Preparation method and application of a near-infrared fluorescent probe with lipid droplet recognition and polarity sensitivity

Abstract

The present invention discloses a preparation method and biological application of a fluorescent probe with lipid droplet recognition and polarity sensitivity. The fluorescent probe CN obtained by the present invention can efficiently and accurately target lipid droplets. The preparation method adopted by the molecule is simple and the conditions are mild. The maximum emission wavelength of the fluorescent probe is at 724nm, which can realize near-infrared I zone fluorescence imaging, and the maximum Stokes shift can reach about 190nm. The maximum emission wavelength and the maximum excitation wavelength have a large wavelength difference, thereby solving the problem of autofluorescence quenching in the measurement, and the background interference is low and the detection sensitivity is high. In the environment inside the lipid droplet, the fluorescence of the probe CN is significantly enhanced, and it can efficiently and accurately target the lipid droplets, detect changes in the number of lipid droplets and changes in polarity in cells. Imaging experiments at the cell and tissue animal level show that it has good biosafety and good photostability. The probe CN provides a reliable tool for further studying the dynamic changes of lipid droplets and the occurrence and development of Parkinson's disease.

Description

Technical Field

[0001] The present invention relates to the application of organic functional molecules and fluorescence diagnostic technology in the early diagnosis of neurodegenerative diseases, and specifically to the preparation of a fluorescent probe with lipid droplet recognition and polarity sensitivity and its application in neurodegenerative disease models. Monitoring the dynamic changes of human lipid droplets is of great significance for understanding the biological characteristics of lipid droplets, the physiological status of cells and the early diagnosis of Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is the second most common major central nervous system degenerative disease, especially common in the elderly. When most Parkinson's patients are diagnosed, the damage and destruction of neurons have reached an incurable level, but the existing detection methods have a high misdiagnosis rate for initial diagnosis. The lack of effective diagnostic methods makes it impossible to diagnose the disease early. Therefore, it is of great significance to develop an accurate tool that can be used for the early diagnosis of PD. At present, the identification of biomarkers is an important step to improve early clinical diagnosis. Lipid droplets (LDs) are involved in various physiological processes. The function of LDs is closely related to its polarity. The polarity of LDs reflects the state and function of LDs. Abnormal LDs polarity state interferes with lipid metabolism, leading to LDs dysfunction, which is related to the development of neurodegenerative diseases.

[0003] The absorption and emission wavelengths of the existing lipid droplet probe BODIPY itself are not in the near-infrared or infrared region, and it is subject to relatively large background interference when used as a fluorescent probe for biological analysis. Summary of the invention

[0004] The purpose of the present invention is to provide a fluorescent probe with lipid droplet recognition and polarity sensitivity and a preparation method. The preparation method adopted by the molecule is simple to operate and the reaction conditions are mild. The fluorescent probe CN obtained by the present invention can efficiently and accurately target lipid droplets. The maximum emission wavelength of the fluorescent probe CN of the present invention is at 724nm, which can realize near-infrared I region fluorescence imaging, and the maximum Stokes shift can reach about 190nm. The maximum emission wavelength has a large wavelength difference with the maximum excitation wavelength, thereby solving the problem of autofluorescence quenching in the measurement, with low background interference and high detection sensitivity.

[0005] The present invention is a new polarity-sensitive fluorescent probe CN with D-π-A structure and lipid droplet targeting, which further studies the dynamic changes of lipid droplets and the occurrence and development of Parkinson's disease. The probe is based on the intramolecular charge transfer effect (ICT) and the D-π-A structure has a strong electron push-pull effect, which makes the probe sensitive to polarity. The probe also has the advantages of strong near-infrared emission, good photostability, large Stokes shift, and low toxicity, showing great application prospects in the early detection of neurodegenerative disease models. In view of the advantages of good biocompatibility, specific targeting of lipid droplets, and low biological background fluorescence, the probe has been successfully applied to the fluorescence imaging of lipid droplets in living cells and nematode and fruit fly brain tissues.

[0006] In order to solve the technical problem of the present invention, the proposed technical solution is: the organic molecule CN with lipid droplet recognition and polarity sensitivity provided by the present invention has the structural formula:

[0007]

[0008] In order to solve the technical problem of the present invention, another technical solution is proposed: a method for preparing a fluorescent probe with lipid droplet recognition and polarity sensitivity, comprising the following steps: dissolving intermediate A and (3,5,5-trimethylcyclohex-2-enylidene)malononitrile in a mixed solution of tetrahydropyrrole and anhydrous ethanol, stirring at 70-80°C for about 7-10 hours. Separating and purifying with a silica gel column, a dark green solid fluorescent probe CN is obtained.

[0009] The reaction route is as follows:

[0010]

[0011] The present invention provides a method for preparing an organic functional molecule CN, comprising the following steps:

[0012] The intermediate A and (3,5,5-trimethylcyclohex-2-enylidene)malononitrile were placed in a 100 mL round-bottom flask, and 1 mL of catalyst tetrahydropyrrole and 20 mL of anhydrous ethanol were added to react, and stirred at 78°C for 9 h. The reaction was monitored by a DCM:MeOH=100:1 spot plate. After confirming that the reaction was complete, the reaction solution was dried and separated and purified by a silica gel column (DCM:MeOH=200:1, v / v). The obtained CN was a dark green solid.

[0013] In order to solve the technical problem of the present invention, another technical solution is proposed: the application of the fluorescent probe CN with lipid droplet recognition and polarity sensitivity, and the probe molecule can be used to prepare reagents for identifying lipid droplets and testing solution polarity.

[0014] Preferably, the maximum emission wavelength of the fluorescent probe CN is at 724 nm, which can realize near-infrared I region fluorescence imaging, and the maximum Stokes shift can reach about 190 nm. There is a large wavelength difference between the maximum emission wavelength and the maximum excitation wavelength, thereby solving the problem of autofluorescence quenching in the measurement, with low background interference and high detection sensitivity.

[0015] Preferably, in the environment inside lipid droplets (high viscosity, low polarity), the fluorescence of the probe is significantly enhanced, and the lipid droplets can be targeted efficiently and accurately to detect changes in the number of lipid droplets and changes in polarity in cells.

[0016] Preferably, the fluorescent probe obtained by the present invention is applied to Drosophila brain tissue and nematode model animals, showing good biosafety and good photostability, and can target lipid droplets efficiently and accurately.

[0017] Beneficial effects:

[0018] The present invention provides a fluorescent probe CN with lipid droplet recognition and polarity sensitivity. The molecule can sensitively and specifically target lipid droplets, and the fluorescence signal is further enhanced under a low-polarity microenvironment. Most probes have poor selectivity for lipid droplets, have relatively short absorption and emission wavelengths, or exhibit severe "self-absorption" due to small Stokes shift. The preparation method used for the probe CN has simple reaction operation, mild conditions, and the fluorophore that absorbs and emits in the near-infrared (NIR) region has the advantages of good tissue penetration, good selectivity for lipid droplets, good photostability, large Stokes shift, high biosafety, and a wide range of applications.

[0019] The absorption and emission wavelengths of the existing lipid droplet probe BODIPY itself are not in the near-infrared or infrared region, and the background interference is relatively large when used as a fluorescent probe for biological analysis. The present invention discloses a preparation method and biological application of a fluorescent probe with lipid droplet recognition and polarity sensitivity. Specifically, the fluorescent probe CN is obtained by condensation reaction of intermediate A and (3,5,5-trimethylcyclohex-2-enylidene)malononitrile. The fluorescent probe CN obtained by the present invention can target lipid droplets efficiently and accurately. The preparation method adopted by the molecule is simple and the conditions are mild. The maximum emission wavelength of the fluorescent probe is at 724nm, which can realize near-infrared I region fluorescence imaging, and the maximum Stokes shift can reach about 190nm. The maximum emission wavelength and the maximum excitation wavelength have a large wavelength difference, thereby solving the problem of autofluorescence quenching in the measurement, and the background interference is low and the detection sensitivity is high.

[0020] In the inner environment of lipid droplets (high viscosity, low polarity), the fluorescence of probe CN is significantly enhanced, and it can efficiently and accurately target lipid droplets, detect changes in the number of lipid droplets and changes in polarity in cells. Imaging experiments at the cell and tissue animal level show that the organic functional molecules obtained by the present invention have good biosafety and good photostability. Excessive accumulation of lipid droplets in specific areas can lead to a variety of diseases, such as obesity, hepatic steatosis, atherosclerosis, Parkinson's disease, etc. In order to gain a deeper understanding of the functions of lipid droplets, appropriate analysis and detection tools must be developed. Probe CN provides a reliable tool for further studying the dynamic changes of lipid droplets and the occurrence and development of Parkinson's disease, and shows great application prospects in the early detection of neurodegenerative disease models.

[0021] The present invention is a new polarity-sensitive fluorescent probe CN with D-π-A structure and lipid droplet targeting, which further studies the dynamic changes of lipid droplets and the occurrence and development of Parkinson's disease. The probe is based on the intramolecular charge transfer effect (ICT) and the D-π-A structure has a strong electron push-pull effect to make the probe sensitive to polarity. The probe also has the advantages of strong near-infrared emission, good photostability, large Stokes shift, and low toxicity, showing great application prospects in the early detection of neurodegenerative disease models. In view of the advantages of good biocompatibility, specific targeting of lipid droplets, and low biological background fluorescence, the probe has been successfully applied to the fluorescence imaging of lipid droplets in living cells and nematode and fruit fly brain tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described below in conjunction with the accompanying drawings.

[0023] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of the probe CN involved in the present invention.

[0024] Figure 2 It is the carbon nuclear magnetic resonance spectrum of the probe CN involved in the present invention.

[0025] Figure 3 This is the mass spectrum of the probe CN involved in the present invention.

[0026] Figure 4 (A) UV normalized spectra of the fluorescent probe CN of the present invention in different solvents, (B) fluorescence emission spectra of the fluorescent probe CN of the present invention in different solvents.

[0027] Figure 5 The fluorescence emission spectra of the fluorescent probe CN of the present invention in 1,4-dioxane-PBS mixed systems with different ratios, and the linear response relationship between LogI724 and 1,4-dioxane content.

[0028] Figure 6 The fluorescence responses of the fluorescent probe CN to interfering substances in PBS and 1,4-dioxane environments, respectively.

[0029] Figure 7 (A) Fluorescence emission spectra of the fluorescent probe CN of the present invention in PBS with different pH values, (B) photostable fluorescence emission spectra of the fluorescent probe CN of the present invention in 1,4-dioxane-PBS mixed systems with different ratios.

[0030] Figure 8 This is a graph showing the effect of the fluorescent probe CN of the present invention on the survival rate of PC12 cells.

[0031] Fig. 9 The fluorescent probe CN of the present invention is used for specific biological imaging of lipid droplets in PC12 cells.

[0032] Fig.10 The fluorescence imaging of lipid droplets in living cells by the fluorescent probe CN of the present invention under the stimulation of oleic acid.

[0033] Fig.11 These are fluorescence imaging images of the fluorescent probe CN of the present invention detecting lipid droplets in different cell models.

[0034] Fig.12 This is a fluorescence imaging diagram of the fluorescent probe CN of the present invention detecting lipid droplets in a nematode model of Parkinson's disease.

[0035] Fig.13 This is a fluorescence imaging diagram of the fluorescent probe CN of the present invention for detecting lipid droplets in a Drosophila brain model of Parkinson's disease. DETAILED DESCRIPTION

[0036] Experimental Materials

[0037] 1. Reagents

[0038]

[0039] 2. Instruments

[0040]

[0041] Example 1

[0042] Synthesis steps of fluorescent probe CN:

[0043] Weigh the intermediate A (660 mg, 2.55 mmol), put the intermediate and (3,5,5-trimethylcyclohex-2-enylidene)malononitrile (521.5 mg, 2.8 mmol) into a 100 mL round-bottom flask with magnetic stirring, add 1 mL of catalyst tetrahydropyrrole and 20 mL of anhydrous ethanol to react, and stir at 78 ° C for 9 h. After the reaction is complete, the reaction solution is spin-dried and separated and purified by silica gel chromatography (DCM: MeOH = 200: 1, v / v), and the fluorescent probe CN is obtained as a black-green solid (0.65 g, 58%).

[0044] The reaction formula is as follows:

[0045]

[0046] Compound CN 1 H NMR data, 1 H NMR (400MHz, DMSO-d6) δ7.50–7.40(m,3H),7.30(d,J=18.8Hz,2H),6.93(d,J=15.2Hz,1H),6.79(s,1H), 6.67(d,J=7.8Hz,2H),3.35(d,J=6.4Hz,4H),3.26–3.24(m,2H),2.56(s,2H),1.07(s,6H),0.98(s,6H).

[0047] Compound CN 13 C NMR data, 13 C NMR (101MHz, DMSO-d6) δ170.25,156.43,148.66,148.16,138.54,133.42,131.93,127.43,127. 36,122.44,122.02,114.76,114.49,112.97,112.08,55.40,44.22,32.13,27.92(s),12.96(s).

[0048] Mass spectrum (HRMS) of compound CN, CN HRMS (m / z):calcd for C 27 H 29 N3S[M+H]+428.2082, found 428.2153, see Figure 1 , Figure 2 , Figure 3 .

[0049] Example 2

[0050] UV and fluorescence spectra of fluorescent probes in solvents of different polarities

[0051] An analytical balance was used to accurately weigh 11 mg of the pure probe prepared in Example 1, and dissolved in 5 mL of dimethyl sulfoxide solvent to prepare a probe stock solution with a final concentration of 5 mM. In this experiment, ten solvents with different dielectric constants were selected to measure the absorption spectra and fluorescence emission spectra of 5 μM probe in 2 mL solution systems of dichloromethane, methanol, water, 1,4-dioxane, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, glycerol, and toluene. The results are shown in Table 1. Figure 4 A. Figure 4 B. As the polarity of the solvent decreases, the position of the fluorescence emission peak changes significantly, and under ultraviolet light (535 nm), the fluorescence intensity of CN increases with the decrease of solvent polarity.

[0052] In this experiment, the ratio of 1,4-dioxane-PBS solution was gradually increased from 0% to 100% to simulate different polar solvent environments, and the fluorescence emission spectra of the probe in different polar solvents were measured. The test results are shown in Figure 5 As the volume content of 1,4-dioxane increases, the polarity of the solution gradually decreases and the fluorescence intensity gradually increases. There is a good linear relationship between LogI724 and the content of 1,4-dioxane, which preliminarily indicates that this probe responds to polarity. The optimal excitation wavelength of the fluorescent probe CN is 535nm, and the optimal emission wavelength is 724nm.

[0053] Example 3

[0054] Selectivity of fluorescent probes to other interfering ions

[0055] The fluorescent probe CN prepared in Example 1 was prepared into a DMSO stock solution with a final concentration of 5 mM, and 100 equivalents of Blank (PBS), H2O2, GSH, AA, ACO were added respectively. - , Cl - , CN - ,CO3 2- , NO2 - , S2O3 2- , HCO3 - , "O2 - , NO3 - ,PO4 3- , Ni 2 + , Fe 2+ , Ca 2+ , Zn 2+ , K + ,Mg 2+ , Na + The samples were diluted with PBS and 1,4-Dioxane to a concentration of 2.5 μM, and their fluorescence spectra were measured (λex = 535 nm). The effects of different ions on the fluorescence intensity of the fluorescent probe were evaluated based on their fluorescence intensity. Figure 6 As shown, it can be seen that the fluorescent probe CN of the present invention hardly responds to other ions at 724 nm, and the fluorescence intensity is only affected by the change of polarity.

[0056] Example 4

[0057] Fluorescent probes are affected by pH environment

[0058] The fluorescent probe CN prepared in Example 1 was prepared into a DMSO stock solution with a final concentration of 5 mM, and diluted to a test concentration of 2.5 μM with PBS of different pH values, and its fluorescence spectra were measured respectively; the effect of different pH environments on its fluorescence intensity was evaluated based on the change in its fluorescence intensity at 724 nm. The results are shown in FIG. Figure 7 As shown in A, within the pH range of 4 to 10, the change in the probe fluorescence intensity is almost unaffected by the pH environment, indicating that the probe has good stability.

[0059] Example 5

[0060] Photostability of fluorescent probes

[0061] Light stability is an important indicator for evaluating probe performance. Therefore, in this experiment, the probe CN was diluted to a test concentration of 2.5 μM in PBS buffer and 40% 1,4-Dioxane-PBS mixture, and its fluorescence spectrum was measured respectively; the results are shown in Figure 7 As shown in B, the fluorescence intensity hardly changed within 1 h, indicating that the probe CN has good photostability.

[0062] Example 6

[0063] Effects of fluorescent probes on cell viability

[0064] PC12 cells were seeded in a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 24 hours until they adhered to the wall. The original culture medium was removed, and probe solutions of different concentrations were added (a control group and a blank group were set up, and the probe concentrations were: 0μM, 2.5μM, 5μM, 10μM, 15μM, 20μM) for 24 hours. The probe solution was removed, and the MTT solution was added to continue culturing for 4 hours. The MTT solution was discarded, DMSO was added, and the cells were shaken for 15 minutes. The absorbance at 490nm was measured on an enzyme reader, and the cell survival rate was calculated. The results are as follows Figure 8 The experimental results show that when the probe concentration is 15 μM, the cell survival rate remains above 80%, which indicates that the probe has low toxicity and good survival rate, and can be used for subsequent biological imaging applications of different cells.

[0065] Example 7

[0066] Fluorescent probes for lipid droplet-specific biological imaging

[0067] PC12 cells were cultured in high-glucose DMEM medium containing 10% FBS and 1% penicillin-streptomycin. Before the experiment, the cells were inoculated in a confocal culture dish and cultured in a 37°C, 5% CO2 incubator for 24 hours until they adhered to the wall. The chemical properties of the probe indicate that CN is a small molecule probe for locating lipid droplets. In order to confirm that the probe CN can target lipid droplets, this experiment used the commercial lipid droplet localization probe BODIPY (1.0 μM; Ex = 488 nm, collection wavelength of 500-540 nm) to verify the ability of the probe CN to locate lipid droplets. The commercial lysosome probe Lyso-Tracker Green and the commercial mitochondrial probe Mito-TrackerGreen were used as controls. Mito-Tracker Green (1.0μM) locates mitochondria, Ex=488nm, and the collection wavelength is 500-540nm; Lyso-Tracker Green (1.0μM) locates lysosomes, Ex=488nm, and the collection wavelength is 500-540nm. The operation steps are as follows: After the cells successfully adhere to the wall, incubate the cells with a cell culture medium containing 10μΜ CN for 30 minutes, aspirate the probe culture medium, rinse three times with PBS, add the same amount of commercial lipid droplet probe BODIPY with a concentration of 1.0μΜ, incubate the cells for 30 minutes, aspirate the culture medium, and rinse three times with PBS. Finally, add an appropriate amount of culture medium to quickly image the cells. The commercial probe BODIPY is excited at 488nm, and the fluorescence is collected at 500-540nm. The CN probe is excited at 561nm, and the fluorescence is collected at 570-670nm (the parameters used in the subsequent confocal experiment of the probe CN are the same as above). The results are as follows Fig. 9 As shown, the red fluorescence of the probe molecule can overlap well with the green fluorescence of BODIPY to form yellow fluorescence, and the Pearson's colocalization coefficient R reaches 0.97, showing good targeting ability to lipid droplets.

[0068] The absorption and emission wavelengths of the existing lipid droplet probe BODIPY itself are not in the near-infrared or infrared region, and the background interference is relatively large when used as a fluorescent probe for biological analysis. Compared with the maximum emission wavelength of the fluorescent probe CN at 724nm, it can achieve near-infrared I region fluorescence imaging, and the maximum Stokes shift can reach about 190nm. There is a large wavelength difference between the maximum emission wavelength and the maximum excitation wavelength, thus solving the problem of autofluorescence quenching in the measurement, with low background interference and high detection sensitivity.

[0069] Example 8

[0070] Fluorescence imaging of lipid droplets in living cells using fluorescent probes stimulated by oleic acid

[0071] Due to the excessive accumulation of neutral lipids in the body, the overexpression and overgrowth of lipid droplets in cells, neurodegenerative diseases are very common. Therefore, we can verify this point by providing exogenous lipid-oleic acid to cells to induce cells to achieve in situ visualization of lipid droplets. Here, we stimulated PC12 cells with oleic acid and then stained the cells with probe CN to show the changes in the number of lipid droplets in living cells. PC12 cells were cultured in high-glucose DMEM medium containing 10% FBS and 1% penicillin-streptomycin. Before the experiment, the cells were seeded in a confocal culture dish and cultured in a 37°C, 5% CO2 incubator for 24 hours until they adhered to the wall. The culture medium was removed, washed with PBS three times, and then the oleic acid induction experiment was performed. The experimental group and the control group were selected for verification, one group was the control group cells, and the three groups were the experimental group cells. After adding oleic acid at 0μM, 100μM, 200μM, and 300μM, the cells were incubated at 37°C for 6h; then, 10μM fluorescent probe CN was added and incubated for 30min, the culture medium was removed, and the cells were washed with PBS three times for cell imaging. Fig.10 It can be seen that under laser confocal microscopy, as the concentration of oleic acid increases, the red channel fluorescence increases, indicating that the number of lipid droplets increases and the cell polarity decreases. Therefore, we can conclude that the increase in the addition of exogenous oleic acid will increase the number of intracellular lipid droplets. The probe can detect the number of lipid droplets in real time, so the probe CN can be used as an effective tool for detecting the number of intracellular lipid droplets.

[0072] Example 9

[0073] Fluorescence imaging of lipid droplets in different cell models

[0074] Parkinson's disease (PD) is a neurodegenerative disease associated with abnormal lipid metabolism. The accumulation of lipid droplets (LDs) in cytoplasmic organelles was observed in PD cell models. Lipid droplets are organelles that maintain cell homeostasis. We first established a PD model by inducing cells with rotenone and observed the changes in the number and brightness of lipid droplets in PC12 cells. Since lipids in cells can be rapidly consumed during glucose starvation, we used the starvation model as a control group to study the changes in the number of lipid droplets under cell starvation. Starvation was induced by incubating cells with culture medium without fetal bovine serum, and the changes in the number and brightness of lipid droplets in PC12 cells were observed. The specific experimental steps are as follows: The cells were divided into three groups, one group was control group cells, and the other two groups were experimental group cells. First, 1.0 mL of culture medium without fetal bovine serum was added to the first group of experimental group cells, and 1.0 mL of normal culture medium containing fetal bovine serum was added to the control group cells, and the two groups of cells were incubated together. After 30 minutes, the culture medium in the two groups of cells was aspirated, and 1.0 mL of 10 μM CN culture medium without fetal bovine serum was added to the cells in the experimental group; 1.0 mL of 10 μM CN culture medium with fetal bovine serum was added to the cells in the control group. After the two groups of cells were incubated together for 30 minutes, they were washed three times with PBS; the second group of experimental cells were treated with 1 μM rotenone culture medium with fetal bovine serum to treat PC12 cells. After 24 hours, the culture medium in the cells was aspirated, and 1.0 mL of 10 μM CN culture medium with fetal bovine serum was added. After incubation for 30 minutes, the cells were washed three times with PBS for cell imaging studies. Fig.11 As shown in the figure, the probe CN was used to observe the changes in the number of lipid droplets in cells after cell starvation and rotenone induction. The experimental results showed that PC12 cells had a small number of intracellular lipid droplets under normal growth conditions, and there was weak fluorescence in the lipid droplets. After the cells were starved for 30 minutes and induced with 1μM rotenone for 24 hours, the cell fluorescence intensity increased significantly compared with the untreated cells. Therefore, the probe CN can be used as an effective tool to detect the number of lipid droplets in cell models, and can distinguish normal cells from Parkinson's disease model cells.

[0075] Example 10

[0076] Fluorescence imaging of lipid droplets in a nematode model of Parkinson's disease

[0077] First, synchronize the nematodes so that the growth cycles of wild-type and Parkinson's model nematodes are consistent. Pick out about 5 nematodes in the egg-laying period from the culture dishes of different types of nematodes and put them into the culture dishes of the corresponding groups, and put them into a constant temperature and humidity incubator at 20°C for culture. On the second day, nematode eggs were observed on the surface of the culture medium. All the mother worms were picked out and placed in the culture medium box for further culture. After 3 to 4 days, it was observed that the nematodes on the surface of the culture medium had grown into adults. Pick out 4 to 5 nematodes from each group of culture medium and soak them in a PBS solution with a concentration of 100 μM probe for 5 minutes. Then pick the nematodes onto the culture medium without OP50. After the food and probes on the surface of the nematodes' bodies were removed, pick the nematodes onto a 2% agar slide for fixation and confocal fluorescence imaging. The wild-type Caenorhabditis elegans (WLZ1) and the Parkinson's disease type Caenorhabditis elegans (WLZ3) were used as research objects to explore the nematode imaging ability of the probe. Fig.12 As shown in the figure, when the probe is added, the wild-type nematode has extremely weak red fluorescence, but emits strong red fluorescence in the nematode Parkinson's disease model. Rosiglitazone is a clinical drug that can reduce triglycerides and regulate lipid metabolism disorders. Therefore, after adding 1mM rosiglitazone, the fluorescence of Parkinson's disease nematodes weakens. This proves that the probe has the ability to image nematodes and can detect and distinguish Parkinson's disease nematodes from wild-type nematodes.

[0078] Embodiment 11

[0079] Fluorescence imaging of lipid droplets in a Drosophila brain model of Parkinson's disease

[0080] First, take the synchronized day 1 to 3 fruit flies to make the growth cycles of wild-type and Parkinson's model fruit flies consistent. Add them separately to the Parkinson's disease (PD) experimental group: containing 1mM rosiglitazone (DMSO concentration is 0.1%), and the normal type (WT) & Parkinson's disease (PD) control group: containing only 0.1% DMSO culture medium, culture at 23 degrees Celsius for 15 days, then peel off the brain tissue and put it into PBS in a 96-well plate, prepare a PBS solution containing 100μM probe, transfer the peeled fruit fly brain into the prepared probe solution, and perform 30 minutes of probe incubation and fluorescence imaging. Fig.13 As shown, the fluorescence of the probe is extremely weak in the wild type, the fluorescence intensity increases in the Parkinson's disease model fruit flies, and the fluorescence of the probe weakens in the Parkinson's disease plus rosiglitazone treatment group. Therefore, the probe CN probe can be used in fruit fly imaging to detect and distinguish wild type and Parkinson's disease fruit flies.

[0081] The present invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent replacement are within the protection scope required by the present invention.

Claims

1. A fluorescent probe with lipid droplet recognition and polarity sensitivity, characterized in that: The polarity-sensitive fluorescent probe CN with lipid droplet targeting connects the donor and the acceptor through a thiophene structure and has a D-π-A structure. The structural formula of the fluorescent probe is as follows: 。 2. The method for preparing a fluorescent probe with lipid droplet recognition and polarity sensitivity according to claim 1, characterized in that: The method comprises the following steps: dissolving intermediate A and (3,5,5-trimethylcyclohex-2-enylidene)malononitrile in a mixed solution of tetrahydropyrrole and anhydrous ethanol, stirring at 70-80° C. for 7-10 h; separating and purifying by silica gel column to obtain a dark green solid fluorescent probe CN; the specific reaction route is as follows: 。 3. The use of the fluorescent probe CN with lipid droplet recognition and polarity sensitivity according to claim 1, characterized in that: The probe molecule can be used to prepare reagents for identifying lipid droplets or testing solution polarity.

4. The use of the fluorescent probe CN according to claim 3, characterized in that: The maximum emission wavelength of the fluorescent probe CN is at 724nm, which can realize near-infrared I region fluorescence imaging, and the maximum Stokes shift can reach 190nm. There is a large wavelength difference between the maximum emission wavelength and the maximum excitation wavelength, which solves the problem of autofluorescence quenching in the measurement, with low background interference and high detection sensitivity.

5. The use of the fluorescent probe CN according to claim 3, characterized in that: The fluorescence of the probe is significantly enhanced in the high viscosity and low polarity environment of lipid droplets, and it can efficiently and accurately target lipid droplets and detect changes in the number of lipid droplets and changes in polarity in cells.

6. The use of the fluorescent probe CN according to claim 3, characterized in that: The fluorescent probe is applied to fruit fly brain tissue and nematode model animals, showing good biosafety and good light stability, and can target lipid droplets efficiently and accurately.

Images