Fluorescent probe based on dicyanomethylene-4H-pyran as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a dicyanomethylene-4H-pyran-based fluorescent probe DCM-Pro as well as a preparation method and application thereof, the probe realizes fluorescence intensity and fluorescence lifetime response to polarity change of a microenvironment by optimizing a molecular structure, and can be covalently bound with protein through a Michael addition reaction. The polarity sensitivity of the probe is adjusted by regulating and controlling the proportion of local excited states in the intramolecular charge transfer process. The fluorescence lifetime of the DCM-Pro is linearly related to the polarity of the microenvironment, and the change of the polarity of the microenvironment in the protein aggregation process can be quantitatively detected. The probe is a fluorescence lifetime probe sensitive to polarity and can be used for visual detection of iron-mediated protein aggregation. The probe has good biocompatibility and specificity, is suitable for living cell imaging, provides a new tool for researching protein aggregation in the ferroptosis process, and also provides a new method and thought for mechanism research and drug screening of neurodegenerative diseases.

Description

Technical Field

[0001] The present invention relates to the technical fields of organic synthesis, chemical analysis, cell biology and protein aggregation, and particularly relates to a fluorescence probe based on dicyanomethylene-4H-pyran, a preparation method and an application thereof, and particularly relates to the monitoring and application of polarity during the protein aggregation process. Background Art

[0002] In the fields of biomedical and chemical research, changes in the microenvironment polarity are closely related to the conformation, function and aggregation behavior of proteins. As a key factor affecting molecular interactions and biological processes, the microenvironment polarity can directly reflect the dynamic characteristics of the intracellular and extracellular environments. Especially in the diseased state, abnormal changes in the microenvironment polarity are often closely related to the pathological process. For example, in neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease), abnormal protein aggregation is one of the core pathological features, and this process is usually accompanied by significant changes in the microenvironment polarity. Therefore, real-time and accurate detection of microenvironment polarity changes and visual monitoring of the protein aggregation process are of great significance for revealing disease mechanisms, developing diagnostic tools and treatment methods.

[0003] Traditional detection methods often have difficulty in real-time and dynamic monitoring of microenvironment polarity changes and protein aggregation processes, which limits the in-depth understanding of their molecular mechanisms. In recent years, fluorescence lifetime imaging technology (FLIM) has gradually become a research hotspot due to its unique advantages. Fluorescence lifetime is an inherent property of the probe itself, which is not affected by the probe concentration and excitation light intensity, and can provide more stable and reliable detection results. The polarity-sensitive fluorescence lifetime probe can reflect the change of microenvironment polarity in real time through the changes of fluorescence intensity and fluorescence lifetime, and can visually track the dynamic process of protein from monomer to aggregate, providing a powerful tool for studying protein aggregation mechanisms and related diseases.

[0004] However, there is still a lack of an efficient and highly specific fluorescence lifetime probe in the prior art that can simultaneously achieve accurate detection of microenvironment polarity changes and visual monitoring of protein aggregation. Especially in complex biological systems, how to improve the sensitivity, selectivity and photostability of the probe remains a current research difficulty. Therefore, the development of a new detection method based on a polarity-sensitive fluorescence lifetime probe can not only promote the in-depth development of microenvironment polarity and protein aggregation research, but also provide important technical support for disease diagnosis, drug screening and treatment, and has broad application prospects and scientific value. Summary of the Invention

[0005] Objective of the Invention: The technical problem to be solved by the present invention is to provide a polarity-sensitive fluorescence lifetime probe small molecule fluorescence probe DCM-Pro that can detect the polarity of the microenvironment and the degree of protein aggregation. This probe can achieve the detection of polarity response and the change of polarity during protein aggregation in both fluorescence intensity and fluorescence lifetime dual modes.

[0006] Another technical problem to be solved by the present invention is to provide a preparation method of the fluorescence probe DCM-Pro.

[0007] Another technical problem to be solved by the present invention is to provide the application of the fluorescence probe DCM-Pro in polarity detection.

[0008] The last technical problem to be solved by the present invention is to provide the application of the fluorescence probe DCM-Pro in the detection of the degree of protein aggregation and / or intracellular fluorescence imaging.

[0009] Technical Solution: To solve the above technical problems, the present invention provides a fluorescence probe DCM-Pro based on dicyanomethylene-4H-pyran. The structural formula of the fluorescence probe DCM-Pro is shown as follows:

[0010]

[0011] Among them, the fluorescence probe DCM-Pro is obtained by the Knoevenagel condensation reaction of the DCM nucleus and 4-(bis(pyridin-2-ylmethyl)amino)benzaldehyde. This probe is extremely sensitive to the change of polarity in the microenvironment and can monitor the degree of protein aggregation through the change of environmental polarity.

[0012] The content of the present invention also includes the preparation method of the fluorescence probe DCM-Pro, which includes the following steps: Weigh the DCM precursor, dissolve it in toluene, add 4-(bis(pyridin-2-ylmethyl)amino)benzaldehyde and stir with a magnetic stirrer. Quickly add p-toluenesulfonic acid and piperidine, set up an oil-water separator and a condenser, stir overnight, remove the solvent under vacuum, and purify the crude product by silica gel column chromatography to obtain the probe molecule DCM-Pro.

[0013] Among them, the molar ratio of the DCM precursor to 4-(bis(pyridin-2-ylmethyl)amino)benzaldehyde is (0.8 - 1.2):(1.8 - 2.2).

[0014] Among them, the silica gel column chromatography purification is carried out with dichloromethane / petroleum ether as the eluent.

[0015] The content of the present invention also includes the application of the fluorescence probe DCM-Pro in polarity detection.

[0016] Among them, the application includes detecting the polarity change of the intracellular microenvironment, and the probe DCM-Pro can respond to the polarity in both fluorescence intensity and fluorescence lifetime dual modes. The fluorescence intensity and fluorescence lifetime of this probe are positively correlated with the polarity change of the microenvironment, and the polarity can be detected.

[0017] Among them, the application includes adjusting the dielectric constant in the solvent of the fluorescent probe DCM-Pro, that is, by increasing the proportion of the locally excited state (LE) during the molecular charge transfer process to regulate the polarity sensitivity of this probe.

[0018] The content of the present invention also includes the application of the fluorescent probe DCM-Pro in the detection of the degree of protein aggregation and / or intracellular fluorescence imaging.

[0019] Among them, the probe DCM-Pro binds to the thiol group on the protein (i.e., the sulfhydryl group of the cysteine residue, -SH) through the Michael Addition reaction, efficiently and specifically fuses to the protein, analyzes the changes in its fluorescence intensity and fluorescence lifetime, and visually detects the iron-mediated protein aggregation.

[0020] Among them, the application includes detecting insoluble aggregated proteins in living cells.

[0021] Among them, the probe DCM-Pro can be used for intracellular fluorescence lifetime imaging, and further monitor the polarity change during the protein aggregation process.

[0022] Among them, the concentration of the fluorescent probe DCM-Pro is 1 - 200 μM.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0024] 1. The present invention utilizes a polarity-sensitive fluorescence lifetime probe to achieve precise detection of the polarity change of the microenvironment. This probe has high sensitivity and specificity, and can accurately reflect the subtle changes in polarity in the microenvironment. At the same time, through fluorescence lifetime imaging technology, real-time visual monitoring of the protein aggregation process is realized. This innovative technology breaks through the limitations of traditional detection methods, provides a new perspective and means for studying biological processes related to protein aggregation, and greatly improves the research efficiency and accuracy.

[0025] 2. The present invention can not only monitor the polarity change of the microenvironment, but also accurately measure the degree of polarity change through quantitative analysis of fluorescence lifetime. Combining image processing technology, it can intuitively display the polarity distribution in different regions during the protein aggregation process, providing a new means for the study of the spatial heterogeneity of protein aggregation.

[0026] 3. The fluorescence lifetime probe used in the present invention has good biocompatibility and photostability, can be compatible with a variety of biological samples, and is suitable for various experimental systems such as living cells. At the same time, this method is easy to operate, without complex sample processing steps, and can be seamlessly docked with the existing fluorescence microscope system, facilitating popularization and application.

[0027] 4. The technology of the present invention can be widely applied in the field of biomedical research, especially in diseases related to protein misfolding and aggregation, such as neurodegenerative diseases like Alzheimer's disease and Parkinson's disease. By monitoring the changes in the microenvironment polarity during the protein aggregation process, it can provide important molecular information for the early diagnosis and treatment of diseases, and has important potential for clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the synthetic method route of the bifunctional fluorescent probe DCM-Pro based on the dicyanomethylene-4H-pyran (DCM) core.

[0029] Figure 2 It is the 1H NMR spectrum of the DCM-Pro probe molecule.

[0030] Figure 3 It is the 13C NMR spectrum of the DCM-Pro probe molecule.

[0031] Figure 4 It is the mass spectrum of the DCM-Pro probe molecule.

[0032] Figure 5 It is the result diagram of the local excited state ratio of the probe during the charge transfer process in five solvents.

[0033] Figure 6 It is the relative fluorescence intensity diagram of the probe in solvents with different dielectric constants.

[0034] Figure 7 It is the relationship diagram between the methanol ratio and the relative fluorescence intensity of the probe in the mixed solvent.

[0035] Figure 8 It is the average fluorescence lifetime diagram of the probe molecule in solvents with different dielectric constants.

[0036] Figure 9 It is the MALDI-TOF mass spectrometry analysis diagram of the compounds SOD1(V4A) (molecular weight: 52088) and DCM-Pro (molecular weight: 493).

[0037] Figure 10 It is the molecular docking result diagram of SOD1 protein and DCM-Pro.

[0038] Figure 11 For Fe at 25 °C 2+Fluorescence and turbidity kinetic diagrams of the inducer-induced aggregation of SOD1(A4V)-Halo.

[0039] Figure 12 is Fe 2+ Protein aggregation induced by... reduces the fluorescence lifetime diagram of DCM-Pro.

[0040] Figure 13 is the transient transfection and expression of α-synuclein(H50Q)-Halo in HEK293T cells. 20 μM Fe 2+ Protein aggregation diagram induced for 2 h.

[0041] Figure 14 is the diagram of the expression results of transient transfection of α-synuclein(H50Q) in HEK293T cells. 20 μM Fe 2+ Concentrations were used to induce protein aggregation for 12 h and 24 h respectively. Detailed implementation manners

[0042] The following further describes the present invention in detail. The Fe used in the present invention 2+ solution is prepared by dissolving ferrous sulfate heptahydrate in water.

[0043] Example 1 Synthesis and characterization of the fluorescent probe molecule DCM-Pro

[0044] 1. Synthesis of the probe DCM-Pro:

[0045] Under the protection of nitrogen, a magnetic stir bar was added to a 100 mL round-bottom flask. 150 mg of 2-(2-methyl-4H-benzo[b]pyran-4-ylidene)malononitrile (1 eq, CAS No.: 15058-15-8) was weighed and dissolved in 10 mL of toluene. 4-(Bis(pyridin-2-ylmethyl)amino)benzaldehyde (2 eq, CAS No.: 946005-73-8) was added and stirred using a magnetic stirrer. 20 mg of toluenesulfonic acid and 250 μL of piperidine were quickly added. An oil-water separator (the upper layer is toluene and the lower layer is water) was set above the round-bottom flask, and a condenser was placed on the oil-water separator. Under a stirring speed of 300 r / min and a temperature of 110 °C, the reaction was carried out overnight. The solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (using dichloromethane / petroleum ether as the eluent), and the probe molecule DCM-Pro was obtained by monitoring through TLC (dichloromethane: petroleum ether = 1:1). The specific synthesis route is as Figure 1 shown.

[0046] 2. Analysis and characterization of the probe DCM-Pro:

[0047] 1H nuclear magnetic resonance spectrum 11H NMR (400 MHz, Chloroform-d) δ 8.90 (d, J = 8.1 Hz, 1H), 8.63 (d, J = 3.7 Hz, 2H), 7.68 (dt, J = 12.2, 7.5 Hz, 4H), 7.54 (d, J = 6.2 Hz, 1H), 7.51 (s, 1H), 7.42 (d, J = 8.7 Hz, 2H), 7.27 (s, 1H), 7.25–7.20 (m, 3H), 6.82–6.72 (m, 3H), 6.56 (d, J = 15.8 Hz, 1H), 4.91 (s, 4H). The spectrum is shown in Figure 2 。

[0048] 13C NMR (101 MHz, CDCl3) δ 190.32, 158.62, 157.66, 157.21, 152.86, 152.37, 150.36, 149.97, 149.93, 139.34, 137.06, 134.29, 130.03, 125.77, 125.70, 123.78, 122.51, 122.44, 120.76, 120.69, 118.49, 117.96, 116.28, 113.84, 112.80, 112.03, 105.61, 57.15, 57.11. The spectrum is shown in Figure 3 。

[0049] HRMS: The spectrum is shown in Figure 4 。

[0050] Example 2 Exploration of the Proportion of the Local Excited State (LE) in the Charge Transfer Process of the Fluorescent Probe Molecule DCM-Pro in Solvents with Different Dielectric Constants

[0051] In this example, the probe DCM-Pro was added to five solvents with different dielectric constants, including ethyl acetate (EA), n-butanol (NBA), isopropanol (IPA), N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO). Density functional theory calculations were performed using the Gaussian 09 software package, and further analysis was carried out using the Multiwfn 3.8 software based on the TD-DFT calculation results. The results show that the CT state dominates during the molecular excitation process, but the proportion of the LE state changes with the increase in the solvent dielectric constant. Among them, the proportion of the LE state of DCM-Pro increases significantly, from 19.017% to 39.969%. The results are as shown in Figure 5 shown. The characteristics of the LE state are high radiative transition rates and photoluminescence efficiencies, which may be the fundamental reason for the high polarity sensitivity of DCM-Pro. Therefore, the polarity sensitivity of the chromophore can be adjusted by increasing the proportion of the LE state during the electron transfer process.

[0052] Example 3: Test the fluorescence intensity and fluorescence lifetime of DCM-Pro in solvents with different polarities to verify its polarity sensitivity experiment

[0053] In this example, a methanol / dioxane (MeOH. / Dioxane.) mixture and 10 solvents with different dielectric constants were selected to measure the polarity sensitivity. The probe DCM-Pro solution was prepared from a 2 mM DMSO stock solution, with a final concentration of 100 μM, and was respectively formulated in MeOH. / Dioxane. mixed solutions with the following different volume ratios, as shown in Table 1. The measured results are as Figure 6 shown.

[0054] Table 1 Dielectric constants of MeOH. / Dioxane. mixed solutions with different volume ratios

[0055] Methanol ratio (%) Dioxane ratio (%) Dielectric constant (ε) 0 100 2.25 10 90 5.30 20 80 8.34 30 70 11.38 40 60 14.43 50 50 17.48 60 40 20.52 70 30 23.56 80 20 26.61 90 10 29.66 100 0 32.70

[0056] The probe DCM-Pro solution was prepared from a 2 mM DMSO stock solution, and the final concentration of the probe DCM-Pro solution was 100 μM in each case. It was respectively formulated in 10 solvents with different dielectric constants: toluene (ε = 2.38), ethyl acetate (ε = 6.02), tetrahydrofuran (ε = 7.58), dichloromethane (ε = 8.93), 1-butanol (ε = 17.8), isopropanol (ε = 19.9), acetone (ε = 20.7), ethanol (ε = 24.55), acetonitrile (ε = 37.5), dimethyl sulfoxide (DMSO, ε = 42.68). The measured results are as Figure 7 shown. The fluorescence intensity was recorded using a Hitachi F-7100 fluorescence and phosphorescence spectrophotometer. The polarity dependence curve was plotted with the solvent polarity (dielectric constant) on the X-axis and the normalized fluorescence intensity on the Y-axis, and the polarity dependence parameter (k) was determined from the slope of the fitted curve. Figure 6 The results show that when the methanol content increases from 0% to 100%, the fluorescence intensity of DCM-Pro increases with the increase in the polarity of the solution, indicating its sensitivity to polarity. Figure 7 It shows that the fluorescence intensity of DCM-Pro increases with the increase in the dielectric constant of the solution; overall, it shows that the DCM-Pro probe has polarity sensitivity.

[0057] The fluorescence lifetime was determined by time - correlated single - photon counting using a PerkinElmer LS - 50B fluorescence spectrophotometer. The DCM - Pro probe solution was prepared from a 2 mM DMSO stock solution, and the concentration of the probe DCM - Pro solution was 200 μM in a series of solvents with different dielectric constants: toluene (ε = 2.38), ethyl acetate (ε = 6.02), 1 - butanol (ε = 17.8), isopropanol (ε = 19.9), N,N - dimethylformamide (ε = 36.7), ethylene glycol / water = 70 / 30 (ε = 38.65), ethylene glycol / water = 70 / 30 (ε = 41.4), dimethyl sulfoxide (ε = 42.68). The obtained fluorescence decay curves were fitted with single - exponential and double - exponential decays respectively. A polarity - dependence curve was plotted with the solvent polarity (dielectric constant) as the X - axis and the average fluorescence lifetime as the Y - axis, and the polarity - dependence parameter (k) was determined from the slope of the fitted curve. The results showed that the fluorescence lifetime of the probe was linearly correlated with the dielectric constant of the microenvironment, as Figure 8 shown, that is, the probe has polarity sensitivity. Therefore, DCM - Pro can be used as a tool to explore the polarity changes in the microenvironment.

[0058] Example 4 Binding of Probe DCM - Pro to Proteins through Michael Addition Reaction and Experiment for Detecting Protein Aggregation

[0059] Since the superoxide dismutase 1 mutant A4V (SOD1(A4V)-Halo) (SOD1(A4V)-Halo was synthesized according to the article A dual-functional probe that allows cascade response to hydrogen peroxide oxidative stress-induced protein aggregation in live cells) is associated with the aggregation of amyotrophic lateral sclerosis (ALS), this mutant was selected for experiments in this example. First, acidic aggregation buffer and denaturing buffer were prepared. Acidic aggregation buffer: 200 mM sodium acetate (NaOAc), 100 mM potassium chloride (KCl), pH adjusted to 6.23 with acetic acid. Denaturing buffer: 50 mM potassium phosphate, 2 mM DTT, 2 mM EDTA, 6 M guanidine hydrochloride, pH 7.00. The protein was aggregated in the acidic aggregation buffer together with the probe DCM-Pro solution at a final concentration of 50 μM. The insoluble fraction was separated by centrifugation, and then the denatured protein solution was diluted to 100 ng·μL-1 after denaturation in 6 M guanidine hydrochloride. Sinapic acid solution was selected as the matrix to assist ionization. 1.0 μL of the protein solution (100 ng·μL-1) and 1.0 μL of the sinapic acid solution at a concentration of 15 mg / L were applied together on a steel target plate, and measured using a Bruker Ultra Flex III MALDI-TOF / TOF mass spectrometer to identify the formation of the SOD1(A4V)-Halo-DCM-Pro covalent adduct ( Figure 9 ). At the same time, molecular docking simulation calculations were carried out, and the binding energy of the probe DCM-Pro binding to the protein was -9.0 kcal / mol ( Figure 10 ), indicating that the probe DCM-Pro can bind to the protein through a Michael addition reaction.

[0060] Protein aggregation is a time-dependent multi-step process, and the turbidity signal can reflect its specific aggregation state. Therefore, in this experiment, the turbidity of SOD1(A4V)-Halo and the fluorescence signal of the probe DCM-Pro were synchronously detected during the protein aggregation process. It was observed that the protein turbidity increased rapidly and reached a plateau at 20 min, while the fluorescence signal gradually decreased and reached a minimum at 60 min ( Figure 11 ). Immediately afterwards, a solution of Fe with a final concentration of 20 μM was added to the DMSO stock solution of the probe DCM-Pro with a final concentration of 50 μM to induce protein aggregation. The blank control (blank) was to add only the probe DCM-Pro solution at the same concentration, and the Fe group was to add only the probe DCM-Pro solution at the same concentration and Fe 2+ solution2+ Solution, for the protein group, only the DCM-Pro probe solution with the same concentration was added to the protein solution. The fluorescence lifetime was measured using an LS-50B fluorescence spectrophotometer from PerkinElmer. It was found that the addition of Fe 2+ solution induced a significant decrease in the fluorescence lifetime of the probe DCM-Pro in the protein solution ( Figure 12 ). The experimental results showed that the fluorescence lifetime of DCM-Pro responded to the aggregation of SOD1(A4V)-Halo. These findings indicated that DCM-Pro could detect protein aggregation by analyzing fluorescence intensity and lifetime.

[0061] Example 5 Experiment on Detecting Intracellular Protein Aggregation with Probe DCM-Pro

[0062] In order to test the ability of DCM-Pro (red, λex = 520 nm) to localize protein aggregates in cells, GFP-Halo (from the article "Installing hydrogen bonds as a general strategy to control viscosity sensitivity of molecular rotor fluorophores") (green, λex = 488 nm) was selected for co-localization. The cells used in the experiment were human embryonic kidney cells HEK293. The plasmid α-synuclein(H50 Q)-Halo (the plasmid sequence is shown in SEQ ID NO.1, and the plasmid was synthesized by Jiangsu Saisuofei Biotechnology Co., Ltd.) was transfected into the cells and co-incubated for 24 h to allow the protein to be fully expressed. Then, an inducer with a final concentration of 20 μM of Fe 2+ was added and induced for 2 h to cause protein aggregation in the cells. Finally, a probe DCM-Pro solution with a final concentration of 1 μM was co-incubated with it. Before imaging, the nuclear dye Hoechst 33342 was allowed to act at 37 °C for 10 min, and the excess probe and dye were rinsed off. The experimental results showed that the probe DCM-Pro maintained bright fluorescence in the protein aggregation region ( Figure 13 ), proving that this probe could effectively detect insoluble aggregated proteins in living cells.

[0063] Example 6 FLIM Monitoring Experiment of Protein Aggregates in Living Cells

[0064] Given the polarity sensitivity shown by DCM-Pro in vitro, we used it for intracellular fluorescence lifetime imaging to study the changes in polarity during protein aggregation. In HEK293T cells expressing α-synuclein(H50 Q)-Halo, with a final concentration of 20 μM Fe 2+The solution was incubated for 12 h and 24 h respectively, and then stained with a probe DCM-Pro solution (red, λex = 520 nm) at a final concentration of 1 μM and GFP-Halo (green, λex = 488 nm) at a final concentration of 1 μM. After incubation at room temperature for 30 min, the cells were washed 3 times with PBS to remove unbound dyes. Then, the stained cells were imaged using a fluorescence lifetime imaging microscope (FLIM). A pseudo-color image was generated in the red channel (DCM-Pro), where the red part represents the region with a longer fluorescence lifetime and the blue part represents the region with a shorter fluorescence lifetime. The fluorescence lifetime values at different positions were quantitatively analyzed by the phasor analysis method. The results showed that in the cells expressing α-synuclein (H50Q)-Halo, with the increase of the induction time, the fluorescence lifetime signal at the protein aggregation site was seen Figure 14 , decreasing from 0.45 ns to 0.35 ns. Multiple parallel experiments were conducted for this experiment. The average value of the fluorescence lifetime signal at the protein aggregation site decreased from 0.48 ns to 0.29 ns. There was a significant difference between the two sets of data, indicating a decrease in polarity in the microenvironment. Combining Figure 8 with the function in it, it can be deduced that the dielectric constant decreased from 13.42 to 8.38. Therefore, the novel analytical tool DCM-Pro developed in the present invention can reflect the change in the degree of protein aggregation through the fluorescence lifetime signal.

Claims

1. A fluorescent probe DCM-Pro based on dicyanomethylene-4H-pyran, characterized in that: The structural formula of the fluorescent probe DCM-Pro is as follows:

2. The method for preparing the fluorescent probe DCM-Pro according to claim 1, characterized in that: The fluorescent probe DCM-Pro is obtained by a Knoevenagel condensation reaction between a DCM precursor and 4-(bis(pyridin-2-ylmethyl)amino)benzaldehyde.

3. The method for preparing the fluorescent probe DCM-Pro according to claim 2, characterized in that: The following steps are involved: Weigh 2-(2-methyl-4H-benzopyran-4-ylidene)malononitrile, dissolve it in toluene, add 4-(bis(pyridin-2-ylmethyl)amino)benzaldehyde and stir with a magnetic stirrer, quickly add p-toluenesulfonic acid and piperidine, set up an oil-water separator and a condenser, stir overnight, remove the solvent in vacuo, and purify the crude product by silica gel column chromatography to obtain the probe molecule DCM-Pro.

4. The method for preparing the fluorescent probe DCM-Pro according to claim 3, characterized in that: The molar ratio of the 2-(2-methyl-4H-benzopyran-4-ylidene)malononitrile to 4-(bis(pyridin-2-ylmethyl)amino)benzaldehyde is (0.8-1.2):(1.8-2.2).

5. Use of the fluorescent probe DCM-Pro described in claim 1 in polarity detection.

6. The use according to claim 5, characterized in that: The applications include detecting changes in the polarity of the intracellular microenvironment.

7. The use according to claim 5, characterized in that: The application includes adjusting the dielectric constant of the fluorescent probe DCM-Pro in the solvent.

8. Use of the fluorescent probe DCM-Pro according to claim 1 in the detection of protein aggregation degree and / or intracellular fluorescence imaging.

9. The use according to claim 7, characterized in that: Such applications include the detection of insoluble aggregated proteins in living cells.

10. The use according to claim 5, characterized in that: The concentration of the fluorescent probe DCM-Pro is 1-200 μM.

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