A multifunctional lipid droplet fluorescent probe and its preparation method and application

Abstract

The present invention discloses a multifunctional fluorescent probe and a preparation method and application thereof, belonging to the technical field of fluorescent probes. The chemical name of the fluorescent probe provided by the present invention is: (2-(Z)-5-(E)-3-(4-(dimethylamino)phenyl)allyl)-3-ethyl-4-oxothiazolidine-2-ylidenemalononitrile). The preparation method of the fluorescent probe has mild reaction conditions and simple process. The fluorescent probe of the present invention has the characteristic of targeting lipid droplets, and can be used as a detection reagent to simultaneously detect viscosity, polarity and HOCl changes in inflammatory cell models or zebrafish models, and the detection means is simple and sensitive.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and specifically relates to a multifunctional lipid droplet fluorescent probe and a preparation method and application thereof. Background Art

[0002] Lipid droplets (LDs) are ubiquitous organelles in almost all eukaryotic cells. They consist of a neutral lipid core and a surrounding phospholipid monolayer membrane. They are recognized as energy storage organelles and are major regulators of cellular metabolism. In the past decade, the study of LDs has become one of the most attractive topics in cell biology. Cell viscosity, as an important microenvironmental parameter, plays a crucial role in many diffusion-mediated biological processes, such as signal transmission and electron transport. Polarity is a feedback of cellular regulatory mechanisms and has been shown to be closely related to various physiological processes, such as membrane fusion, protein denaturation, signal transduction, and peptide aggregation. However, abnormal LD ​​polarity or viscosity changes may disrupt lipid metabolism, leading to LD dysfunction and many diseases such as inflammation, diabetes, neurodegenerative diseases, and even cancer. In order to better understand the intrinsic relationship between LD viscosity, polarity, and hypochlorous acid (HOCl) levels and LD-related diseases, it is urgent to develop a simple, reliable, and precise method that can simultaneously detect changes in LD viscosity, polarity, and HOCl.

[0003] Fluorescence imaging technology has become a powerful tool for direct imaging and monitoring of important biomolecules and their related processes. Small molecule fluorescent probes have been widely used in fluorescence imaging due to their high sensitivity, real-time detection, rapid and non-destructive analysis, etc. Developing a small molecule fluorescent probe targeting lipid droplets to explore the relationship between lipid droplets and diseases has great application value.

[0004] CN113651834A discloses a fluorescent probe based on a dithiophene acene derivative for specifically labeling lipid droplets in HeLa cells; CN 114262272 A discloses a naphthalene-indane dione donor-acceptor compound, a preparation method thereof, and its application in a lipid droplet wash-free fluorescent probe.

[0005] Currently, many fluorescent probes for the single detection of lipid droplet viscosity, lipid droplet polarity or HOCl have been reported. However, due to the lack of suitable tools, the interrelated roles of these parameters (including HOCl, viscosity and polarity) in many complex physiological and pathological environments have not been fully studied. Compared with traditional fluorescent probes or single-function sensors, multifunctional probes with more than two reporting parts can not only complete many tasks at the same time, but also reduce mutual interference and reduce toxicity to cells. Therefore, the development of multifunctional lipid droplet fluorescent probes to detect changes in multiple parameters such as viscosity, polarity and HOCl levels during ferroptosis and in zebrafish inflammation models is of great significance for in-depth research on lipid droplet-related diseases. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a multifunctional lipid droplet fluorescent probe and its preparation method and application in view of the deficiencies in the prior art.

[0007] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0008] The present invention provides a multifunctional lipid droplet fluorescent probe, the structural formula of which is as follows:

[0009]

[0010] The fluorescent probe NCN of the present invention has a chemical name: (2-(Z)-5-(E)-3-(4-(dimethylamino)phenyl)allyl)-3-ethyl-4-oxothiazolidine-2-ylidenemalononitrile).

[0011] The present invention also provides a method for preparing the multifunctional lipid droplet fluorescent probe, comprising the following steps:

[0012]

[0013] (1) dissolving 2-(3-ethyl-4-oxothiazolidin-2-ylidene)malononitrile and 4-dimethylaminocinnamaldehyde NCM in anhydrous ethanol, slowly adding piperidine to the reaction solution, and heating to react; after the reaction is completed, cooling to room temperature, filtering and collecting solids to obtain a crude product;

[0014] (2) The crude product obtained in step (1) is purified by silica gel column chromatography to obtain a red solid, namely the fluorescent probe NCN, chemical name: (2-(Z)-5-(E)-3-(4-(dimethylamino)phenyl)allyl)-3-ethyl-4-oxothiazolidin-2-ylidenemalononitrile).

[0015] The fluorescent probe prepared by the invention uses N, N-dimethylamino as an electron-donating group (D) and a rhodanine derivative of a dicyanomethylene unit as an electron-withdrawing group (A).

[0016] Preferably, in step (1), the molar ratio of 2-(3-ethyl-4-oxothiazolidin-2-ylidene)malononitrile to 4-dimethylaminocinnamaldehyde NCM is 1:1.

[0017] Preferably, in step (1), the reaction temperature is reflux temperature and the reaction time is 8 hours.

[0018] Preferably, in step (1), the molar ratio of piperidine to 4-dimethylaminocinnamaldehyde NCM is 1:20.

[0019] Preferably, in step (2), the silica gel column chromatography eluent is dichloromethane.

[0020] The present invention also provides application of the multifunctional lipid droplet fluorescent probe in lipid droplet imaging.

[0021] The multifunctional lipid droplet fluorescent probe simultaneously detects the viscosity, polarity and hypochlorous acid level of the lipid droplets.

[0022] The near-infrared (NIR) fluorescence intensity of the fluorescent probe of the present invention gradually increases with the increase of the environmental viscosity, and the green fluorescence intensity gradually increases with the increase of the HOCl concentration.

[0023] The detection of polarity change of the probe of the present invention: in the 1,4-dioxane / water system, when the solution polarity Δf increases from 0.229 to 0.3200, the near-infrared fluorescence intensity of the probe is significantly enhanced; and I max There is a good linear relationship with Δf (R 2 =0.9933), which has the characteristics of highly sensitive quantitative or qualitative detection of environmental polarity.

[0024] The recognition principle of viscosity change by the probe described in the present invention is as follows: in a dissolved state or a low-viscosity solution, due to the free rotation of the ethylene single bond within the molecule, the entire molecule has a TICT effect and the probe fluorescence is very weak; when in a solution with a higher viscosity, the free rotation movement within the molecule is restricted, the TICT effect is suppressed, making the entire molecule coplanar, and the probe will emit very strong near-infrared fluorescence.

[0025] The probe of the present invention detects viscosity changes: in a water / glycerol system, when the solution viscosity coefficient η increases from 0.59 cP to 945 cP, the near-infrared fluorescence intensity at 660 nm is significantly enhanced; and log I 660 There is a good linear relationship with logη (R 2 =0.9951), the slope is 0.3867, and it has the characteristics of highly sensitive quantitative or qualitative detection of environmental viscosity.

[0026] The recognition principle of the probe of the present invention for the change of HOCl is as follows: HOCl can react with the C=C of the probe to remove the 2-(3-ethyl-4-oxothiazolidine-2-ylidene)malononitrile connected to the double bond, thereby releasing a new fluorophore NCM with strong green fluorescence emission.

[0027] The probe of the present invention detects changes in HOCl: the molecular probe is dissolved in a mixed system of ethanol (EtOH) and phosphate buffer solution (PBS), and as the concentration of HOCl increases, the green fluorescence at 550nm is significantly enhanced; the linear range of HOCl detection is 0-40μM (R 2=0.9946), and the detection limit is 11.84 nM. Therefore, the probe of the present invention is very suitable for HOCl detection in water and biological systems.

[0028] The present invention also provides a detection reagent containing the multifunctional lipid droplet fluorescent probe, wherein the detection reagent is used to detect at least one of lipid droplet polarity, lipid droplet viscosity or hypochlorous acid level.

[0029] The detection reagent can be used to simultaneously detect changes in viscosity, polarity and HOCl during ferroptosis; the detection reagent can also be used to simultaneously detect changes in viscosity, polarity and HOCl in an inflammatory zebrafish model.

[0030] Beneficial effects:

[0031] (1) The fluorescent probe of the present invention can simultaneously detect the viscosity, polarity and hypochlorous acid level of lipid droplets, has the characteristics of highly sensitive quantitative or qualitative detection of environmental viscosity and environmental polarity, and is suitable for HOCl detection in water bodies and biological systems.

[0032] (2) The maximum fluorescence emission wavelengths of the fluorescent probe for viscosity and HOCl detection differ by about 110 nm, which can effectively avoid spectral interference in dual-channel simultaneous detection and improve detection sensitivity.

[0033] (3) The detection reagent containing the multifunctional lipid droplet fluorescent probe NCN of the present invention has a simple detection method, which only requires a fluorescence spectrophotometer and a laser confocal microscope.

[0034] (4) The fluorescent probe of the present invention has a good lipid droplet imaging effect.

[0035] (5) The preparation method of the fluorescent probe of the present invention has mild reaction conditions and simple process. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 For the NMR characterization of the fluorescent probe NCN, 1 H-NMR spectrum;

[0037] Figure 2 For the NMR characterization of the fluorescent probe NCN, 13 C-NMR spectrum;

[0038] Figure 3 The fluorescence emission spectrum of the fluorescent probe NCN in a 1,4-dioxane / water mixed solvent changes with the water volume content;

[0039] Figure 4 I of the fluorescent probe NCN max Linear relationship diagram with the change of Δf in 1,4-dioxane / water mixed system;

[0040] Figure 5is the fluorescence emission spectrum of the fluorescent probe NCN in a water / glycerol mixture system as the volume content of glycerol changes;

[0041] Figure 6 is the log I of the fluorescent probe NCN 660 Linear relationship diagram with logη in water / glycerol mixed system;

[0042] Figure 7 The fluorescence emission spectrum of the fluorescent probe NCN in the EtOH / PBS mixed system changes with the HOCl concentration;

[0043] Figure 8 is the fluorescence intensity of the fluorescent probe NCN (I 550 ) and the linear relationship between HOCl concentration;

[0044] Fig. 9 The selectivity of the fluorescent probe NCN for viscosity;

[0045] Fig.10 Mass spectrometry analysis of the fluorescent probe NCN identifying HOCl;

[0046] Fig.11 This is the fluorescence co-localization imaging of the fluorescent probe NCN and the commercially available lipid droplet-specific dye BODIPY 493 / 503;

[0047] Fig.12 This is a fluorescence imaging diagram of the changes in viscosity / polarity and HOCl level using dual channels to detect the fluorescent probe NCN during ferroptosis.

[0048] Fig.13 Fluorescence imaging of the changes in viscosity / polarity and HOCl levels using dual channels for the fluorescent probe NCN in a zebrafish inflammation model. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0050] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0051] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0052] Example 1 Preparation of fluorescent probe

[0053]

[0054] 2-(3-ethyl-4-oxothiazolidin-2-ylidene)malononitrile (193.0 mg, 1 mmol) and compound NCM (175.1 mg, 1 mmol) were dissolved in 25 mL of anhydrous ethanol, and piperidine (5 μL, 0.05 mmol) was added as a catalyst, and stirred at reflux (80°C) for 8 h. The red solid was collected by filtration and washed with cold ethanol and water. Finally, the crude product was purified by silica gel column chromatography (eluent: dichloromethane) to obtain NCN (278.4 mg, yield 79.55%).

[0055] Figure 1 This is the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe NCN. Figure 2 This is the carbon NMR spectrum of the fluorescent probe NCN.

[0056] 1 H NMR(300MHz, CDCl3)δ(ppm)7.64(dd,J=11.8,0.8Hz,1H),7.49-7.40(m,2H),7.09(d,J=14.8Hz,1H),6.72( d,J=8.7Hz,2H),6.57(dd,J=14.8,11.9Hz,1H),4.26(q,J=7.1Hz,2H),3.09(s,6H),1.38(t,J=7.1Hz,3H);

[0057] 13 C NMR(75MHz,CDCl3)δ(ppm):165.61,152.30,148.21,138.11,132.04,130.59,130 .40,122.98,117.29,113.85,113.56,112.00,53.44,50.87,40.27,40.13,14.20;

[0058] HR-MS: m / z: calculated value 351.1200; found value 351.1276 [M+H] + .

[0059] Example 2 Fluorescence response characteristics of fluorescent probe NCN to polarity in 1,4-dioxane / water mixed solvent

[0060] The probe in Example 1 was diluted to a final concentration of 5 μmol / L with a 1,4-dioxane / water mixed solvent, the excitation wavelength was fixed at 560 nm, and the fluorescence emission spectrum of the probe was recorded as the volume content of 1,4-dioxane (or polarity coefficient Δf) varied (see Figure 3 ), and plot the fluorescence intensity value of the probe at the maximum fluorescence (I max), the linear relationship with the viscosity coefficient (Imax) in the 1,4-dioxane / water mixed system is as follows Figure 4 shown.

[0061] As the water volume ratio increased from 10% (polarity coefficient Δf 0.229) to 100% (polarity coefficient Δf 0.3200), the near-infrared fluorescence emission at 660 nm decreased successively, indicating that the probe can achieve highly sensitive detection of environmental polarity.

[0062] Example 3 Fluorescence response characteristics of fluorescent probe NCN to viscosity in water / glycerol mixed solvent

[0063] The probe in Example 1 was diluted with a water / glycerol mixed solvent to a final concentration of 5 μmol / L, the excitation wavelength was fixed at 560 nm, and the fluorescence emission spectrum of the probe was recorded as the volume content of glycerol (or viscosity coefficient η) varied (see Figure 5 ), and plot the fluorescence intensity of the probe at 660 nm (logI 660 ), the linear relationship diagram of the viscosity coefficient (logη) in the water / glycerol mixed system is as follows Figure 6 shown.

[0064] As the volume ratio of glycerol increased from 0% (viscosity coefficient η 0.59 cP) to 99% (viscosity coefficient η 945 cP), the near-infrared fluorescence emission at 660 nm increased successively, indicating that the probe can achieve highly sensitive detection of environmental viscosity.

[0065] Example 4 Fluorescence response characteristics of fluorescent probe NCN in EtOH / PBS mixed solvent as HOCl concentration changes

[0066] The probe NCN in Example 1 was diluted to a final concentration of 5 μmol / L using a mixed solvent of EtOH / PBS (4 / 6, v / v, pH 7.4), the excitation wavelength was fixed at 410 nm, and the fluorescence emission spectrum of the probe was recorded as the concentration of HOCl changed (see Figure 7 ), as the concentration of HOCl increased from 0 μmol / L to 350 μmol / L, the green fluorescence emission at 550 nm increased successively; the linear range of HOCl detection was 0-40 μM (R 2 =0.9946), such as Figure 8 As shown, the detection limit is 11.84 nM, indicating that the probe can achieve high-sensitivity detection of HOCl.

[0067] Example 5 Selectivity of Fluorescent Probe for Viscosity

[0068] The concentration of the fluorescent probe NCN in Example 1 was kept at 5 μmol / L (EtOH / PBS mixed solvent: 4 / 6, v / v, pH 7.4), and the probe was investigated for common ions and bioactive small molecules (for specific substances, see Fig. 9 ) in the presence of 5% paraformaldehyde and 1% paraformaldehyde to investigate the selectivity of the probe for viscosity. Fig. 9 Among the substances shown (200 μmol / L), only gly can significantly enhance the fluorescence intensity of the probe, indicating that the probe has good selectivity for viscosity.

[0069] Example 6 Verification of the action mechanism of fluorescent probe NCN and HOCl

[0070] 50 μmol of probe NCN was reacted with 3.5 mM HOCl solution at room temperature for 1 h.

[0071] The fluorescent probe NCN in Example 1, the solution after the probe NCN reacted with HOCl for 1 hour, and the compound NCM were respectively subjected to mass spectrometry to verify the action mechanism of the probe NCN and HOCl.

[0072] like Fig.10 As shown, the peak at m / z=351.1276 is the mass spectrum peak of the probe NCN (theoretical value: 351.1200), and the peak at m / z=176.0088 can be attributed to the mass spectrum peak of the new fluorophore released after the probe NCN reacts with HOCl for 1 hour, and is almost consistent with the mass spectrum peak of the pure compound NCM (m / z=176.1066) (theoretical value: 176.1000), indicating that after the reaction of the probe NCN with HOCl, 2-(3-ethyl-4-oxothiazolidine-2-ylidene)malononitrile is removed, thereby releasing a new fluorophore, which is the compound NCM.

[0073] Example 7 Co-localization experiment of fluorescent probe NCN and commercially available lipid droplet-specific dye BODIPY493 / 503

[0074] In order to observe whether the probe NCN can be targeted and localized in lipid droplets, a co-localization experiment between the probe and commercially available lipid droplet-specific dye BODIPY493 / 503 (Anjieke Biopharmaceuticals) was performed.

[0075] The adherent HepG2 cells (from ATCC) were incubated with BODIPY493 / 503 (dissolved in DMSO to a final concentration of 0.1 μmol / L) at pH 7.4 in an incubator at 37°C and 5% CO2 for 30 minutes, and then gently washed three times with phosphate buffer (pH 7.4) to remove excess dye. Then the probe NCN (NCN was dissolved in DMSO to a final concentration of 5 μmol / L) was added and incubated for another 30 minutes, and the co-localization of the two was observed under a laser confocal microscope. The fixed excitation wavelength of the probe NCN was 561 nm, and the red channel range was 600-700 nm; the fixed excitation wavelength of BODIPY493 / 503 was 488 nm, and the green channel range was 500-550 nm. The fluorescence co-localization imaging of the fluorescent probe NCN and the commercially available lipid droplet-specific dye BODIPY 493 / 503 is shown in Fig.11 .

[0076] Depend on Fig.11 It can be seen that the fluorescent probe NCN and BODIPY493 / 503 can overlap well to obtain yellow overlapping fluorescence, and the Pearson coefficient of the fluorescent probe NCN and BODIPY493 / 503 is as high as 0.98 after processing with image J software. This shows that the fluorescent probe NCN and BODIPY493 / 503 have significant co-localization imaging and can be targeted in lipid droplets.

[0077] Example 8 Dual-channel fluorescence imaging of fluorescent probe NCN

[0078] The adherent HepG2 cells were incubated with the fluorescent probe NCN in Example 1 (NCN was dissolved in DMSO to a final concentration of 5 μmol / L) at pH 7.4 in an incubator at 37°C and 5% CO2 for 30 min, and the dual-channel fluorescence imaging of the probe was observed under a laser confocal microscope, where: the green channel (E x =405nm, Em = 500 600nm) is used to observe the detection of HOCl by the probe; the NIR channel (E x =561nm, E m =600-700nm) is used to observe the probe's detection of viscosity / polarity.

[0079] Fluorescence imaging of the changes in viscosity / polarity and HOCl levels using the fluorescent probe NCN during ferroptosis. Fig.12 . Fig.12(A) Fluorescence dual-channel images of HepG2 cells incubated with NCN (5 μM) as a control; pretreated with Erastin (10 μM) and then treated with NCN (5 μM); pretreated with Fer-1 (15 μM) and Erastin (10 μM) and then treated with NCN (5 μM). Green channel (HOCl channel): λex = 405 nm, λem = 500-600 nm. Red channel (viscosity / polarity channel): λex = 561 nm, λem = 600-750 nm. Scale bar: 20 μm. Fig.12 (B) is the relative fluorescence intensity of the image shown in (A), ****P<0.0001.

[0080] like Fig.12 As shown, in the control group where only the probe NCN was added, weak fluorescence was emitted in both channels. Then Erastin (Shanghai Yuanye Biotechnology Co., Ltd.) was added to stimulate the cells for 30 minutes, and it was observed that the fluorescence emission of both channels in the cells was enhanced, indicating that Erastin can induce a significant increase in intracellular viscosity and HOCl levels. After adding Fer-1 (Shanghai Yuanye Biotechnology Co., Ltd.) for 30 minutes, Erastin was added to stimulate the cells for 30 minutes, and it was observed that the fluorescence emission of both channels in the cells was weakened, indicating that the probe NCN can achieve the simultaneous detection of viscosity / polarity and HOCl in ferroptotic cells.

[0081] Example 9 Dual-channel fluorescence imaging of fluorescent probe NCN

[0082] Zebrafish were purchased from Shanghai Feixi Biotechnology Co., Ltd., and then transferred to culture dishes and cultured in E3 embryo culture medium. The culture dishes were cultured in an incubator at 28°C for four days, and new E3 embryo culture medium was replaced every day. Subsequently, 10 zebrafish were grouped into one group. The three experimental groups were added with PBS-dissolved lipopolysaccharide LPS (1, 2, 5 μg / ml) and incubated at 28°C for 24 hours. Then, they were incubated for 30 minutes with the fluorescent probe NCN in Example 1 (NCN was dissolved in DMSO to a final concentration of 5 μmol / L). The control group only added the fluorescent probe NCN (NCN was dissolved in DMSO to a final concentration of 5 μmol / L) and incubated for 30 minutes. Each group was washed three times with PBS before imaging to remove background interference. Afterwards, the dual-channel fluorescence imaging of the probe was observed under a laser confocal microscope, where: the green channel (E x =405nm,E m =500-600nm) is used to observe the detection of HOCl by the probe; NIR channel (E x =561nm,E m =600-700nm) is used to observe the probe's detection of viscosity / polarity.

[0083] Fluorescent probe NCN dual-channel detection of viscosity / polarity and HOCl level fluorescence imaging changes in zebrafish inflammation model Fig.13 . NCN fluorescence imaging after LPS stimulation in zebrafish, Fig.13 (A) The first to third columns: Zebrafish were pretreated with LPS (0, 0.5, 1, 2 μg / mL) and then incubated with the probe NCN (5 μM). Green channel (HOCl channel): λex = 405 nm, λem = 500-600 nm. Red channel (viscosity / polarity channel): λex = 561 nm, λem = 600-750 nm. Scale bar: 500 μm. Fig.13 (B) is the relative fluorescence intensity of the image shown in (A), ****P<0.0001.

[0084] like Fig.13 As shown, the probe emits weak fluorescence in both channels without LPS. However, as the LPS concentration increases, bright fluorescence emission can be observed in both channels, indicating that LPS can induce a significant increase in viscosity (polarity reduction) and HOCl levels in zebrafish, and the probe NCN can achieve simultaneous detection of viscosity / polarity and HOCl in the inflammatory zebrafish model.

[0085] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A multifunctional lipid droplet fluorescent probe, characterized in that: Its structural formula is as follows:

2. A method for preparing a multifunctional lipid droplet fluorescent probe, characterized in that: The following steps are involved: (1) dissolving 2-(3-ethyl-4-oxothiazolidin-2-ylidene)malononitrile and 4-dimethylaminocinnamaldehyde NCM in anhydrous ethanol, slowly adding piperidine to the reaction solution, heating to 80° C. for reaction; cooling to room temperature after the reaction, filtering and collecting the solid to obtain a crude product; (2) The crude product obtained in step (1) is purified by silica gel column chromatography to obtain a red solid, namely the fluorescent probe NCN, chemical name: (2-(Z)-5-(E)-3-(4-(dimethylamino)phenyl)allyl)-3-ethyl-4-oxothiazolidin-2-ylidenemalononitrile).

3. The preparation method according to claim 2, characterized in that: In step (1), the molar ratio of 2-(3-ethyl-4-oxothiazolidine-2-ylidene)malononitrile to 4-dimethylaminocinnamaldehyde NCM is 1:

1.

4. The preparation method according to claim 2, characterized in that: In step (1), the reaction temperature is reflux temperature and the reaction time is 8 hours.

5. The preparation method according to claim 2, characterized in that: In step (1), the molar ratio of piperidine to 4-dimethylaminocinnamaldehyde NCM is 1:

20.

6. The preparation method according to claim 2, characterized in that: In step (2), the silica gel column chromatography eluent is dichloromethane.

7. Use of the multifunctional lipid droplet fluorescent probe according to claim 1 in lipid droplet imaging.

8. The use according to claim 7, characterized in that: The multifunctional lipid droplet fluorescent probe simultaneously detects the viscosity, polarity and hypochlorous acid level of the lipid droplets.

9. A detection reagent comprising the multifunctional lipid droplet fluorescent probe according to claim 1, characterized in that: The detection reagent is used to detect at least one of lipid droplet polarity, lipid droplet viscosity or hypochlorous acid level.

10. The detection reagent according to claim 9, characterized in that The detection reagent simultaneously detects changes in viscosity, polarity and HOCl during ferroptosis; the detection reagent simultaneously detects changes in viscosity, polarity and HOCl in an inflammatory zebrafish model.

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