Fluorescent probe for detecting tension of cell membrane as well as preparation method and application of fluorescent probe

Abstract

The invention provides a fluorescent probe for detecting tension of a cell membrane and a preparation method and application thereof, the fluorescent probe has a structural formula as shown in formula I: # imgabs0 #, and the invention also provides a detection method for tension change of the cell membrane, and the detection method comprises the following steps: S1) co-incubating the fluorescent probe and cells; s2) providing stimulation influencing cell membrane tension change for the cells, and respectively detecting the fluorescence lifetime of the fluorescent probe before and after the stimulation by adopting an imaging system; and S3) based on the fluorescence lifetime data obtained in the step S2), calculating the relative variation of the cell membrane tension so as to determine the dynamic change trend of the cell membrane tension. The fluorescent probe disclosed by the invention is low in cost and good in biocompatibility, and can be used for carrying out long-time imaging observation on cells. Meanwhile, the probe has cell membrane targeting property, can be specifically positioned on a cell membrane, and can be used for microimaging of the cell membrane.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and particularly to a fluorescent probe for detecting cell membrane tension, a preparation method thereof, and an application thereof. Background Art

[0002] Cell membrane tension is one of the indispensable factors for cell morphology and function. Under various physiological and pathological conditions, changes in cell membrane tension can alter cell morphology and function, which is an important way to study and intervene in various cell behaviors and physiological responses. Therefore, obtaining the spatial and temporal distribution information of cell membrane tension in real time is of great significance for deeply understanding the biophysical mechanism from the perspective of cell mechanics, revealing the intracellular homeostasis and kinetic processes, and studying the pathogenesis and treatment methods of related diseases.

[0003] Existing measurement methods include physical mechanics methods: (1) Micropipette technique: By aspirating the cell membrane and measuring the deformation to calculate the tension, it is only applicable to non-adherent cells and has strong invasiveness. (2) Atomic force microscope (AFM): Using a cantilever probe to contact the membrane surface to measure the mechanical response, but the single-point detection efficiency is low and it may damage the membrane structure. (3) Optical tweezers technique: By capturing microspheres to apply tension, it requires complex calibration and cannot achieve multi-target synchronous detection.

[0004] In the currently reported schemes, although the physical mechanics measurement method can provide quantitative measurement, the operation difficulty is very high. In addition, each measurement can only evaluate the mechanical properties of a single target or a single site, and these measurement methods based on cell membrane contact cannot measure the mechanical properties inside the cell, so the application scenarios are limited.

[0005] Fluorescence resonance energy transfer technique (FRET), by inserting a pair of fluorescent proteins that can undergo energy transfer into the phospholipid bilayer, detects the ratio change of the fluorescence intensities emitted at two wavelengths after being excited by one excitation wavelength. The efficiency of energy transfer between the two fluorescent proteins is inversely proportional to the sixth power of the distance between them, making it very sensitive to changes in distance. FRET technology provides molecular-level resolution for membrane tension research, but its disadvantages mainly focus on probe dependence, environmental sensitivity, spatio-temporal resolution limitation, and complex data processing.

[0006] The disadvantages of FRET technology are as follows: 1. Uneven probe expression: When relying on genetic engineering to express probes, differences in probe concentrations in different cells or membrane regions may lead to signal deviations, especially in heterogeneous regions such as lipid rafts. 2. Limitations in sensitivity, dynamic range, and effective distance: The efficiency of FRET highly depends on the distance between the donor and the acceptor (usually 2 - 10 nm). If the change in probe spacing caused by membrane tension changes exceeds this range, the signal may not respond linearly. 3. Photobleaching: Prolonged illumination causes fluorescence signal attenuation, affecting long-term dynamic monitoring. Frequent calibration or the use of anti-bleaching agents is required, which may interfere with the cell physiological state. 4. Interference from non-specific signals: Changes in intracellular pH, ion concentration, temperature, or other fluorescent molecules may interfere with the FRET efficiency, and strict control experiments are needed. 5. Multiple parameters affecting the signal: In addition to distance, factors such as probe orientation and environmental polarity also affect the FRET efficiency, and multi-dimensional calibration is required. 6. Application limitations: It relies on genetic manipulation and requires transfection or transgenic expression of probes, which limits its application in primary cells or cells that are difficult to transfect. In addition, FRET imaging in deep tissues or living animals may be affected by tissue scattering and absorption.

[0007] Existing fluorescent probes (such as ) although having molecular specificity, their excitation wavelength (488 nm) is easily interfered by the autofluorescence of biological tissues, and the signal decays severely during the penetration process in deep tissues, making it difficult to achieve in-situ detection at the living body level. Summary of the Invention

[0008] In view of the above-mentioned requirements in the prior art, namely a method for real-time detecting the spatial distribution and dynamic changes of cell membrane tension, as well as the problems, the present invention provides a cell membrane-targeted organic small molecule probe, which establishes a fluorescence lifetime-based membrane tension visualization detection method by utilizing the principle of restricted molecular motion luminescence combined with fluorescence lifetime imaging technology.

[0009] One aspect of the present invention provides a fluorescent probe having a structural formula shown in Formula I:

[0010]

[0011] Another aspect of the present invention provides a preparation method of the above fluorescent probe; the preparation method includes the following steps:

[0012] S1) Prepare the intermediate shown in Formula II: Condense the compound shown in Formula III and 4-(4-methylpiperazine) benzaldehyde to obtain the intermediate shown in Formula III;

[0013] S2) The intermediate shown in II undergoes a nucleophilic substitution reaction with an alkyl halide to obtain the compound shown in Formula I; where

[0014]

[0015]

[0016] Further, in step S1), the catalyst for the condensation reaction is a weak base; the weak base is preferably piperidine, and more preferably a mixture of acetic acid and piperidine.

[0017] Further, in step S1), the conditions for the condensation reaction are heating under reflux at 120 - 150 °C.

[0018] Further, in step S1) and / or S2), a purification step is further included.

[0019] Another aspect of the present invention provides the use of the above-mentioned fluorescent probe as a detection reagent for detecting cell membrane tension.

[0020] Further, the cell membrane is the cell membrane of in vitro living cells, the cell membrane of cells in vivo, and a cell membrane-like membrane having cell membrane properties. The cell membrane-like membrane having cell membrane properties, for example, liposomes, artificial cell membranes - lipid vesicles.

[0021] Another aspect of the present invention provides a method for detecting changes in cell membrane tension, the method comprising the following steps:

[0022] S1) Co-incubating the above-mentioned fluorescent probe with cells;

[0023] S2) Providing a stimulus to the cells that affects the change in cell membrane tension, and using an imaging system to separately detect the fluorescence lifetime of the fluorescent probe before and after the stimulus is given;

[0024] S3) Based on the fluorescence lifetime data obtained in step S2), calculating the relative change amount of the cell membrane tension, thereby determining the dynamic change trend of the cell membrane tension.

[0025] Further, the imaging system is selected from a fluorescence lifetime imaging microscope, a fluorescence microscope, a confocal microscope, a two-photon microscope, a small animal in vivo optical imaging system, and a time-correlated single photon counting system.

[0026] Further, the stimulus in step S2) is adding a reagent, changing the cell incubation environment, or increasing physical stimulation.

[0027] Another aspect of the present invention provides a method for visualizing changes in cell membrane tension, the method comprising the following steps:

[0028] S1) Co-incubating the above-mentioned fluorescent probe with cells;

[0029] S2) Providing a stimulus to the cells that affects the change in cell membrane tension, and using an imaging system to separately observe the fluorescence change of the fluorescent probe before and after the stimulus is given, to achieve visualization of the change in cell membrane tension.

[0030] In another aspect of the present invention, there is provided a detection reagent for cell membrane tension, and the detection reagent contains the above-mentioned fluorescent probe.

[0031] In another aspect of the present invention, there is provided a real-time monitoring system for cell membrane tension, including: the above-mentioned fluorescent probe; an imaging system for detecting the fluorescence lifetime of the fluorescent probe, which is used to detect the fluorescence lifetime of the fluorescent probe; and an algorithm module for analyzing the fluorescence lifetime data.

[0032] In another aspect of the present invention, there is provided the use of the above-mentioned fluorescent probe in the preparation of a detection reagent for cell membrane imaging.

[0033] In another aspect of the present invention, there is provided a method for cell membrane imaging, and the method includes the following steps:

[0034] S1) Co-incubate the above-mentioned fluorescent probe with cells;

[0035] S2) Use the imaging system to image the fluorescence image

[0036] Further, the imaging system is selected from a fluorescence lifetime imaging microscope, a fluorescence microscope, a confocal microscope, a two-photon microscope, a small animal in vivo optical imaging system, and a time-correlated single photon counting (TCSPC) system.

[0037] In another aspect of the present invention, there is provided a detection reagent for cell membrane imaging, and the detection reagent contains the above-mentioned fluorescent probe.

[0038] In another aspect of the present invention, there is provided a cell membrane imaging system, including: the above-mentioned fluorescent probe; a fluorescence imaging module for collecting fluorescence imaging.

[0039] Beneficial effects

[0040] The fluorescent probe of the present invention has low cost and good biocompatibility, and can perform long-term imaging observation on cells.

[0041] Meanwhile, it has cell membrane targeting property, can specifically localize on the cell membrane, and can be used for microscopic imaging of the cell membrane.

[0042] The optimal excitation light of the fluorescent probe of the present invention is 570 nm, and the emission is at 650 nm, which can effectively avoid the influence of the autofluorescence of biological samples and has a wide application scenario in the study of cell mechanical mechanisms. Description of the drawings

[0043] Figure 1 It is a synthetic route diagram of the fluorescent probe molecule MEM-NIR1.

[0044] Figure 2The UV-visible absorption spectrum and fluorescence emission spectrum of the fluorescent probe molecule MEM-NIR1.

[0045] Figure 3 The result graph of the cytotoxicity test of the fluorescent probe molecule MEM-NIR1.

[0046] Figure 4 The laser confocal image of the fluorescent probe molecule MEM-NIR1 incubated with Hela cells for 15 min.

[0047] Figure 5 The co-localization image of the fluorescent probe molecule MEM-NIR1 and DIO (cell membrane dye).

[0048] Figure 6 The FLIM image and fluorescence lifetime of the fluorescent probe molecule MEM-NIR1 in GUVs with different components (membrane tension).

[0049] Figure 7 The fluorescence lifetime image of Hela of the fluorescent probe molecule MEM-NIR1 under different osmotic shocks.

[0050] Figure 8 The relationship between the fluorescence lifetime of the fluorescent probe molecule MEM-NIR1 and the osmotic pressure applied to cells. Specific implementation mode

[0051] Specifically, the present invention provides a cell membrane fluorescent probe, which has hydrophobicity and can target and anchor at the hydrophobic end of phospholipid molecules. The tightness of the arrangement between phospholipid molecules and the force between molecules will affect the rotation of the rotor of the probe, thereby causing a change in the fluorescence lifetime of the probe. Moreover, the fluorescence lifetime of the fluorescent probe is positively correlated with the magnitude of the force in the surrounding environment. That is, when the cell membrane tension is large, the squeezing effect of phospholipid molecules on the fluorescent probe is stronger, and the fluorescence lifetime of the probe is larger. Therefore, by establishing a model of cell membrane tension and fluorescence lifetime with this probe, measuring the magnitude of the fluorescence lifetime in the cell membrane can reflect the membrane tension situation. Combining with fluorescence lifetime imaging microscopy (FLIM) for fluorescence lifetime-resolved imaging, the spatial distribution and dynamic changes of cell membrane tension in living cells can be monitored in real time.

[0052] The solution adopted in the present invention is the fluorescence probe method. Fluorescence probes are tools for bioimaging and biorecognition. They can target specific structures or biomolecules in cells and produce fluorescence under the excitation of light in a specific wavelength band, thereby realizing the tracking and research of biomolecules or cell structures at the micro-nano scale. Fluorescence lifetime refers to the average residence time of a molecule in the excited state before returning to the ground state after being excited by a light pulse. When a fluorophore is excited by an excitation light, it will jump to the excited (S1) state. When the excitation light is removed, the fluorescent molecule will fall back to the ground state (S0) from the S1 state. Fluorescence lifetime refers to the time it takes for the fluorescence intensity of the molecule to decrease to 1 / e. Generally speaking, fluorescence lifetime is different from fluorescence intensity. It is an inherent property of the fluorophore, independent of the excitation light wavelength, insensitive to the concentration of the fluorophore (low concentration), the intensity of the excitation light or the duration of illumination, and not affected by photobleaching. It only depends on the structure of the fluorophore molecule itself and the environment it is in. Therefore, fluorescence lifetime imaging technology has high accuracy in the quantitative analysis of intracellular membrane tension.

[0053] By designing a fluorescence probe that can target the cell membrane and has mechanical force sensitivity, staining the cells, and the mechanical characteristics of the membrane affect the fluorescence lifetime of the probe, the magnitude of the cell membrane tension can be reflected by the fluorescence lifetime characteristics exhibited by the fluorescence probe under FLIM. The solution most similar to the present invention is Probe: It senses the change in the lipid bilayer membrane structure through the torsion angle and polarization of two twisted dithienothiophenes on the mechanical carrier. This probe spontaneously inserts into the plasma membrane of the cell and only emits fluorescence when inserted into the lipid membrane. By detecting the intensity and color of the fluorescence with a fluorescence lifetime imaging microscope (FLIM), the magnitude of the membrane tension can be judged. However, the excitation wavelength of this molecule is fixed (488 nm), and the emission band (575 - 625 nm) is easily interfered by the autofluorescence of biological samples.

[0054] A fluorescence probe for the spatial distribution and dynamic change of force, combined with FLIM imaging, establishes a relationship model between membrane tension and fluorescence lifetime, and constructs a detection method for the magnitude of cell membrane tension based on the fluorescence lifetime of the probe. This method is simple to operate, low in cost, and has a wide range of application scenarios. The novel cell membrane-targeted organic small molecule probe designed and synthesized in the present invention has excellent cell membrane targeting and mechanical force sensitivity, good biocompatibility, an optimal excitation light of 570 nm, and an emission at 650 nm, which can effectively avoid the influence of autofluorescence of biological samples. It has a wide range of application scenarios in the study of cell mechanical mechanisms.

[0055] Example 1 Synthesis of Fluorescence Probe Molecule

[0056] Take ph-BDP (1eq) and 4-(4-methylpiperazine) benzaldehyde (1-4eq) were dissolved in anhydrous N,N-dimethylformamide (20 mL per mmol of ph-BDP). Glacial acetic acid: piperidine (1:1, V / V) (1 mL per mmol of ph-BDP) was added to the reaction mixture. Then, the reaction mixture was heated under reflux at 130 °C for 1-3 hours. After cooling to room temperature, the mixture was poured into water and extracted three times with dichloromethane. The organic layers were combined, dried over sodium sulfate, and then filtered. The solvent was evaporated, and the residue was purified by silica gel chromatography. The polarity of the solvent was gradually increased using a ratio of dichloromethane and methanol (0:1 - 10:1), and the green solid, Mono-pd-BDP, was isolated.

[0057]

[0058] Mono-pd-BDP (50 mg, 0.01 mmol) was dissolved in acetonitrile (10 mL), and then methyl iodide (0.5 mL) was added. The reaction mixture was stirred at 80 °C for 12 hours and then evaporated to dryness. The residual solid was washed with dichloromethane to remove impurities, and then the blue solid (38 mg) MEM-NIR1 was collected.

[0059]

[0060] The successful synthesis of the material was verified by NMR. The NMR results were 1H NMR (400 MHz, Chloroform-d) δ 7.58–7.47 (m, 6H), 7.33 (dt, J = 6.9, 2.1 Hz, 2H), 7.23 (d, J = 16.1 Hz, 1H), 6.74–6.68 (m, 2H), 6.62 (s, 1H), 5.99 (s, 1H), 3.05 (d, J = 1.8 Hz, 6H), 2.61 (s, 3H), 1.42 (d, J = 17.7 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 154.83, 152.92, 142.86, 140.83, 138.91, 137.76, 135.42, 133.09, 131.22, 129.27, 128.99, 128.77, 128.42, 124.69, 120.45, 117.71, 114.43, 112.04, 77.28, 77.03, 76.77, 40.27, 14.68, 14.61, 14.23, 0.01.

[0061] Example 2 Detection of Spectral Characteristics of Fluorescent Probe Molecules

[0062] After synthesis, the absorption and emission of MEM-NIR1 were measured using an ultraviolet-visible spectrophotometer and a fluorescence spectrophotometer.

[0063] The test results are as Figure 2 shown. It can be Figure 2 seen that the optimal excitation wavelength is 570 nm, and the corresponding emission wavelength is 650 nm. This wavelength can effectively avoid the influence of the autofluorescence of biological samples and is beneficial for applications in the cell mechanics application scenario.

[0064] Example 3 Artificial cell membrane - giant unilamellar vesicle simulation detection test

[0065] Construct artificial cell membranes - giant unilamellar vesicles (GUVs) to simulate cell membranes with different membrane tensions and verify the responsiveness of MEM - NIR1 to changes in membrane tension.

[0066] Prepare GUVs by the W / O emulsion centrifugation method:

[0067] Prepare a 20 μL inner solution containing 200 mM sucrose under ice - bath conditions and temporarily store it under ice - bath conditions. Subsequently, prepare a 500 μL outer solution containing 200 mM glucose, also for ice - bath standby.

[0068] Prepare lipid membranes with different components to simulate different cell membrane tensions. Transfer 100 μL of lipid solutions with different ratios to glass tubes cleaned with chloroform - n - hexane. Among them, the first group of lipids is dioleoylphosphatidylcholine (DOPC), the second group of lipids is dioleoylphosphatidylcholine and cholesterol (DOPC–CL), and the third group of lipids is dipalmitoylphosphatidylcholine (DPPC). Dry the solvent with nitrogen and assist with vortexing. Optionally, further reduce the pressure to dry to remove residual moisture. At the same time, the fluorescent probe molecule MEM - NIR1 prepared in Example 1 is added to the lipid solution and protected from light. After the lipid membrane is completed, add 500 μL of mineral oil and vortex vigorously for 20 seconds, then heat twice at 70 °C for 1 minute each time, and immediately vortex after each heating until cooled to room temperature. Subsequently, add the inner solution to the lipids and vortex for 30 seconds to form homogeneous water - in - oil (W / O) droplets, transfer them to the surface of the outer solution, and centrifuge at 4 °C for 5 minutes. After centrifugation, carefully aspirate the upper oil phase and debris, slowly collect 20–40 μL of giant vesicle precipitate from the bottom of the tube with a new pipette tip and transfer it to a new tube. Its morphology can be observed through a microscope. If necessary, add the outer solution to wash and repeat centrifugation to purify the vesicles. During the operation, the pipette tip needs to be frequently changed to prevent the residual oil phase from damaging the vesicles. Aspirate a small amount of the prepared vesicles into an eight - well chamber, let them stand on the stage for 30 min until the vesicles settle to the bottom, and take fluorescence lifetime images with FLIM to obtain fluorescence lifetime data.

[0069] The test results are shown in Figure 6, the fluorescence lifetime of MEM-NIR1 shows a significant positive correlation with the change in lipid membrane tension. Among GUVs with three different membrane tensions, the fluorescence lifetime of MEM-NIR1 gradually increases from 2.0123 ± 0.1 ns in the disordered membrane (DOPC) to 3.2145 ± 0.12 ns in the DOPC membrane containing cholesterol, and finally reaches 3.6587 ± 0.16 ns in the highly ordered DPPC membrane. This trend is consistent with the increase in membrane tension during the transition of the lipid membrane from loose and disordered (Ld phase) to tight and ordered (Lo phase). The possible mechanism is that as the saturation of lipid tails increases and cholesterol is inserted, the arrangement of membrane molecules becomes more compact, reducing the lateral diffusion ability of lipid molecules and the rotational freedom of the probe, thereby reducing non-radiative energy dissipation and prolonging the fluorescence lifetime. At the same time, the above fluorescence probe aggregates on the cell membrane and can be applied to cell membrane imaging.

[0070] Example 4 Cytotoxicity Test

[0071] Hela cell culture: Cultured in DMEM complete medium in a cell culture incubator (37 °C, 5% CO2). 24 hours before the imaging experiment, the cells were seeded on a 35 mm glass-bottom cell culture dish (NEST), and then placed in the cell culture incubator for storage. Before imaging, observe the cell morphology, density, adhesion, and growth status under the microscope to see if they are good.

[0072] Cell viability experiment: Cell viability was used to evaluate cytotoxicity using the standard CCK-8. HeLa cells were seeded in a 96-well plate at a density of 7×10 3 cells per well and cultured for 24 h. The 10 mM probe stock solution in DMSO was diluted to different concentrations (0.5, 1, 2, 3, 4, 5, and 10 μM) in fresh medium, and DMSO was used as a vehicle control. After incubating the cells with the above solutions for 24 hours, 10 μL of CCK-8 PBS stock solution was added to each well, and the cells were incubated for another 4 hours. The absorbance was measured at a wavelength of 450 nm using a microplate reader, and the percentage of cell viability was calculated based on the optical density.

[0073] The results of the cytotoxicity test are shown in Figure 3 , according to the results using the content, it can be seen that as the concentration increases, the cell activity hardly changes, indicating that MEM-NIR1 is non-toxic and harmless to cells within the working concentration range and does not affect cell activity. This is a prerequisite for the probe to be used in cell experiments.

[0074] Example 5 Target Region Test of Fluorescent Probe Molecules

[0075] Fluorescent staining: Hela cells were incubated with 1 μM of the dye and 1 μM of DIO (cell membrane dye) at 37 °C for 15 minutes, then washed 3 times with phosphate-buffered saline (PBS), and then imaged with a laser confocal microscope.

[0076] The test results are shown in Figure 4 , and the test results show that the targeting region of the fluorescent probe MEM-NIR1 is the cell membrane.

[0077] Furthermore, through the co-action of DIO (cell membrane dye) and the fluorescent probe MEM-NIR1, HeLa cells were seeded in an eight-well chamber (a confocal special culture dish) and cultured at 37 °C and 5% CO2 for 24 hours to allow the cells to adhere. Then, the cells were washed, the culture medium was aspirated, and the cells were gently washed 3 times with pre-warmed 1×PBS. Next, the cells were stained. 1 mL of pre-warmed culture medium was added, followed by 5 μM of DIO (commercial cell membrane probe), and incubated at 37 °C in the dark for 10 minutes. Then, 1 μM of the MEM-NIR1 probe was added, and incubation was continued at 37 °C in the dark for 5 minutes. After that, the cells were washed with PBS buffer to remove the unbound probes. Microscope observation parameter settings: The excitation wavelength of DIO is 488 nm, and the emission wavelength is 501 nm (green channel); the excitation wavelength of MEM-NIR1 is 570 nm, and the emission wavelength is 630 - 650 nm (red channel). A 63× oil immersion lens was used to collect dual-channel images simultaneously.

[0078] The test results are shown in Figure 5 , and the test results show that the targeting region of the fluorescent probe MEM-NIR1 is the cell membrane.

[0079] Example 6 Cell Membrane Tension Detection Experiment

[0080] HeLa cells were stained with the fluorescent probe MEM-NIR1, and then the culture medium was replaced with solutions of different osmotic pressures. Fluorescence lifetime imaging was performed immediately after cell osmotic shock. The Hepes-buffered DMEM with an osmolarity of approximately 310 mOsm / L was regarded as the isotonic culture medium; the hypotonic culture media (77.5 and 155 mOsm / L) were prepared by diluting Hepes-buffered DMEM with Milli-Q water; the hypertonic culture media (530 and 750 mOsm / L) were prepared by adding NaCl to Hepes-buffered DMEM. Fluorescence lifetime images were taken with a fluorescence lifetime imaging microscope FLIM to obtain fluorescence lifetime data. Since the cell membrane tension decreases with the increase in osmotic pressure, analyzing the relationship between fluorescence lifetime and osmotic pressure can obtain the relationship model between fluorescence lifetime and cell membrane osmotic pressure, and verify the membrane tension detection ability of the probe.

[0081] The test results are shown in Figures 7 - 8 , where Figure 7Fluorescence lifetime images of Hela under different osmotic shocks. It can be seen from the figure that the fluorescence lifetime of the fluorescent probe increases with the increase of membrane tension. Figure 8 Relationship between the fluorescence lifetime of MEM-NIR1 and the osmotic pressure applied to cells. Among them, a is the histogram of fluorescence lifetime distribution under different osmotic shocks, indicating that there are obvious differences in fluorescence lifetime. b is the relationship between the specifically fitted average fluorescence lifetime and osmotic pressure, and three groups of data are measured at each osmotic pressure. c is the linear fitting of b, indicating that the fluorescence lifetime has an inverse linear relationship with the osmotic pressure of osmotic shock.

Claims

1. A fluorescent probe, characterized in that, It has a structural formula shown in Formula I:

2. The preparation method of the fluorescent probe according to claim 1, characterized in that, The preparation method includes the following steps: S1) Prepare the intermediate shown in Formula II: Condense the compound shown in Formula III and 4-(4-methylpiperazinyl)benzaldehyde to obtain the intermediate shown in Formula III; S2) The intermediate shown in II undergoes a nucleophilic substitution reaction with a haloalkane to obtain the compound shown in Formula I; wherein Preferably, in step S1), the catalyst for the condensation reaction is a weak base; the weak base is preferably piperidine, more preferably a mixture of acetic acid and piperidine; Preferably, in step S1), the conditions for the condensation reaction are heating under reflux at 120 - 150 °C.

3. Use of the fluorescent probe according to claim 1 as a detection reagent for detecting cell membrane tension; Preferably, the cell membrane is the cell membrane of living cells in vitro, the cell membrane of cells in vivo, and a cell membrane-like structure having cell membrane properties. The cell membrane-like structure having cell membrane properties.

4. A method for detecting changes in cell membrane tension, characterized in that, The detection method includes the following steps: S1) Co-incubate the cell with the fluorescent probe according to claim 1; S2) Provide a stimulus to the cell that affects the change in cell membrane tension, and use an imaging system to separately detect the fluorescence lifetime of the fluorescent probe before and after the stimulus is given; S3) Based on the fluorescence lifetime data obtained in step S2), calculate the relative change amount of the cell membrane tension, thereby determining the dynamic change trend of the cell membrane tension.

5. The detection method of cell membrane tension change according to claim 4, characterized in that, The imaging system is selected from a fluorescence lifetime imaging microscope, a fluorescence microscope, a confocal microscope, a two-photon microscope, a small animal in vivo optical imaging system, a time-correlated single photon counting system; Preferably, the stimulus in step S2) is adding a reagent, changing the cell incubation environment, or increasing physical stimulation.

6. A visualization method for changes in cell membrane tension, characterized in that, The method includes the following steps: S1) Co-incubate the cell with the fluorescent probe according to claim 1; S2) Provide a stimulus to the cell that affects the change in cell membrane tension, and use an imaging system to separately observe the fluorescence change of the fluorescent probe before and after the stimulus is given to visualize the change in cell membrane tension.

7. A detection reagent for cell membrane tension, characterized in that, The detection reagent contains the fluorescent probe according to claim 1.

8. A real-time monitoring system for cell membrane tension, characterized in that, It includes: The fluorescent probe according to claim 1; An imaging system for detecting the fluorescence lifetime of the fluorescent probe, used to detect the fluorescence lifetime of the fluorescent probe; An algorithm module for analyzing the fluorescence lifetime data.

9. Use of the fluorescent probe according to claim 1 in the preparation of a detection reagent for cell membrane imaging.

10. A method for cell membrane imaging, the method includes the following steps: S1) Co-incubate the cell with the fluorescent probe according to claim 1; S2) Use an imaging system to image the fluorescence image; Preferably, the imaging system is selected from a fluorescence lifetime imaging microscope, a fluorescence microscope, a confocal microscope, a two-photon microscope, a small animal in vivo optical imaging system, a time-correlated single photon counting (TCSPC) system.

Images