Two-color fluorescent probe for monitoring polarity and viscosity fluctuation of lysosome in cell apoptosis process as well as preparation method and application of two-color fluorescent probe

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly relates to a double-color fluorescent probe for monitoring polarity and viscosity fluctuation of lysosome in a cell apoptosis process as well as a preparation method and application of the double-color fluorescent probe. The novel two-color fluorescent probe Lyso-DCPV is successfully synthesized, and the probe can respond to dynamic changes of polarity and viscosity at the same time. In the probe, N, N-diethyl coumarin serves as a polarity sensitive module (emitting green fluorescence), and N, N-diethyl coumarin and N-propyl quinoline are connected through a twisted double bond to serve as a viscosity sensitive module (emitting red fluorescence). The green fluorescence of the Lyso-DCPV is gradually weakened along with the increase of the polarity, and the red fluorescence of the Lyso-DCPV is remarkably enhanced along with the increase of the viscosity, so that the Lyso-DCPV can simultaneously track the change of the polarity and the viscosity. In addition, the Lyso-DCPV has remarkable lysosome targeting capacity in living cells, and the capacity of tracking polarity and viscosity changes in the living cell lysosome in real time in the drug-induced apoptosis process is achieved for the first time.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a dual-color fluorescent probe for monitoring lysosome polarity and viscosity fluctuations during cell apoptosis, and a preparation method and application thereof. Background Art

[0002] Cell biology is an important discipline in the field of biology. It meticulously studies the complex mechanisms of cell structure, function, physiology, and metabolism. Studying cellular processes is essential for revealing the basic principles of life activities. Among them, apoptosis is a unique form of cell death that regulates individual development and maintains homeostasis through characteristics such as cell shrinkage, chromatin condensation, and nuclear fragmentation. The importance of apoptosis provides an important biological guide for understanding the development and dynamic changes in organisms. In addition, apoptosis is closely related to a variety of pathological conditions, and its complex mechanisms further highlight its research significance. Anti-cancer therapy usually eliminates cancer cells by introducing apoptosis and regulating the expression of genes associated with treatment resistance. Therefore, monitoring the process of cell apoptosis has become an important means of early disease diagnosis and evaluation of the efficacy of apoptotic drugs.

[0003] The complex mechanisms in the apoptotic process inevitably affect multiple factors of the cell microenvironment, such as dynamic characteristics such as pH, polarity and viscosity. Changes in microenvironmental factors can serve as subtle indicators of the degree of apoptosis. At the cellular and subcellular levels, polarity, as a key factor, plays an important role in the differentiation and maintenance of specific regions of cells, and changes in polarity can have a profound impact on a variety of biological activities. Disorders in polarity can cause dysfunction of subcellular organelles, leading to a series of physiological and pathological consequences. Similarly, viscosity, as another important microenvironmental factor, closely regulates the dynamic balance within the cell. Abnormal viscosity can disrupt the intracellular balance, leading to the rupture of subcellular structures and organ dysfunction. Therefore, real-time monitoring of changes in intracellular polarity and viscosity can help reveal the deep mechanisms of cell physiology and pathology.

[0004] Bioimaging using fluorescent imaging tools has become mainstream because of its non-invasiveness, high sensitivity, and superior spatiotemporal resolution. The innovative design of dual-color fluorescent probes can overcome the limitations of single-color probes in monitoring a single microenvironmental parameter and can detect multiple targets simultaneously, thus avoiding potential signal interference. Dual-color fluorescent probes can respond to both polarity and viscosity, making them very suitable for advancing the monitoring of apoptotic cells. However, there are few reports of dual-color fluorescent probes for monitoring lysosomal polarity and viscosity fluctuations during apoptosis. Summary of the invention

[0005] In view of the problems and shortcomings in the prior art, the purpose of the present invention is to provide a dual-color fluorescent probe for monitoring the fluctuation of lysosomal polarity and viscosity during cell apoptosis, and a preparation method and application thereof.

[0006] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a dual-color fluorescent probe for monitoring the fluctuation of lysosomal polarity and viscosity during cell apoptosis. The structural formula of the dual-speed fluorescent probe is as follows:

[0008]

[0009] The second aspect of the present invention provides a method for preparing the above-mentioned dual-color fluorescent probe for monitoring the fluctuation of lysosomal polarity and viscosity during cell apoptosis, comprising adding 2-methyl-1-propylquinolinium and 7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde to piperidine, heating for reaction, and after the reaction is completed, post-treating the reaction solution to obtain;

[0010] Wherein, the structure of 2-methyl-1-propylquinolinium is shown in Formula 1, and the structure of 7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde is shown in Formula 2:

[0011]

[0012] Preferably, the molar ratio of 2-methyl-1-propylquinolinium, 7-(diethylamino)-2-oxo-2H-chromene-3-carbaldehyde and piperidine is 1:(1-1.2):(1-1.2).

[0013] Preferably, the heating reaction temperature is 80-90° C., and the heating reaction time is 6-8 h.

[0014] Preferably, the specific process of the post-treatment includes: cooling the reaction solution to room temperature, removing the solvent and then performing column chromatography separation.

[0015] Preferably, the preparation method of 2-methyl-1-propylquinolinium comprises: adding 2-methylquinoline and iodopropane to ethanol, heating for reaction, cooling to room temperature and filtering, collecting the product, washing and drying.

[0016] Preferably, the molar ratio of 2-methylquinoline to iodopropane is 1:(2.3-2.5).

[0017] Preferably, the temperature of the heating reaction is 80-85°C, and the time of the heating reaction is 8-9h.

[0018] The third aspect of the present invention provides an application of the above-mentioned dual-color fluorescent probe in monitoring the fluctuation of lysosome polarity and viscosity during cell apoptosis.

[0019] Preferably, the fluorescent probe can simultaneously respond to changes in polarity and viscosity in lysosomes with dual-color fluorescence. As the degree of apoptosis deepens, the green fluorescence of the fluorescent probe gradually weakens and the red fluorescence significantly increases, indicating that both polarity and viscosity in lysosomes increase.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention successfully synthesized a new dual-color probe Lyso-DCPV, which can respond to the dynamic changes of polarity and viscosity at the same time. The probe integrates N,N-diethylcoumarin as a polarity-sensitive module (emitting green fluorescence), and N,N-diethylcoumarin and N-propylquinoline connected by a torsional double bond as a viscosity-sensitive module (emitting red fluorescence). The interaction of the two modules gives Lyso-DCPV a dual function: as the polarity increases, its green fluorescence gradually weakens, while as the viscosity increases, its red fluorescence significantly increases, allowing Lyso-DCPV to simultaneously track changes in polarity and viscosity.

[0022] (2) The Lyso-DCPV prepared by the present invention has significant lysosomal targeting ability in living cells, and for the first time realizes the ability to track the changes in polarity and viscosity in the lysosomes of living cells in real time during drug-induced apoptosis. The research results of the present invention show that as the degree of apoptosis deepens, the polarity and viscosity in the lysosomes increase. In addition, the Lyso-DCPV probe also demonstrates its wide applicability by dynamically monitoring the changes in polarity and viscosity during peroxide-induced apoptosis.

[0023] (3) The Lyso-DCPV prepared by the present invention can be used to explore the complex relationship between microenvironment polarity and viscosity changes, which not only expands the understanding of cell dynamics during apoptosis, but also opens up a new direction for the evaluation and development of apoptosis-related therapeutic agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The Lyso-DCPV synthesized by the present invention 1 H NMR spectra;

[0025] Figure 2 The Lyso-DCPV synthesized by the present invention 13 C NMR spectra;

[0026] Figure 3 is the mass spectrum of Lyso-DCPV synthesized by the present invention;

[0027] Figure 4 A is the fluorescence intensity of Lyso-DCPV synthesized by the present invention in 0% 1,4-dioxane (ethanol) and 100% 1,4-dioxane solution, Figure 4 B is the fluorescence intensity of Lyso-DCPV in solvents of different polarities;

[0028] Figure 5 A is the fluorescence intensity of Lyso-DCPV synthesized by the present invention in 0% glycerol (dimethyl sulfoxide) and 100% glycerol solution, Figure 5 B is the fluorescence intensity of Lyso-DCPV in solvents with different viscosities;

[0029] Figure 6 Confocal imaging of live HeLa cells prestained with Lyso-DCPV (10 μM) and then co-incubated with Lyso-Tracker Red, where A is the confocal imaging of Lyso-DCPV, B is the confocal imaging of Lyso-Tracker Red, C is the bright field, D is the confocal fluorescence overlay of Lyso-DCPV and Lyso-Tracker Red, E is the Pearson correlation coefficient, and F is the intensity profile of ROI in Lyso-DCPV and Lyso-Tracker Red images, scale bar: 25 μm;

[0030] Figure 7 Confocal images of HeLa cells treated with different concentrations of cisplatin (0-50 μM) for 12 hours and stained with 10 μM Lyso-DCPV for 2 hours, as well as the relative fluorescence intensity of the green and red channels;

[0031] Figure 8 Confocal photographs of HeLa cells stained with 10 μM Lyso-DCPV and the relative fluorescence intensity of the green and red channels after being treated with cisplatin (30 μM) for different time periods (0-36 hours);

[0032] Fig. 9 These are fluorescence images of HeLa cells after they were pre-stained with 10 μM Lyso-DCPV for 2 hours and then treated with 4 mM hydrogen peroxide for different time periods, as well as the relative fluorescence intensity change curves of the green channel and the red channel. DETAILED DESCRIPTION

[0033] The following examples are only for further explanation of the present invention. It should be noted that all technical and scientific terms used in the present invention have the same meanings as in the technical field to which the present invention belongs unless otherwise specified. The experimental methods in the following examples without specifying specific conditions are all based on conventional techniques in the technical field, or according to the conditions recommended by the manufacturer; the reagents or instruments used without specifying the manufacturer are all conventional products that can be purchased commercially.

[0034] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0035] Example 1

[0036] A dual-color fluorescent probe for monitoring lysosomal polarity and viscosity fluctuations during cell apoptosis, the preparation process of which comprises the following steps:

[0037] (1) Preparation of 2-methyl-1-propylquinolinium

[0038] 2-Methylquinoline (8.58 g, 60.0 mmol) was dissolved in anhydrous ethanol (20 mL), and iodopropane (23.7 g, 140 mmol, 2.3 equiv) was added. The reaction mixture was heated at 80°C for 8 h, cooled to room temperature, filtered, washed with ethanol (3×5 mL), and dried in vacuo to obtain a white solid compound 1, i.e., 2-methyl-1-propylquinolinium.

[0039] The specific reaction formula is as follows:

[0040]

[0041] (2) Preparation of 7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde

[0042] 4-Diethylaminosalicylaldehyde (2.01 g, 10 mmol), piperidine (1 mL) and diethyl malonate (3.20 g, 20 mmol) were added to anhydrous ethanol (30 mL) and stirred at 80 ° C for 6 h. After removing the ethanol, concentrated hydrochloric acid (20 mL) and glacial acetic acid (20 mL) were added for hydrolysis reaction. Stir for another 6 h, cool to room temperature, pour the solution into ice water, add NaOH solution to adjust the pH to 5. After filtration, washing with water, drying, and purification by silica gel chromatography (EtOAc: PE = 1: 10), intermediate 1 (2, 0.81 g, yield 37.3%) was obtained;

[0043] The above reaction formula is:

[0044]

[0045] DMF (1 mL) was added dropwise to POCl3 (1.5 mL) and stirred at room temperature under nitrogen for 20 minutes. Intermediate 1 (0.77 g, 3.5 mmol) dissolved in 4 mL DMF was then added dropwise to the above solution. After stirring at 60 ° C for 10 h, the mixture was poured into ice water and NaOH solution was added to adjust the pH to 7. The precipitate was filtered, washed with water and ethanol, and vacuum dried to obtain an orange-yellow solid (3, 0.50 g, yield 58.3%), i.e. 7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde.

[0046] The above reaction formula is as follows:

[0047]

[0048] (3) Preparation of Lyso-DCPV

[0049] 2-Methyl-1-propylquinolinium (157 mg, 0.5 mmol), 7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde (123 mg, 0.5 mmol) and piperidine (43 mg, 0.5 mmol) were prepared into a solution and refluxed at 80°C for 6 hours. After the reaction mixture was cooled to room temperature, the solvent was removed by rotary evaporation. A purple solid (yield 35%) was separated by column chromatography (DCM:EtOH=50:1, volume ratio), which is a dual-color fluorescent probe for monitoring lysosomal polarity and viscosity fluctuations during cell apoptosis, named Lyso-DCPV.

[0050] The above reaction formula is:

[0051]

[0052] Lyso-DCPV 1 H NMR Figure 1 As shown, 1 H NMR(400MHz,DMSO-d6)δ8.99(d,J=9.0Hz,1H),8.54(dd,J=9.0,3.9Hz,2H),8.4 2(s,1H),8.33(d,J=7.9Hz,1H),8.21-8.12(m,3H),7.92(t,J=7.5Hz,1H),7.62( d,J=9.0Hz,1H),6.86(dd,J=9.0,2.0Hz,1H),6.67-6.63(m,1H),4.99-4.72(m,2 H),3.56-3.50(m,4H),2.04-1.98(m,2H),1.24-1.22(m,3H),1.19-1.16(m,6H).

[0053] Lyso-DCPV 13 C NMR Figure 2 As shown, 13C NMR(101MHz,DMSO-d6)δ160.17,157.26,156.04,154.73,144.23,144.07,135.42,133.93,132.12,130.76, 130.75,129.18,128.22,120.97,119.34,117.40,110.98,96.74,45.00,31.62,30.29,21.94,12.89,11.24.

[0054] The mass spectrum of the above Lyso-DCPV is as follows Figure 3 As shown, MALDI-TOF MS m / z[M] + =calcd.ForC 27 H 29 N2O2413.2223, found 413.2401.

[0055] Example 2

[0056] A dual-color fluorescent probe for monitoring lysosomal polarity and viscosity fluctuations during cell apoptosis, the preparation process of which comprises the following steps:

[0057] (1) Preparation of 2-methyl-1-propylquinolinium

[0058] 2-Methylquinoline (8.58 g, 60.0 mmol) was dissolved in anhydrous ethanol (20 mL), and iodopropane (25.5 g, 150 mmol, 2.5 equiv) was added. The reaction mixture was heated at 85 ° C for 9 h, cooled to room temperature, filtered, washed with ethanol (3×5 mL), and vacuum dried to obtain a white solid compound 1, i.e., 2-methyl-1-propylquinolinium.

[0059] (2) Preparation of 7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde

[0060] 4-Diethylaminosalicylaldehyde (2.01 g, 10 mmol), piperidine (1 mL) and diethyl malonate (3.20 g, 20 mmol) were added to anhydrous ethanol (30 mL) and stirred at 80 ° C for 6 h. After removing the ethanol, concentrated hydrochloric acid (20 mL) and glacial acetic acid (20 mL) were added for hydrolysis reaction. Stir for another 6 h, cool to room temperature, pour the solution into ice water, add NaOH solution to adjust the pH to 5. After filtration, washing with water, drying, and purification by silica gel chromatography (EtOAc: PE = 1: 10), intermediate 1 (2, 0.81 g, yield 37.3%) was obtained;

[0061] DMF (1 mL) was added dropwise to POCl3 (1.5 mL) and stirred at room temperature under nitrogen for 20 minutes. Intermediate 1 (0.77 g, 3.5 mmol) dissolved in 4 mL DMF was then added dropwise to the above solution. After stirring at 60 °C for 10 h, the mixture was poured into ice water and NaOH solution was added to adjust the pH to 7. The precipitate was filtered, washed with water and ethanol, and vacuum dried to obtain an orange-yellow solid, i.e., 7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde.

[0062] (3) Preparation of Lyso-DCPV

[0063] 2-Methyl-1-propylquinolinium (157 mg, 0.5 mmol), 7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde (147 mg, 0.6 mmol) and piperidine (52 mg, 0.6 mmol) were prepared into a solution and refluxed at 90°C for 8 hours. After the reaction mixture was cooled to room temperature, the solvent was removed by rotary evaporation. A purple solid was separated by column chromatography (DCM: EtOH = 50: 1, volume ratio), which is a dual-color fluorescent probe for monitoring lysosomal polarity and viscosity fluctuations during cell apoptosis.

[0064] Performance Testing

[0065] (I) Optical response of Lyso-DCPV to polarity

[0066] The present invention studies the optical response behavior of Lyso-DCPV in solvents of different polarities. The specific method is as follows: Lyso-DCPV (2 μM) prepared in Example 1 is added to 0% 1,4-dioxane (100% ethanol) and 100% 1,4-dioxane solution, respectively, and fluorescence measurement is performed in the range of 400-800 nm using a QuantaMasterTM fluorescence spectrophotometer (PTI, Canada). The excitation and emission slit widths of 2.5 nm are used for spectrum recording, and a quartz cuvette of 2 mm×10 mm is used. The results are shown in FIG. Figure 4 As shown in A.

[0067] from Figure 4 A can be seen that at an excitation wavelength of 390nm, the green fluorescence (470nm) of Lyso-DCPV in a 100% 1,4-dioxane solution is significantly enhanced (about 1.64 times) compared to that in a 0% 1,4-dioxane solution, while its red fluorescence (660nm) remains essentially unchanged in solvents of different polarities. This indicates that the green fluorescence of Lyso-DCPV increases with decreasing polarity, while the red fluorescence is insensitive to changes in polarity.

[0068] The present invention further studies the fluorescence changes of Lyso-DCPV in the range of dielectric constant (ε) from 2.21 (ethanol: 1,4-dioxane volume ratio = 100:0) to 24.30 (ethanol: 1,4-dioxane volume ratio = 100:0). Figure 4 As shown in B. Figure 4 B shows that with the increase of dielectric constant, the green fluorescence intensity of Lyso-DCPV decreases significantly. This result further indicates that the green fluorescence of Lyso-DCPV is very sensitive to changes in polarity.

[0069] (II) Optical response of Lyso-DCPV to viscosity

[0070] The present invention studies the response of the Lyso-DCPV probe to changes in microenvironmental viscosity. The method is as follows: Lyso-DCPV prepared in Example 1 is added to 0% glycerol (dimethyl sulfoxide) and 100% glycerol, and fluorescence measurement is performed in the range of 400-800nm ​​using a QuantaMasterTM fluorescence spectrophotometer (PTI, Canada). The results are shown in Figure 5 A.

[0071] from Figure 5 A It can be seen that the green fluorescence (470nm) of Lyso-DCPV did not change significantly in 0% glycerol (dimethyl sulfoxide) and 100% glycerol solutions. However, the red fluorescence (660nm) of Lyso-DCPV was significantly enhanced in 100% glycerol solution, which was increased by about 6 times compared to 0% glycerol solution. The above results show that the red fluorescence of Lyso-DCPV is highly sensitive to changes in viscosity.

[0072] In order to further quantify the relationship between the red fluorescence of Lyso-DCPV and viscosity, the present invention also conducted fluorescence measurement experiments in dimethyl sulfoxide-glycerol mixed solutions with different volume ratios. The results are as follows: Figure 5 As shown in B. Figure 5 The results of B show that the red fluorescence intensity of Lyso-DCPV increases significantly with the increase of viscosity.

[0073] (III) Targeting of Lyso-DCPV

[0074] The present invention further studied the cellular localization of Lyso-DCPV by confocal laser scanning microscopy (CLSM). The specific method is: add Lyso-Tracker Red or Mito-TrackerRed (100nM) to the confocal culture dish containing HeLa cells for pre-incubation. Before imaging, wash 3 times with PBS, then add culture medium containing 10μM Lyso-DCPV (1% dimethyl sulfoxide) for 2 hours, and wash again with PBS 3 times. Live cell imaging was performed at room temperature using a Nikon confocal laser scanning microscope (Nikon) and a 60x oil objective (Olympus). The fluorescence intensity of 20 cells in each image was analyzed and quantified using ImageJ software. The results are shown in the figure. Figure 6 shown.

[0075] from Figure 6 It can be seen that the green fluorescence signal of Lyso-DCPV highly overlaps with the fluorescence signal of Lyso-Tracker Red in living cells ( Figure 6 AE), and the Pearson correlation coefficient was 0.88 ( Figure 6 F). The above results show that Lyso-DCPV is mainly localized in the lysosomes of cells and is an ideal molecular tool for studying the dynamic changes of lysosomes.

[0076] (IV) Imaging of polarity and viscosity changes of Lyso-DCPV during cell apoptosis

[0077] (1) Imaging of polarity and viscosity changes during cisplatin-induced apoptosis

[0078] HeLa cells were treated with different concentrations of cisplatin (0-50 μM) for 12 hours, or 30 μM cisplatin for different time periods (0-36 hours) to induce apoptosis, and then washed three times with PBS. They were then stained with 10 μM Lyso-DCPV (containing 1% dimethyl sulfoxide) at 37°C for 2 hours, and washed three times with PBS for imaging. The results are shown in Figure 2. Figure 7 and Figure 8 shown.

[0079] Figure 7 A is a confocal image of HeLa cells stained with 10 μM Lyso-DCPV for 2 hours after treatment with different concentrations of cisplatin (0-50 μM) for 12 hours, including red channel, green channel, merged imaging and bright field. Cisplatin concentrations: (A1-A4) 0 μM, (B1-B4) 20 μM, (C1-C4) 30 μM, (D1-D4) 40 μM and (E1-E4) 50 μM. Scale bar: 25 μm. Figure 7B and 7C are the relative fluorescence intensities of the green and red channels after HeLa cells were incubated in different concentrations of cisplatin (AE) using Image J.

[0080] Figure 8 A is a confocal image of HeLa cells stained with 10 μM Lyso-DCPV after treatment with cisplatin (30 μM) for different time periods (0-36 hours). Cisplatin incubation time: (A1-A4) 0 hours, (B1-B4) 6 hours, (C1-C4) 12 hours, (D1-D4) 24 hours, and (E1-E4) 36 hours. Scale bar: 25 μm. Figure 8 B and 8C are the relative fluorescence intensities of the green channel and the red channel in HeLa cells after cisplatin culture for different time periods (0-36 hours).

[0081] Figure 7 and Figure 8 The results showed that with the increase of cisplatin concentration and treatment time, the green fluorescence of Lyso-DCPV gradually weakened, while the red fluorescence significantly enhanced, indicating that Lyso-DCPV can monitor the changes in lysosome polarity and viscosity during cisplatin-induced cell apoptosis.

[0082] (2) Imaging of polarity and viscosity changes during hydrogen peroxide-induced apoptosis

[0083] HeLa cells were treated with 4 mM hydrogen peroxide for 0-8 minutes to induce apoptosis and washed three times with PBS. They were then stained with 10 μM Lyso-DCPV (containing 1% dimethyl sulfoxide) at 37°C for 2 hours and washed three times with PBS for imaging. Fig. 9 shown.

[0084] Fig. 9 A is the fluorescence image of HeLa cells after being stained with 10 μM Lyso-DCPV for 2 hours and then treated with 4 mM hydrogen peroxide for (A1-A4) 0 minutes, (B1-B4) 1 minute, (C1-C4) 2 minutes, (D1-D4) 4 minutes, and (E1-E4) 8 minutes, respectively. Scale bar = 25 μm. Fig. 9 B is the relative fluorescence intensity change curve of the green channel and the red channel in (A1-E1) and (A2-E2) with the incubation time of hydrogen peroxide.

[0085] Fig. 9 The results showed that with the increase of hydrogen peroxide treatment time, the green fluorescence of Lyso-DCPV gradually weakened, while the red fluorescence significantly increased, indicating that Lyso-DCPV can monitor the changes in lysosome polarity and viscosity during hydrogen peroxide-induced cell apoptosis.

[0086] The above embodiments are specific implementation methods of the present invention, but the implementation methods of the present invention are not limited to the above embodiments. Any other combination, change, modification, substitution and simplification that does not exceed the design concept of the present invention shall fall within the protection scope of the present invention.

Claims

1. A dual-color fluorescent probe for monitoring lysosomal polarity and viscosity fluctuations during cell apoptosis, characterized in that: The structural formula of the fluorescent probe is as follows:

2. The method for preparing a dual-color fluorescent probe for monitoring lysosomal polarity and viscosity fluctuations during cell apoptosis according to claim 1, characterized in that: 2-methyl-1-propylquinolinium and 7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde are added to piperidine, heated for reaction, and after the reaction is completed, the reaction solution is post-treated to obtain; Wherein, the structure of 2-methyl-1-propylquinolinium is shown in Formula 1, and the structure of 7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde is shown in Formula 2:

3. The method for preparing a dual-color fluorescent probe for monitoring lysosomal polarity and viscosity fluctuations during cell apoptosis according to claim 2, characterized in that: The molar ratio of 2-methyl-1-propylquinolinium, 7-(diethylamino)-2-oxo-2H-chromene-3-carbaldehyde and piperidine is 1:(1-1.2):(1-1.2).

4. The method for preparing a dual-color fluorescent probe for monitoring lysosomal polarity and viscosity fluctuations during cell apoptosis according to claim 2, characterized in that: The temperature of the heating reaction is 80-90° C., and the time of the heating reaction is 6-8 hours.

5. The method for preparing a dual-color fluorescent probe for monitoring lysosomal polarity and viscosity fluctuations during cell apoptosis according to claim 2, characterized in that: The specific process of the post-treatment includes: cooling the reaction solution to room temperature, removing the solvent and then performing column chromatography separation.

6. The method for preparing a dual-color fluorescent probe for monitoring lysosomal polarity and viscosity fluctuations during cell apoptosis according to claim 2, characterized in that: The preparation method of 2-methyl-1-propylquinolinium comprises: adding 2-methylquinoline and iodopropane into ethanol, heating for reaction, cooling to room temperature and filtering, collecting the product, washing and drying.

7. The method for preparing a dual-color fluorescent probe for monitoring lysosomal polarity and viscosity fluctuations during cell apoptosis according to claim 6, characterized in that: The molar ratio of the 2-methylquinoline to iodopropane is 1:(2.3-2.5).

8. The method for preparing a dual-color fluorescent probe for monitoring lysosomal polarity and viscosity fluctuations during cell apoptosis according to claim 6, characterized in that: The temperature of the heating reaction is 80-85° C., and the time of the heating reaction is 8-9 hours.

9. Use of the dual-color fluorescent probe according to claim 1 in monitoring the fluctuations of lysosomal polarity and viscosity during cell apoptosis.

Images