A membrane-anchored fluorescent probe, and a preparation method and application thereof

Abstract

The application relates to the technical field of fluorescent probe preparation and application, in particular to a membrane-anchored fluorescent probe and a preparation method and application thereof. The membrane-anchored fluorescent probe provided by the application can specifically image the cell membranes of living cells, can quantitatively detect the pH of different kinds of cell membranes, and can successfully distinguish non-alcoholic fatty liver tissues and detect non-alcoholic fatty liver tissues with different degrees of pathological changes. The membrane-anchored fluorescent probe has the structural formula:

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fluorescent probe preparation and application, and particularly relates to a membrane-anchored fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the background of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art that is already commonly known to a person of ordinary skill in the art.

[0003] In recent years, with the continuous development of biochemistry, people's research on active species of living cells, cell signaling and apoptosis is becoming more and more in-depth. Cell membrane plays a very important role in the life activities of the body. First of all, it provides a relatively stable internal environment for the life activities of the cell. Secondly, it provides a recognition site for the cell and completes the transmembrane conduction of substances inside and outside the cell. Thirdly, it provides a binding site for various enzymes, so that the enzyme reaction can proceed efficiently and orderly. The cell membrane is composed of a phospholipid bilayer and membrane proteins. The fluctuation of the pH value of the cell membrane can lead to changes in the structure of the phospholipid bilayer and the permeability of the cell membrane. The pH value disorder of the cell membrane can seriously affect the receptors such as ion channels and transport proteins on the cell membrane, trigger oxidative stress and apoptosis, affect normal lipid and energy metabolism, and lead to further imbalance of the pH value of the cell membrane. In addition, the fluctuation of the pH value of the cell membrane affects enzyme activity, membrane potential and cytoskeleton stability. Therefore, it is particularly important to expand and research new cell membrane pH value sensors.

[0004] Non-alcoholic fatty liver has become the most common chronic liver disease, accounting for about 25% of the total population. If timely and effective intervention cannot be carried out, non-alcoholic fatty liver is easy to develop into cirrhosis and even liver cancer. Generally speaking, non-alcoholic fatty liver is a curable disease, and early diagnosis and timely treatment can cure it, so it is of great significance to realize early diagnosis of non-alcoholic fatty liver.

[0005] In recent years, fluorescence imaging technology based on fluorescent probes has been widely used in cell imaging and biological detection due to its high sensitivity, easy operation and real-time in-situ detection. SUMMARY

[0006] In order to overcome the above problems, the present application provides a membrane-anchored fluorescent probe and a preparation method and application thereof. The membrane-anchored fluorescent probe provided by the present application can specifically image the cell membrane of living cells and quantitatively detect the pH values of different types of cell membranes and successfully distinguish non-alcoholic fatty liver tissues and detect non-alcoholic fatty liver tissues of different degrees of pathological changes.

[0007] To achieve the above technical purposes, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a membrane-anchored fluorescent probe, which has a structure as shown in formula (I):

[0009]

[0010] In a second aspect, the present application provides a preparation method of the membrane-anchored fluorescent probe, which comprises:

[0011] Compound 1 is dissolved in a solvent, and compound 2 is added to synthesize the membrane-anchored fluorescent probe shown in formula (I) under catalysis of a catalyst;

[0012] wherein the structure of compound 1 is shown in formula (II):

[0013]

[0014] The structure of compound 2 is shown in formula (III):

[0015]

[0016] In one or more embodiments, the solvent comprises anhydrous methanol.

[0017] In one or more embodiments, the molar ratio of compound 1 to compound 2 is 0.8-1.2:1, preferably 1:1.

[0018] In one or more embodiments, the concentration of compound 1 in the solvent is 27-40 mM, preferably 34 mM.

[0019] In one or more embodiments, the catalyst comprises piperidine.

[0020] In one or more embodiments, the reaction temperature is 90-100℃, preferably 95℃; and the reaction time is 40-60 h, preferably 48 h.

[0021] In a third aspect, the present application provides an application of the membrane-anchored fluorescent probe of the first aspect and / or the membrane-anchored fluorescent probe prepared by the preparation method of the second aspect in cell membrane positioning fluorescent imaging.

[0022] In one or more embodiments, the cells comprise MIHA, RAW, A549, HeLa and SiHa cells.

[0023] In a fourth aspect, the present application provides an application of the membrane-anchored fluorescent probe of the first aspect and / or the membrane-anchored fluorescent probe prepared by the preparation method of the second aspect in detecting cell membrane pH value.

[0024] In one or more embodiments, the cells include: MIHA, RAW, A549, HeLa, and SiHa cells.

[0025] In a fifth aspect of the present application, the membrane-anchored fluorescent probe of the first aspect and / or the membrane-anchored fluorescent probe prepared by the preparation method of the second aspect is used for preparing a diagnostic product for non-alcoholic fatty liver.

[0026] In one or more embodiments, the diagnostic product includes a detection reagent and a detection kit.

[0027] In a sixth aspect of the present application, a diagnostic product for non-alcoholic fatty liver is provided, which includes the membrane-anchored fluorescent probe of the first aspect and / or the membrane-anchored fluorescent probe prepared by the preparation method of the second aspect.

[0028] In one or more embodiments, the diagnostic product includes a detection reagent and a detection kit.

[0029] The present application has the following beneficial effects:

[0030] In the present application, a membrane-anchored fluorescent probe is designed and synthesized, which can be used for fluorescence imaging of cell membranes. Due to the lipophilic effect of the long alkyl chain of the probe on the phospholipid of the cell membrane and the electrostatic effect between the positive charge pyridine salt of the probe and the negative charge phosphate of the cell membrane, the probe can specifically target the cell membrane. At the same time, the probe shows excellent pH sensitivity and reversibility in in vitro and cell imaging experiments, and the pH response mechanism is confirmed by H NMR spectrum and Gaussian simulation. 1 The highest occupied molecular orbital (HOMO) of the membrane-anchored fluorescent probe is mainly concentrated in the methoxy and naphthalene ring fluorophore region before protonation, while the lowest unoccupied molecular orbital (LUMO) is significantly distributed on the pyridine ring. The energy gap between HOMO and LUMO is calculated to be 2.37 eV. The separation distribution of such frontier orbitals indicates the presence of intramolecular charge transfer (ICT) transition in the membrane-anchored fluorescent probe. After protonation, the HOMO energy decreases and the distribution shifts to the alkyl chain, and the electron-donating ability of the methoxy group decreases, which blocks the ICT process and leads to a decrease in fluorescence intensity. Furthermore, the membrane-anchored fluorescent probe can not only detect the pH values of different types of cell membranes and analyze the changes in cell membrane pH values during different physiological activities, but also can detect different degrees of non-alcoholic fatty liver by imaging the clarity of the boundary of liver cell membranes, and can visualize their pH heterogeneity, thereby realizing the early detection of non-alcoholic fatty liver. BRIEF DESCRIPTION OF DRAWINGS

[0031] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated herein by reference. The illustrations are of exemplary embodiments of the application and explain the principles of the application, but do not limit the application.

[0032] Figure 1 Preparation route of membrane-anchored fluorescent probe;

[0033] Figure 2 Absorption and fluorescence spectra of probe Mem-pH (10 μM) as a function of pH value (a, b), (c) plot of fluorescence intensity of Mem-pH as a function of pH value, (d) pH reversibility of Mem-pH (10 μM) between pH 3.0 and 12.0;

[0034] Figure 3 Photostability of probe Mem-pH;

[0035] Figure 4 HOMO-LUMO energy levels (a) and electrostatic potential (b) of Mem-pH before and after protonation;

[0036] Figure 5 MTT results of HeLa (a), A549 (b), SiHa (c), RAW (d), MIHA (e) and RAW (f) after incubation in different concentrations of Mem-pH for 24 hours; black columnar chart in (a)-(f) represents the control group, and red columnar chart represents Mem-pH treatment group;

[0037] Figure 6 Fluorescence images of GUVs of different pH values stained with 10 mM Mem-pH (a-c); d) average fluorescence intensity obtained in the circular area of a, b, c, Ex = 405 nm, Em = 550-620 nm, scale bar = 20 μm;

[0038] Figure 7 Fluorescence, DIC and merged images of HeLa cells stained with Mem-pH (2 uM, 10 min) and Dil (0.2 uM, 10 min); Merged 1 is the merged image of Mem-pH and DIC; Merged 2 is the merged image of Mem-pH and Dil channels; Mem-pH: Ex = 405 nm, Em = 550-620 nm; Dil: Ex = 532 nm, Em = 550-580 nm; scale bar = 20 μm;

[0039] Figure 8A is the fluorescence and merged images of SiHa and HeLa cells stained by Mem-pH (2 μΜ, 20 min), Lyso-Red (0.5 μΜ, 10 min) and MTDR (0.5 μΜ, 10 min), Mem-pH: Ex = 405 nm, Em = 550-620 nm; Lyso-Red: Ex = 552 nm, Em = 570-610 nm; MTDR: Ex = 635 nm, Em = 650-680 nm; scale = 20 μιη; B is the fluorescence distribution along the white arrow direction in the merged image of stained SiHa cells; C is the fluorescence distribution along the white arrow direction in the merged image of stained HeLa cells;

[0040] Figure 9 are the fluorescence, DIC, merged and quantification pseudo-color images of HeLa cells stained by Mem-pH (2 μΜ, 20 min) at different pH values, Ex = 405 nm, Em = 550-620 nm, scale = 20 μιη;

[0041] Figure 10 are the fluorescence, DIC, merged and quantification pseudo-color images of different types of cells stained by Mem-pH (2 μΜ, 20 min), Ex = 405 nm, Em = 550-620 nm, scale = 20 μιη;

[0042] Figure 11 are the fluorescence, DIC and their merged images of HeLa cells stained by Mem-pH (2 μΜ, 20 min) under normal condition and after LPS incubation for different time, Ex = 405 nm, Em = 550-620 nm, scale = 20 μιη;

[0043] Figure 12 are the fluorescence, DIC and their merged images of HeLa cells stained by Mem-pH (2 μΜ, 20 min) under normal condition and after H2O2 incubation for different time, Ex = 405 nm, Em = 550-620 nm, scale = 20 μιη;

[0044] Figure 13 are the fluorescence, DIC, merged and quantification pseudo-color images of normal and NAFLD tissues stained by Mem-pH (2 μΜ, 20 min), Ex = 405 nm, Em = 550-620 nm, scale = 20 μιη. DETAILED DESCRIPTION

[0045] It should be noted that the following detailed description is merely exemplary and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0046] It is to be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments consistent with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, devices, components and / or combinations thereof, but do not preclude the presence or addition of one or more other features, steps, operations, devices, components and / or combinations thereof.

[0047] In order to enable a person skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific examples.

[0048] The cells used in the following examples were all cultured in a 37℃ 5% CO2 incubator. HeLa, SiHa, MIHA and A549 cells were cultured in f12k medium containing 10% fetal bovine serum FBS and 1% penicillin / streptomycin, and RAW cells were cultured in RPMI1640 medium containing 10% FBS and 1% penicillin / streptomycin.

[0049] Example 1

[0050] Figure 1 For the preparation route of the membrane-anchored fluorescent probe, reference is made to Figure 1 , to synthesize the membrane-anchored fluorescent probe (hereinafter referred to as Mem-pH).

[0051] Preparation of Mem-pH:

[0052] Compound 1 (0.400 g, 0.845 mmol) and anhydrous methanol (25 mL) were added to a three-necked flask, heated to complete dissolution at 55℃, then compound 2 (0.198 g, 0.845 mmol) and piperidine (6 drops) were added. The temperature was raised to 95℃ and refluxed for 48h until red-brown solid precipitated. After the reaction was completed, the system was cooled to room temperature, and Mem-pH, a membrane-anchored fluorescent probe, was obtained by filtration. 1H NMR (DMSO-d6, 300 MHz): δ (ppm): 8.83 (d, J = 8 Hz, 2H), 8.13 (d, J = 8 Hz, 2H), 8.04 (d, J = 16 Hz, 2H), 7.79 (s, 3H), 7.47 (m, 1H), 7.28 (d, 1H), 7.09 (s, 1H), 4.37 (d, J = 16 Hz, 2H), 3.78 (s, 2H), 1.79 (s, 2H), 1.13 (m, 30H), 0.72 (d, J = 12 Hz, 3H).13C NMR (101 MHz, DMSO-d6): δ (ppm): 158.55, 152.92, 144.08, 141.11, 135.36, 130.47, 130.11, 129.68, 128.17, 127.58, 124.14, 123.54, 122.32, 119.33, 106.30, 59.58, 55.31, 31.19, 30.38, 28.94, 28.89, 28.77, 28.65, 28.60, 28.26, 25.29, 21.99, 13.85. HRMS (m / z): calcd for C 36 H 52 INO: 641.30; found: 514.41 (M-I) + 。

[0053] Example 2

[0054] To verify whether the probe Mem-pH can respond to pH, pH titration experiments were carried out, and the absorption and fluorescence spectra of Mem-pH are shown in Figure 2 As shown in a~b of Figure 2 , the maximum absorption and emission peaks of the probe Mem-pH are about 390 nm and 580 nm, respectively. In addition, the fluorescence intensity of Mem-pH gradually increases with the increase of pH value, indicating that the ICT process of the probe is restored in the alkaline environment. In addition, by fitting the fluorescence intensity at different pH values to prove the linear fluorescence response of the probe Mem-pH to pH Figure 2 c). More importantly, after several acid-base cycles, the fluorescence intensity of Mem-pH almost does not change, which proves that Mem-pH has excellent reversible pH response. Figure 2 d). In addition, the light stability of Mem-pH was studied, as shown in Figure 3 , even after 20 minutes of continuous xenon lamp irradiation, the fluorescence intensity of Mem-pH is still stable, which is conducive to the long-time imaging of Mem-pH to cells. These experimental results show that Mem-pH can respond to different pH values and can be well fitted to a linear relationship, which provides a basis for detecting and calculating the pH changes of the cell membrane.

[0055] In addition, to understand the relationship between optical properties and electronic transition, the energy and electrostatic potential of Mem-pH were calculated using density functional theory (DFT). Figure 4 a shows that the highest occupied molecular orbital (HOMO) of Mem-pH is mainly concentrated in the methoxy group and naphthalene fluorophore region before protonation, while the lowest unoccupied molecular orbital (LUMO) is significantly distributed on the pyridine ring. The energy gap between HOMO and LUMO is calculated to be 2.37 eV. The separate distribution of this frontier orbital indicates the presence of an ICT transition in Mem-pH. After protonation, the HOMO energy decreases and the distribution shifts to the alkyl chain, weakening the methoxy electron donor and blocking the ICT process and resulting in a decrease in fluorescence intensity. Figure 4 b further shows that the potential energy of the protonated methoxy molecule increases, thereby inhibiting the electron donor ability of the methoxy group and the ICT process, ultimately leading to a decrease in its fluorescence intensity.

[0056] Example 3

[0057] The cytotoxicity of the probe is an important parameter before cell staining and imaging, so the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was used to quickly and easily identify the cytotoxicity of Mem-pH. As shown in Figure 5 After incubation with different concentrations of Mem-pH values (2, 4, 6, 8 and 10 μM) for 24 hours, the cell viability of HeLa, A549, SiHa, RAW and MIHA was still higher than 85%. Therefore, the cytotoxicity of Mem-pH at short time and low dose can be ignored.

[0058] Example 4

[0059] Giant unilamellar vesicles (GUVs) are a model membrane commonly used to simulate cell membranes to analyze the physical properties of probes. Here, the probe Mem-pH was first tested for its ability to detect membrane pH using GUVs with different pH values. As shown in Figure 6 The probe Mem-pH can clearly image GUVs, and its fluorescence intensity gradually increases with increasing pH value. In addition, as shown in Figure 6 As the pH value of GUVs increased from 4.00 to 9.18, the fluorescence intensity of Mem-pH increased by nearly 10-fold. Therefore, the probe Mem-pH can visualize GUVs composed of phospholipids and give different fluorescence intensity signals according to different pH values.

[0060] Then, the probe Mem-pH was used to stain living cells to verify whether it can target the cell membrane. As shown in Figure 7As shown, the Mem-pH fluorescence from the merged images of differential interference microscopy (DIC) and fluorescence completely outlined the cell profile. In addition, the fluorescence of probe Mem-pH can be completely covered by the commercial cell membrane probe Dil, and the colocalization coefficient reaches 0.89. In addition, Mem-pH was co-stained with commercial lysosome probe Lyso-Red and mitochondrial probe MTDR to further confirm that it can stain the cell membrane. As shown in FIG. 2B, the fluorescence of probe Mem-pH was distributed around Lyso-Red and MTDR, and was completely non-overlapping with them. Therefore, probe Mem-pH can specifically stain the cell membrane. Figure 8

[0061] Example 5

[0062] To prove that Mem-pH can detect the pH value of the cell membrane, HeLa cells were selected to be stained with Mem-pH in different acid-base environments. As shown in FIG. 3A, the fluorescence of Mem-pH was completely analyzed on all cells, which indicated that Mem-pH had the ability to stain the cell membrane of normal cells. However, since the pH value of the cell membrane of normal cells (pH = 7.2-7.4) was not much different from that of the cell membrane of cancer cells (pH = 6.2-6.8), the fluorescence intensity of Mem-pH had little change with the fluorescence channel of Figure 9 As shown, as the pH value increased from 4.00 to 9.18, the Mem-pH emitted cell membrane fluorescence gradually increased. This may be because the ICT process of Mem-pH was inhibited by the protonation of methoxy in the acidic environment, and was restored in the alkaline environment. According to the linear relationship between the maximum emission of Mem-pH and the pH mentioned in c, the corresponding fluorescence intensity was calculated, and the pseudo-color gradually changed from purple to yellow as the pH increased, indicating that the fluorescence intensity gradually increased. These data clearly show that Mem-pH can image and monitor the pH value of the cell membrane using fluorescence intensity. Figure 2

[0063] Example 6

[0064] Mem-pH probe can distinguish normal cells, cancer cells and study cell physiological activities:

[0065] According to relevant reports, the cell membrane will be acidified after carcinogenesis, so the probe Mem-pH can be used to distinguish and detect normal cells and cancer cells. For this purpose, five kinds of cells such as MIHA, RAW, A549, HeLa and SiHa cells were selected for Mem-pH staining. As shown in FIG. 4A, the fluorescence of Mem-pH was completely analyzed on all cells, which indicated that Mem-pH had the ability to stain the cell membrane of normal cells. However, since the pH value of the cell membrane of cancer cells (pH = 6.2-6.8) was not much different from that of the cell membrane of normal cells (pH = 7.2-7.4), the fluorescence intensity of Mem-pH had little change with the fluorescence channel of Figure 10 Figure 10 Figure 2 ​​​​The equation in c calculates the pH value of different cell membranes. By comparing the pH value pseudo-color bands, the probe Mem-pH mainly shows yellow fluorescence in normal cell membranes, while it shows purple fluorescence in cancer cell membranes. This indicates that the pH value of cancer cell membranes is lower than that of normal cell membranes. Therefore, the probe Mem-pH successfully distinguishes and detects the pH values of cancer cell membranes and normal cell membranes by fluorescence intensity.

[0066] During the physiological process of cell inflammation, the pH value of the cell membrane will be affected by the increase in cell metabolic activity. For this reason, HeLa cells were selected to construct an inflammation model by treating them with lipopolysaccharide (LPS) to study this cell behavior. As shown in Figure 11 , with the increase of LPS treatment time, the Mem-pH fluorescence intensity of the cell membrane continuously decreased. This should be due to the dysfunction of ion channels, transporters and proton pumps on the cell membrane caused by inflammation, which in turn led to the outflow of metabolites such as hydrogen ions, organic acids and bicarbonate, thus reducing the pH value of the cell membrane. In addition, apoptosis is also an important physiological activity that plays a key role in processes such as cell development, tissue repair and immune response. Therefore, HeLa cells were then treated with H2O2 to induce the apoptosis process. As shown in Figure 12 , with the increase of H2O2 treatment time, the cell membrane began to swell and deform, and the fluorescence intensity of Mem-pH continuously decreased. Therefore, during the apoptosis process, due to the dysfunction of the permeability and the imbalance of the cell membrane, the pH value of the cell membrane also decreased. In summary, the probe Mem-pH can not only detect the pH values of normal cells and cancer cells, but also analyze the changes in cell membrane pH values during different physiological activities.

[0067] Example 6

[0068] Non-alcoholic fatty liver is a typical liver steatosis disease, which can be cured by early diagnosis and timely treatment. The metabolites produced during the lipid deacidification process will reduce the pH value, so the tissue acidification of the liver is also an important parameter to reflect the degree of non-alcoholic fatty liver disease, so it is very meaningful to detect the pH value changes of fatty liver. Considering the pH response characteristics of Mem-pH, it was therefore used to detect the pH value changes of non-alcoholic fatty liver. As shown in Figure 13As shown, the probe Mem-pH can visualize the outline of the liver tissue, and the outline gradually blurs as the fatty liver worsens. In addition, the fluorescence of Mem-pH in non-alcoholic fatty liver tissue is weaker than that in normal tissue, and gradually decreases as the fatty liver worsens. According to the pH value pseudo-color test paper, the probe Mem-pH shows yellow fluorescence at the clear and regular boundary of the hepatocyte membrane, and shows blue fluorescence at the blurred boundary. These data show that the deterioration of non-alcoholic fatty liver increases the acidification of liver tissue and disrupts the cell boundary of the tissue. Therefore, the probe Mem-pH can not only detect different degrees of non-alcoholic fatty liver by imaging the clarity of the hepatocyte membrane boundary, but also visualize the pH heterogeneity of them. Therefore, the probe Mem-pH can not only distinguish non-alcoholic fatty liver tissues, but also detect early fatty liver lesions, providing hope for timely diagnosis of non-alcoholic fatty liver.

[0069] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A membrane anchored fluorescent probe, characterized in that, The structural formula is shown as formula (I): Formula (I).

2. The method for preparing the membrane-anchored fluorescent probe as described in claim 1, characterized in that, Comprising: The compound 1 is dissolved in a solvent, compound 2 is added, and a membrane anchoring fluorescent probe shown in formula (I) is synthesized under the catalysis of a catalyst; the catalyst is piperidine; The structural formula of compound 1 is shown as formula (II): Formula (II); The structural formula of compound 2 is shown as formula (III): Formula (III).

3. The method of claim 2, wherein, The solvent includes anhydrous methanol; Or, the molar ratio of compound 1 to compound 2 is 0.8-1.2:1; Or, the concentration of compound 1 in the solvent is 27-40 mM; Or, the temperature of the reaction is 90-100 ℃; the time of the reaction is 40-60 h.

4. The method of claim 3, wherein, The molar ratio of compound 1 to compound 2 is 1:1; the concentration of compound 1 in the solvent is 34 mM; the temperature of the reaction is 95 ℃; and the time of the reaction is 48 h.

5. The membrane anchoring fluorescent probe of claim 1 and / or the membrane anchoring fluorescent probe prepared by the preparation method of any one of claims 2-3 is applied to cell membrane positioning fluorescent imaging for non-disease diagnosis and treatment purposes.

6. The use according to claim 5, wherein the compound is ###0002### The cells are selected from the group consisting of MIHA, RAW, A549, HeLa and SiHa cells.

7. The membrane anchoring fluorescent probe of claim 1 and / or the membrane anchoring fluorescent probe prepared by the preparation method of any one of claims 2-3 is applied to detecting cell membrane pH value for non-disease diagnosis and treatment purposes.

8. Use according to claim 7, wherein the compound is ###0002### The cells are selected from the group consisting of MIHA, RAW, A549, HeLa and SiHa cells.

9. The membrane anchoring fluorescent probe of claim 1 and / or the membrane anchoring fluorescent probe prepared by the preparation method of any one of claims 2-3 is applied to preparing a diagnostic product for non-alcoholic fatty liver.

10. A diagnostic product for non-alcoholic fatty liver disease, characterized by comprising the polypeptide of claim 1 or 2. The diagnostic product comprises the membrane anchoring fluorescent probe of claim 1 and / or the membrane anchoring fluorescent probe prepared by the preparation method of any one of claims 2-5.

11. The diagnostic product of claim 10, wherein, The diagnostic product comprises a detection reagent and a detection kit.

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