzaldehyde in anhydrous ethanol, add piperidine, and cool and reflux at 78°C for 12 hours. After the reaction is completed, the solvent is removed under reduced pressure. Purify by column chromatography to obtain a red solid DBD.

[0007] Furthermore, in step S1, the volume ratio of the elution phase for column chromatography purification is methanol: dichloromethane = 1:100, and the molar ratio of dimethylaminocinnamaldehyde: quinaldine iodoethane is 1:1.

[0008] Furthermore, in step S2, the volume ratio of the elution phase for column chromatography purification is methanol: dichloromethane = 1:100, and the molar ratio of quinaldine iodoethane: 4-(dimethylamino)benzaldehyde is 1:1.

[0009] Furthermore, in step S3, the volume ratio of the elution phase for column chromatography purification is methanol: dichloromethane = 1:100, and the molar ratio of 4-methylpyridinium iodide: dimethylaminocinnamaldehyde is 1:1.

[0010] Furthermore, in step S4, the volume ratio of the elution phase for column chromatography purification is methanol: dichloromethane = 1:100, and the molar ratio of 4-methylpyridinium iodide: 4-(dimethylamino)benzaldehyde is 1:1.

[0011] Furthermore, in steps S1 to S4, the reactions are all carried out under nitrogen protection.

[0012] According to the third object of the present invention, the present invention provides a method for detecting the above-mentioned fluorescent probe for detecting viscosity changes, comprising the following steps: The probes SKL, DKL, SBD and DBD were dissolved in DMSO and prepared at 1×10 -3mol / L stock solution; PBS solution and glycerol were mixed in different volume ratios to prepare test systems with different viscosities for fluorescence testing.

[0013] Furthermore, the method further comprises the following steps: L-O2 cells were preincubated with 1 μg / mL lipopolysaccharide for 12 h and then incubated with 10 μM probe for 30 min as a cellular inflammation model; L-O2 cells were incubated with 10 μM probe SKL in culture medium for 30 min as a control group of the cell inflammation model; L-O2 cells were incubated with monensin and nystatin (10 μM) for 30 min and then incubated with SKL for 30 min to serve as the apoptosis model group; L-O2 cells were incubated with 10 μM SKL for 30 minutes as a control group for the apoptosis model; biological cell experiments were performed.

[0014] Beneficial effects: The fluorescent probe provided by the present invention is simple to prepare, low in cost, has high detection sensitivity, good selectivity and good biocompatibility; the fluorescent probe can detect changes in mitochondrial viscosity in apoptotic and inflammatory cells; the detection method provided by the present invention is simple to operate, uses a low-cost solvent and is convenient for post-processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The synthetic route of the fluorescent probes SKL, DKL, SBD, and DBD of the present invention is shown in FIG. Figure 2 is the hydrogen nuclear magnetic resonance spectrum of the fluorescent probe SKL of the present invention; Figure 3 is the carbon NMR spectrum of the fluorescent probe SKL of the present invention; Figure 4 is the mass spectrum of the fluorescent probe SKL of the present invention; Figure 5 The fluorescence intensity diagrams of the fluorescent probes DBD, DKL, SBD, and SKL of the present invention in solvents of different polarities are shown; Figure 6 This is a fluorescence intensity diagram of the fluorescent probe SKL of the present invention in different viscosity systems; Figure 7 This is a graph showing changes in mitochondrial viscosity in inflammatory cells detected by the fluorescent probe SKL of the present invention; Figure 8 This is a comparison chart of the fluorescence intensity of the fluorescent probe SKL of the present invention; Figure 9 This is a graph showing changes in mitochondrial viscosity in apoptotic cells detected by the fluorescent probe SKL of the present invention; Figure 10This is a comparison chart of the fluorescence intensity of the fluorescent probe SKL of the present invention. DETAILED DESCRIPTION

[0016] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0017] Example 1 A fluorescent probe for detecting viscosity changes, the specific chemical structure is shown below: .

[0018] Example 2 like Figure 1-Figure 4 As shown, a method for preparing a fluorescent probe for detecting viscosity changes includes the following steps: S1. Under nitrogen atmosphere, dissolve dimethylaminocinnamaldehyde (175 mg, 1 mmol) and quinaloxaline iodide (299 mg, 1 mmol) in 20 ml of anhydrous ethanol, add 2 drops of piperidine, and reflux at 78°C for 12 h. After the reaction is complete, the solvent is removed under reduced pressure to obtain a crude product. The crude product is separated by thin-layer chromatography in methanol and dichloromethane to obtain SKL as a black solid. S2. Under a nitrogen atmosphere, dissolve quinaldine iodoethane (299 mg, 1 mmol) and 4-(dimethylamino)benzaldehyde (149 mg, 1 mmol) in 20 ml of anhydrous ethanol, add 2 drops of piperidine, and reflux at 78°C for 12 h. After the reaction is complete, the solvent is removed under reduced pressure to obtain a crude product. The crude product is separated by thin-layer chromatography in methanol and dichloromethane to obtain DKL as a dark green solid. S3. Under a nitrogen atmosphere, dissolve 4-methylpyridinium iodide (235 mg, 1 mmol) and dimethylaminocinnamaldehyde (175 mg, 1 mmol) in 20 ml of anhydrous ethanol, add 2 drops of piperidine, and reflux at 78°C for 12 h. After the reaction is complete, the solvent is removed under reduced pressure to obtain a crude product. The crude product is separated by thin-layer chromatography in methanol and dichloromethane to obtain SBD as a purple solid. S4. Under a nitrogen atmosphere, dissolve 4-methylpyridinium iodide (235 mg, 1 mmol) and 4-(dimethylamino)benzaldehyde (149 mg, 1 mmol) in 20 ml of anhydrous ethanol, add 2 drops of piperidine, and cool and reflux at 78°C for 12 h. After the reaction is completed, the solvent is removed under reduced pressure to obtain a crude product. The crude product is separated by thin layer chromatography in methanol and dichloromethane solutions to obtain a red solid DBD.

[0019] During the above preparation process: In step S1, the volume ratio of the elution phase for column chromatography purification is methanol: dichloromethane = 1:100.

[0020] In step S2, the volume ratio of the elution phase for column chromatography purification is methanol: dichloromethane = 1:100.

[0021] In step S3, the volume ratio of the elution phase for column chromatography purification is methanol: dichloromethane = 1:100.

[0022] In step S4, the volume ratio of the elution phase for column chromatography purification is methanol: dichloromethane = 1:100.

[0023] Example 3 A method for detecting changes in mitochondrial viscosity in apoptotic and inflammatory cells using a fluorescent probe comprises the following steps: The probes SKL, DKL, SBD and DBD were dissolved in DMSO and prepared at 1×10 -3 mol / L stock solution; PBS solution and glycerol were mixed at different volume ratios to prepare test systems with different viscosities for fluorescence testing. Fluorescence spectra were tested at excitation wavelengths of 482 nm, 501 nm, 535 nm, and 550 nm, with both the Ex site and Em site at 5 nm. L-O2 cells were preincubated with 1 μg / mL lipopolysaccharide for 12 h and then incubated with 10 μM probe for 30 min as a cellular inflammation model; L-O2 cells were incubated with 10 μM probe SKL in culture medium for 30 min as a control group of the cell inflammation model; L-O2 cells were incubated with monensin and nystatin (10 μM) for 30 min and then incubated with SKL for 30 min to serve as the apoptosis model group; L-O2 cells were incubated with 10 μM SKL for 30 minutes as a control group for the apoptosis model; biological cell experiments were performed.

[0024] like Figure 5 As shown in Figure 3, the fluorescence emission spectra of four fluorescent probes in solvents with different viscosities and polarities were tested, and it was found that SKL was least affected by the polarity of the solvent.

[0025] like Figure 6 As shown in Figure 1, the fluorescence response of SKL in different viscosity environments was tested, and the results showed that the fluorescence intensity increased with increasing viscosity.

[0026] like Figure 7 and Figure 8As shown, SKL detects changes in mitochondrial viscosity in inflammatory cells.

[0027] like Figure 9 and Figure 10 As shown, SKL detected changes in mitochondrial viscosity in apoptotic cells.

[0028] The fluorescent probe of the present invention is simple to prepare, low in cost, has high detection sensitivity, good selectivity, and good biocompatibility, and can detect changes in mitochondrial viscosity in apoptotic and inflammatory cells. The detection method provided by the present invention is simple to operate, uses inexpensive solvents, and is convenient for post-processing.

[0029] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fluorescent probe for detecting viscosity changes, characterized in that: The specific chemical structure is shown below: .

2. The method for preparing a fluorescent probe for detecting viscosity changes according to claim 1, wherein: The following steps are involved: S1. Dissolve dimethylaminocinnamaldehyde and quinaldine iodoethane in anhydrous ethanol, add piperidine, and reflux under cooling at 78°C for 12 h. After the reaction is complete, remove the solvent under reduced pressure and purify by column chromatography to obtain SKL as a black solid. S2. Dissolve quinaldine iodoethane and 4-(dimethylamino)benzaldehyde in anhydrous ethanol, add piperidine, and reflux under cooling at 78°C for 12 hours. After the reaction is complete, remove the solvent under reduced pressure and purify by column chromatography to obtain a dark green solid DKL. S3. Dissolve 4-methylpyridinium iodide and dimethylaminocinnamaldehyde in anhydrous ethanol, add piperidine, and reflux at 78°C for 12 hours. After the reaction is complete, remove the solvent under reduced pressure. Purify by column chromatography to obtain a purple solid SBD. S4. Dissolve 4-methylpyridinium iodide and 4-(dimethylamino)benzaldehyde in anhydrous ethanol, add piperidine, and cool and reflux at 78°C for 12 hours. After the reaction is completed, the solvent is removed under reduced pressure. Purify by column chromatography to obtain a red solid DBD.

3. The method for preparing a fluorescent probe for detecting viscosity changes according to claim 2, wherein: In step S1, the volume ratio of the elution phase for column chromatography purification is methanol: dichloromethane = 1:100, and the molar ratio of dimethylaminocinnamaldehyde: quinaldine iodoethane is 1:

1.

4. The method for preparing a fluorescent probe for detecting viscosity changes according to claim 2, wherein: In step S2, the volume ratio of the elution phase for column chromatography purification is methanol: dichloromethane = 1:100, and the molar ratio of quinaldine iodoethane: 4-(dimethylamino)benzaldehyde is 1:

1.

5. The method for preparing a fluorescent probe for detecting viscosity changes according to claim 2, wherein: In step S3, the volume ratio of the elution phase for column chromatography purification is methanol:dichloromethane=1:100, and the molar ratio of 4-methylpyridinium iodide:dimethylaminocinnamaldehyde is 1:

1.

6. The method for preparing a fluorescent probe for detecting viscosity changes according to claim 2, wherein: In step S4, the volume ratio of the elution phase for column chromatography purification is methanol:dichloromethane=1:100, and the molar ratio of 4-methylpyridinium iodide:4-(dimethylamino)benzaldehyde is 1:

1.

7. The method for preparing a fluorescent probe for detecting viscosity changes according to claim 2, wherein: In steps S1 to S4, the reactions are all carried out under nitrogen protection.

8. The method for detecting viscosity changes using a fluorescent probe according to claim 1, wherein: The following steps are involved: The probes SKL, DKL, SBD and DBD were dissolved in DMSO and prepared at 1×10 -3 mol / L stock solution; PBS solution and glycerol were mixed in different volume ratios to prepare test systems with different viscosities for fluorescence testing.

9. The method for detecting viscosity changes using a fluorescent probe according to claim 8, wherein: The following steps are also included: L-O2 cells were preincubated with 1 μg / mL lipopolysaccharide for 12 h and then incubated with 10 μM probe for 30 min as a cellular inflammation model; L-O2 cells were incubated with 10 μM probe SKL in culture medium for 30 min as a control group of the cell inflammation model; L-O2 cells were incubated with monensin and nystatin (10 μM) for 30 min and then incubated with SKL for 30 min to serve as the apoptosis model group; L-O2 cells were incubated with 10 μM SKL for 30 minutes as a control group for the apoptosis model; biological cell experiments were performed.

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