Fluorescent dye as well as preparation method and application thereof

Abstract

The invention provides a fluorescent dye and a preparation method and application thereof, the fluorescent dye has the following chemical structural formula: wherein R1 and R2 are respectively and independently selected from H, Cl, Br and I; r3 is selected from alkyl, and n is a positive integer. The problems that a traditional dye is low in binding selectivity and overlapped in life fingerprints in a complex lipid system are solved, and high-resolution recognition of lipid types such as CE, TG and PL in a serum environment is achieved. Meanwhile, extraction-free in-situ classification detection can be carried out on the lipid, when the fluorescent dye is used for the probe, dependence of chromatography and mass spectrometry technologies on tedious pretreatment can be broken through based on the synergistic effect of the fluorescent probe and the fluorescence lifetime imaging technology, and serum lipid spatial distribution analysis and relative quantification of components are achieved. In addition, a standardized classification criterion can be established, and a quantifiable lipid subtype analysis tool is provided for clinic.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biomedical detection materials and analysis technology, and particularly relates to a fluorescent dye as well as a preparation method and application thereof. BACKGROUND

[0002] Serum lipid subtype typing has key significance for the accurate diagnosis of cardiovascular diseases and metabolic syndrome. In the related technology, the dye is not sensitive enough to the lipid microenvironment in the application of fluorescence lifetime imaging (FLIM), so that the lifetime difference after binding with different lipids is weak and the distribution is highly overlapped (typical as the characteristic lifetime values of cholesterol ester (CE) and triglyceride (TG) are close), and it is difficult to establish an effective classification threshold system. SUMMARY

[0003] The technical purpose of the present application is to provide a fluorescent dye as well as a preparation method and application thereof, aiming at solving the technical problem that the dye in the related technology is not sensitive enough to the lipid microenvironment, so that the lifetime difference after binding with different lipids is weak and the distribution is highly overlapped, and it is difficult to establish an effective classification threshold system.

[0004] To solve the above technical problems, the present application is realized by providing a fluorescent dye, which has the following chemical structure formula: wherein R1, R2 are independently selected from H, Cl, Br and I; R3 is selected from alkyl, , and wherein n is a positive integer.

[0005] Further, in some embodiments, the alkyl includes CH3 and C12-18 alkyl.

[0006] The second aspect of the present application provides a preparation method of the fluorescent dye, comprising: Step 1, adding phenothiazine and a first substance to an organic solvent and stirring; obtaining a first intermediate by column chromatography separation; the first substance is selected from any one of halogenated alkyl, 2-(2-(2-methoxyethoxy)ethoxy)ethyl p-toluenesulfonate, 1,3-propane sulfolane, brominated PEG2000 and a second substance; the second substance includes 3-bromo-1-propanol and phosphorus oxychloride; Step 2, under an inert atmosphere, cooling the phosphorus oxychloride to -5-5℃ by ice bath, adding an equal amount of DMF, stirring for 30 min, then adding the DMF solution of the first intermediate, warming to 80-100℃, and stirring until the first intermediate is completely reacted; adding ice water to the mixture and adjusting the pH to 7-8; extracting, washing and concentrating; obtaining a second intermediate by column chromatography separation; Step 3, under inert atmosphere, the second intermediate is dissolved in dichloromethane, drop trifluoroacetic acid, add 2,4-dimethylpyrrole in dichloromethane solution under light-proof condition, stir the solution; add 2,3-dichloro-5,6-dicyano-benzoquinone and stir; under ice bath, add triethylamine and boron trifluoride ether solution and stir; after concentration, column chromatography is used to separate to obtain the fluorescent dye.

[0007] Further, in some embodiments, when the first substance in step 1 is selected from any one of halogenated alkyl, 2-(2-(2-methoxyethoxy)ethoxy)ethyl p-toluenesulfonate, 1,3-propane sulfolactone and bromo-PEG2000, the step 1 comprises: placing phenothiazine, the first substance and KOH solution in dimethyl sulfoxide, heating to 55-65℃, stirring until the phenothiazine is completely consumed; adding ice water, extracting, washing and concentrating; purifying by column chromatography to obtain the first intermediate.

[0008] Further, in some embodiments, when the first substance in step 1 is 3-bromo-1-propanol and phosphorus oxychloride, the step 1 comprises: placing phenothiazine, 3-bromo-1-propanol and KOH solution in dimethyl sulfoxide, heating to 55-65℃, stirring until the phenothiazine is completely consumed; adding ice water, extracting, washing and concentrating; purifying by column chromatography to obtain the third intermediate; dissolving the third intermediate and pyridine in chloroform and cooling to-20--10℃; adding chloroform solution of phosphorus oxychloride, stirring and heating to 10-30℃; distilling under reduced pressure and washing to obtain the fourth intermediate; adding the fourth intermediate into deionized water, stirring the solution; extracting, washing and drying; distilling under reduced pressure to obtain the first intermediate.

[0009] Further, in some embodiments, before the operation step of placing phenothiazine, the first substance and KOH solution in dimethyl sulfoxide in step 1, it further comprises: drying dimethyl sulfoxide by a drying agent or activated molecular sieve.

[0010] Further, in some embodiments, before the operation step of dissolving the second intermediate in dichloromethane under inert atmosphere in step 3, it further comprises: drying dichloromethane by re-distillation.

[0011] Further, in some embodiments, the step 2 uses saturated sodium carbonate to adjust pH value.

[0012] The third aspect of the embodiment of the present application provides application of the fluorescent dye to in-situ detection and analysis of lipids in serum.

[0013] The fourth aspect of the embodiment of the present application provides application of the fluorescent dye to preparation of a fluorescent probe targeting lipids.

[0014] The fluorescent dye, the preparation method and the application thereof in the application have the beneficial effects that, compared with related technologies, the problems of low binding selectivity and overlapping of life fingerprints of traditional dyes in a complex lipid system are solved, high-resolution recognition of lipids such as cholesteryl ester (CE), triglyceride (TG) and phospholipid (PL) in a serum environment is achieved. Meanwhile, the lipids can be classified and detected in situ without extraction, when the fluorescent dye is used as a probe, the fluorescent dye can break through the dependence of chromatography and mass spectrometry on complicated pretreatment based on the synergistic effect of the fluorescent probe and fluorescence lifetime imaging (FLIM) technology, realizes spatial distribution analysis and relative quantification of serum lipids, and establishes a standardized classification criterion to solve the classification failure problem of FLIM technology in related technologies due to overlapping of life distribution, thereby providing a quantifiable lipid subtype analysis tool for clinics. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a fluorescence spectrum diagram of the fluorescent dye in the embodiment of the application in tetrahydrofuran-methanol solutions with different volume ratios; Figure 2 is a fluorescence lifetime decay diagram of the fluorescent dye in the embodiment of the application in tetrahydrofuran-methanol solutions with different volume ratios; Figure 3 is a flowchart of the fluorescent dye for pure lipid detection in the embodiment of the application; Figure 4 is a comparison of lifetimes of the fluorescent dye and traditional dyes in combination with pure lipids in the embodiment of the application; Figure 5 is a flowchart of the fluorescent dye for serum detection in the embodiment of the application; Figure 6 is a serum FLIM diagram and data result diagram of normal people and hyperlipidemia patients in the embodiment of the application. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and advantages of the application clearer, the application is further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the application, and are not used to limit the application.

[0017] Serum lipid subtype classification is of great significance for the precise diagnosis of cardiovascular diseases and metabolic syndrome. However, the existing detection technologies still have systematic limitations. Traditional biochemical methods (such as enzyme method and colorimetric method) are limited to total amount analysis (such as total cholesterol and total triglyceride), and cannot realize specific analysis of lipid components; although the combination of chromatography and mass spectrometry (LC-MS / GC-MS) can provide component information at the molecular level, it still has the problems of complicated sample pretreatment process (including lipid extraction and derivatization steps), high equipment cost and long analysis period, which cannot meet the needs of clinical instant detection. The alternative solution based on optical imaging, especially fluorescence analysis technology, also faces multiple bottlenecks: first, the emission spectra of traditional lipid probes (such as Nile red and conventional BODIPY series dyes) after binding with different lipids are highly overlapped, which makes the classification strategy based on fluorescence emission wavelength invalid; second, the fluorescence intensity is significantly dependent on the concentration, which is easily disturbed by the local enrichment concentration gradient and photobleaching effect, and does not have the ability of quantification; third, the complex matrix of serum (such as albumin, apolipoprotein and metabolites) causes non-specific adsorption of the probe, resulting in a sharp increase in background noise and a decrease in signal-to-noise ratio; it is particularly important to note that in the related technology, the dye used in fluorescence lifetime imaging (FLIM) application is not sensitive enough to the lipid microenvironment, so that the lifetime difference after binding with different lipids is weak and the distribution is highly overlapped (for example, the characteristic lifetime values of cholesterol ester (CE) and triglyceride (TG) are close), which makes it difficult to establish an effective classification threshold system.

[0018] The first aspect of the embodiment of the present application provides a fluorescent dye, which has the following chemical structural formula: wherein R1 and R2 are independently selected from H, Cl, Br and I; R3 is selected from alkyl, , and wherein n is a positive integer.

[0019] In the embodiment of the present application, the advantages of the fluorescent dye are as follows: Strong microenvironment responsiveness: a phenothiazine-BODIPY fluorescent molecular system is constructed, so that the microenvironment responsiveness of the fluorescent dye can be strengthened. The intramolecular electronic structure of phenothiazine can be used to adjust the intramolecular electronic push-pull ability, significantly enhance the sensitivity of fluorescence lifetime to local polarity and viscosity, and make the lifetime value τ become a specific reporter factor of the lipid microenvironment.

[0020] Serum compatibility regulation: different linear chains (alkyl chains and PEG) and ionized groups (sulfonic acid groups and phosphate groups) are modified on the periphery, so that the non-specific adsorption of serum proteins can be inhibited by the steric hindrance effect and electrostatic repulsion, and the anti-serum interference ability is strong; compared with traditional dyes, the signal-to-noise ratio is at least improved by 3 times.

[0021] Target structure directional optimization: the phenothiazine derivative is used to construct a large planar rigid hydrophobic skeleton, and the dense alkyl chain region of cholesteryl ester is embedded by Van der Waals force, so that the lifetime value tau can be greater than 3.5 ns, and the long lifetime characteristic can be realized; and the BODIPY derivative containing C12-C18 alkyl chain can be designed in the fluorescent dye, and the flexible structure of the C12-C18 alkyl chain can be adapted to the liquid core of the triglyceride, so that the lifetime value tau can be between 2.5-3 ns, so that the medium lifetime response is generated; furthermore, the fluorescent dye can also construct an amphiphilic structure (such as a phosphatidylcholine mimic), wherein the hydrophilic head group (phosphocholine) can anchor the surface polarity region of the lipid bilayer, and induce a short lifetime state (tau < 2.0 ns); in this way, the problem of insufficient lipid FLIM resolution in serum can be solved, and the lifetime difference is significant.

[0022] Excitation spectrum synergy: by adjusting the power supply group and the branch type, the fluorescence lifetime response range and biocompatibility of the fluorescent dye are extended, so that when the fluorescent dye is used as a probe, the maximum absorption peaks of all the fluorescent probes converge in the range of 530-560 nm, which is suitable for the standard laser light source of 488 nm, and the fluorescence lifetime of the fluorescent probe is adjusted in the range of 1.7-5 ns.

[0023] Therefore, the embodiment of the present application solves the problems of low binding selectivity and overlapping lifetime fingerprint of traditional dyes in a complex lipid system, and realizes high-resolution identification of cholesterol ester (CE), triglyceride (TG), phospholipid (PL) and other lipid categories in serum environment. At the same time, the lipid can be classified and detected in situ without extraction, and when the fluorescent dye is used as a probe, the synergistic effect of the fluorescent probe and the fluorescence lifetime imaging (FLIM) technology can be used to break through the dependence of chromatography and mass spectrometry technology on complicated pretreatment, realize spatial distribution analysis and relative quantification of serum lipid components. In addition, a standardized classification criterion can be established, the classification failure problem caused by overlapping lifetime distribution in the related technology can be solved by presetting the lifetime threshold, and a quantifiable lipid subtype analysis tool is provided for clinical use.

[0024] The second aspect of the embodiment of the present application provides a preparation method of a fluorescent dye, comprising: Step 1, phenothiazine and a first substance are added to an organic solvent for stirring; a first intermediate is obtained by column chromatography separation; the first substance is selected from any one of halogenated alkyl, 2-(2-(2-methoxyethoxy)ethoxy)ethyl p-toluenesulfonate, 1,3-propane sulfolane, bromo-PEG2000 and a second substance; the second substance includes 3-bromo-1-propanol and phosphorus oxychloride; Step 2, under inert atmosphere, cool phosphorus oxychloride to -5~5℃ by ice bath, add DMF (N,N-dimethylformamide) and stir for 30 min, then add the DMF (N,N-dimethylformamide) solution of the first intermediate, warm to 80~100℃, and stir until the first intermediate reaction is complete; add ice water to the mixture and adjust the pH to 7~8; extract, wash and concentrate; column chromatography to separate the second intermediate; Step 3, under inert atmosphere, dissolve the second intermediate in dichloromethane, drop trifluoroacetic acid, add 2,4-dimethylpyrrole in dichloromethane solution under light shielding condition, stir the solution; add 2,3-dichloro-5,6-dicyanoquinone and stir; under ice bath, add triethylamine and boron trifluoride ether solution and stir; after concentration, column chromatography to separate the fluorescent dye.

[0025] The fluorescent dye can be obtained by the above preparation method, which can solve the problems of low binding selectivity and overlapping of lifetime fingerprints of traditional dyes in complex lipid systems, and realize high-resolution recognition of lipid categories such as cholesteryl ester (CE), triglyceride (TG), phospholipid (PL) and the like in serum environment. At the same time, the lipid can be classified and detected in situ without extraction, and when the fluorescent dye is used as a probe, based on the synergistic effect of fluorescent probe and fluorescence lifetime imaging (FLIM) technology, the dependence on tedious pretreatment of chromatography and mass spectrometry technology can be broken through, and the spatial distribution analysis and relative quantification of serum lipids can be realized. In addition, a standardized classification criterion can be established, and the classification failure problem caused by overlapping of lifetime distribution in related technologies can be solved by presetting the lifetime threshold, thereby providing a quantifiable lipid subtype analysis tool for clinical use.

[0026] Further, in some embodiments, the first substance in step 1 can be selected from any one of halogenated alkyl, 2-(2-(2-methoxyethoxy)ethoxy)ethyl p-toluenesulfonate, 1,3-propane sulfolane and brominated PEG2000, at this time, the step 1 specifically comprises: placing phenothiazine, the first substance and KOH solution in dimethyl sulfoxide, warming to 55~65℃, and stirring until the phenothiazine is completely consumed; add ice water, extract, wash and concentrate; purify by column chromatography to obtain the first intermediate.

[0027] Specifically, step 1 is a step of preparing the first intermediate. The phenothiazine, the first substance and dimethyl sulfoxide can be mixed in an alkaline environment, and the temperature is raised at the same time, so that the whole reaction is carried out at 55-65℃, and the specific reaction temperature can be 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65℃, etc. In the actual preparation process, the reaction can be monitored by TLC until the phenothiazine is completely consumed, that is, the reaction is completed. In addition, after the reaction is completed, it can be cooled to room temperature (10-30℃) first, and then the reaction mixture is poured into ice water, extracted with dichloromethane, washed with water and concentrated. In addition, the crude product obtained after concentration is purified by silica gel column chromatography to obtain the first intermediate.

[0028] It can be understood that the first intermediate prepared by the above steps is a fluorescent dye, and the corresponding fluorescent dye can be: 、 、 、 、 、 Or .

[0029] Further, in some embodiments, when the first substance of step 1 is 3-bromo-1-propanol and phosphorus oxychloride, step 1 comprises: placing phenothiazine, 3-bromo-1-propanol and KOH solution in dimethyl sulfoxide, heating to 55-65℃, stirring until the phenothiazine is completely consumed; add ice water, extract, wash and concentrate; purify the third intermediate by column chromatography; dissolve the third intermediate and pyridine in chloroform, and cool to -20 to -10℃; slowly add a chloroform solution of phosphorus oxychloride, stir and heat to 10-30℃; distill under reduced pressure, and clean to obtain the fourth intermediate; add the fourth intermediate to deionized water, stir the liquid; extract, wash and dry; distill under reduced pressure to obtain the first intermediate.

[0030] Specifically, step 1 is a step of preparing the first intermediate, which includes three processes.

[0031] The first process: the phenothiazine, the first substance and dimethyl sulfoxide can be mixed in an alkaline environment, and the temperature is raised at the same time, so that the whole reaction is carried out at 55-65℃, and the specific reaction temperature can be 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65℃. In the actual preparation process, the reaction can be monitored by TLC until the phenothiazine is completely consumed, that is, the reaction is completed. In addition, after the reaction is completed, it can be cooled to room temperature (10-30℃) first, and then the reaction mixture is poured into ice water, extracted with dichloromethane, washed with water and concentrated. In addition, the crude product obtained after concentration is purified by silica gel column chromatography to obtain the third intermediate.

[0032] The second process: the third intermediate and phosphorus oxychloride are further reacted, the third intermediate and pyridine can be first dissolved in chloroform and cooled to -20~ -10℃, which can be -20, -19, -18, -17, -16, -15, -14, -13, -12, -11 or -10℃, etc.; then slowly add the chloroform solution of phosphorus oxychloride, keep stirring at the cooling temperature for a period of time; finally, slowly warm the whole system to room temperature, continue to stir; remove chloroform by reduced pressure distillation, and wash the crude product with toluene to obtain the fourth intermediate.

[0033] The third process: the fourth intermediate is added to deionized water, and the solution is stirred at room temperature; the obtained crude product is extracted with ethyl acetate; after washing with water, it is dried with anhydrous sodium sulfate; then remove ethyl acetate by reduced pressure distillation to obtain the first intermediate.

[0034] It can be understood that the first intermediate obtained by the above steps is used to prepare a fluorescent dye, and the corresponding fluorescent dye can be: .

[0035] Further, in some embodiments, before the phenothiazine, the first substance and the KOH solution are placed in dimethyl sulfoxide in step 1, the dimethyl sulfoxide can be dried by a desiccant or activated molecular sieve, so that the water and impurities in the dimethyl sulfoxide can be removed, so that the dimethyl sulfoxide can be kept dry and pure, and the subsequent steps will not be disturbed by water and impurities, which is beneficial to the preparation of fluorescent dyes.

[0036] Further, in some embodiments, in step 2, under an inert atmosphere, the phosphorus oxychloride is cooled to -5~5℃ by ice bath, DMF (N, N-dimethylformamide) is added and stirred for 30 min, the reaction system is discolored, then the DMF solution of the first intermediate is added, and the temperature is raised to 80~100℃ and stirred until the first intermediate is completely reacted; add ice water to the mixture and adjust the pH to 7~8; extract, wash and concentrate; column chromatography is used to separate the second intermediate.

[0037] Specifically, DMF and phosphorus oxychloride are reacted at low temperature to generate Vilsmeier reagent, which is then subjected to electrophilic substitution with the first intermediate, followed by hydrolysis to generate the formylated product. Additionally, by way of example, the inert gas can be argon, helium, or the like. Understandably, in some embodiments, the reaction of Step 2 can also be carried out under nitrogen atmosphere. Step 2 can be carried out under ice bath condition with phosphorus oxychloride cooling, which can be at -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, or 5°C, or the like. DMF is added dropwise at low temperature until color change; then the temperature is slowly increased and the DMF solution of the first intermediate is slowly added dropwise, which can be at 80, 80, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 100°C, or the like, and the reaction is carried out at the increased temperature, which can be monitored by thin layer chromatography (TLC) until the first intermediate is completely reacted. After the reaction is completed, the mixture is poured into ice water as a whole while hot, which can be adjusted to pH value using saturated sodium carbonate solution; then extracted with dichloromethane, washed with water, and concentrated. The crude product after concentration is purified by silica gel column chromatography to obtain the second intermediate.

[0038] It should be understood that the TLC monitoring of the reaction is a routine technical means in the art, which will not be described here.

[0039] Further, in some embodiments, the operation step of adding triethylamine and boron trifluoride ether solution under ice bath in Step 3 includes: slowly adding triethylamine under ice bath, stirring for 15-45 min, and continuously adding boron trifluoride ether solution, stirring for 8-12 h.

[0040] Specifically, triethylamine and boron trifluoride ether solution can be added in Step 3 in sequence, so that the triethylamine added first can neutralize the acid in the reaction system and form an alkaline environment in the reaction system, promoting the coordination of the substrate and boron trifluoride. At this time, Step 3 can be specifically as follows: under inert atmosphere, the second intermediate is dissolved in dichloromethane, trifluoroacetic acid is added, 2,4-dimethylpyrrole in dichloromethane is slowly added dropwise under light shielding condition, and the liquid is stirred; 2,3-dichloro-5,6-dicyanoquinone (DDQ) is added and stirred; triethylamine is slowly added dropwise under ice bath, stirring for 15-45 min, boron trifluoride ether (BF3·Et2O) solution is continuously added dropwise, stirring for 8-12 h; after concentration, column chromatography is used for separation to obtain the fluorescent dye. By way of example, the inert gas can be argon, helium, nitrogen, or the like.

[0041] In addition, in step 3, before the second intermediate is dissolved in dichloromethane under inert atmosphere, the dichloromethane is dried by re-evaporation. In this way, the water and impurities in the dichloromethane can be removed, so that the dichloromethane can be kept dry and pure, and the subsequent steps will not be disturbed by water and impurities, which is beneficial to the preparation of the fluorescent dye.

[0042] Further, for the preparation method of the fluorescent dye, the following is further illustrated by specific examples, and it should be understood that the specific examples described herein are only used to explain the present application, but not to limit the present application.

[0043] Example 1 Step i, 1 mmol of phenothiazine (A0), 1.5 mmol of iodomethane and 3 mmol of KOH were stirred in 50 ml of dry dimethyl sulfoxide, and the reaction was monitored by TLC until the phenothiazine was completely consumed. After cooling to room temperature, it was poured into ice water, extracted with dichloromethane, washed with water and concentrated. The crude product was purified by silica gel column chromatography (a mixed system of dichloromethane and petroleum ether mixed at a volume ratio of 1:2) to obtain the first intermediate (A1).

[0044] Step ii, under argon, 10 mmol of phosphorus oxychloride was cooled to 0°C in an ice bath, 50 mmol of DMF was slowly added, and the reaction system was stirred for 30 minutes until it turned light yellow. Then 1 mmol of the first intermediate was added in DMF solution, and the temperature was slowly raised to 90°C. The reaction was monitored by TLC until the first intermediate was completely consumed. The mixture was quickly poured into ice water, the pH was adjusted to 7.5 with saturated sodium carbonate solution, extracted with dichloromethane, washed with water and concentrated. The crude product was purified by silica gel column chromatography (a mixed system of dichloromethane and petroleum ether mixed at a volume ratio of 1:2) to obtain the second intermediate (A2).

[0045] Step iii, under argon, 1 mmol of the second intermediate was dissolved in dry dichloromethane, 3 drops of trifluoroacetic acid (TFA) were added, 2.1 mmol of 2,4-dimethylpyrrole in dichloromethane was slowly added under light protection, and the solution was stirred at room temperature. 1 mmol of 2,3-dichloro-5,6-dicyanoquinone was added and stirred for 4 hours. 5 mL of triethylamine (TEA) was slowly added under ice bath, stirred for 30 minutes, and then 5 mL of boron trifluoride etherate (BF3·Et2O) solution was added, and stirred for 10 hours. The reaction mixture was concentrated and purified by silica gel column chromatography (a mixed system of dichloromethane and petroleum ether mixed at a volume ratio of 1:2) to obtain the fluorescent dye (PTZ-BDP-C1).

[0046] In which, the chemical reaction formula involved in Example 1 is as follows:

[0047] Example 2 Step i, Take 1 mmol phenothiazine (A0), 1.5 mmol bromo dodecane and 3 mmol KOH in 50 mL dry dimethyl sulfoxide, stir, heat to 60°C, TLC monitor the reaction until the phenothiazine is completely consumed, the reaction is complete. Cool to room temperature, pour into ice water, dichloromethane extraction, then washed with water and concentrated. The crude product is purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether mixed according to the volume ratio of 1:2) to obtain the first intermediate (B1).

[0048] Step ii, under argon, ice bath, 10 mmol phosphorus oxychloride is cooled to 0°C, slowly drop 50 mmol DMF, stir for 30 minutes until the reaction system turns light yellow, slowly drop 1 mmol first intermediate DMF solution, and slowly heat to 90°C. Stir until the first intermediate is completely reacted by TLC monitoring. Pour the mixture into ice water quickly, adjust the pH to 7 with saturated sodium carbonate solution, extract with dichloromethane, then wash with water and concentrate. The crude product is purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether mixed according to the volume ratio of 1:2) to obtain the second intermediate (B2).

[0049] Step iii, under argon, 1 mmol of the second intermediate is dissolved in dry dichloromethane, 3 drops of trifluoroacetic acid (TFA) are added, 2.1 mmol of 2, 4-dimethylpyrrole in dichloromethane is slowly added in the dark, and the solution is stirred at room temperature. Add 1 mmol 2,3-dichloro-5,6-dicyanoquinone and continue to stir for 4 hours, slowly add 5 mL triethylamine (TEA) under ice bath, stir for 30 minutes, continue to add 5 mL boron trifluoride etherate (BF3·Et2O) solution, stir for 10 hours. The reaction mixture is concentrated and purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether mixed according to the volume ratio of 1:2) to obtain the fluorescent dye (PTZ-BDP-C12).

[0050] Wherein, the chemical reaction formula involved in example 2 is as follows:

[0051] Example 3 Step i, Take 1 mmol phenothiazine (A0), 1.5 mmol bromo dodecane and 3 mmol KOH in 50 mL dry dimethyl sulfoxide, stir, heat to 60°C, TLC monitor the reaction until the phenothiazine is completely consumed, the reaction is complete. Cool to room temperature, pour into ice water, dichloromethane extraction, then washed with water and concentrated. The crude product is purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether mixed according to the volume ratio of 1:2) to obtain the first intermediate (B1).

[0052] Step ii, 10 mmol of phosphorus oxychloride was cooled to 0 °C under argon, 50 mmol of DMF was added slowly dropwise, and the mixture was stirred for 30 min until the reaction system turned light yellow. 1 mmol of the first intermediate was added slowly dropwise in DMF, and the temperature was slowly raised to 90 °C. The reaction was stirred until the first intermediate was completely consumed, as monitored by TLC. The mixture was quickly poured into ice water, and the pH was adjusted to 7.5 with a saturated sodium carbonate solution. After extraction with dichloromethane, the organic phase was washed with water and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the second intermediate (C2).

[0053] Step iii, 1 mmol of the second intermediate was dissolved in dry dichloromethane, 3 drops of trifluoroacetic acid (TFA) were added, and 2.1 mmol of 2,4-dimethylpyrrole in dichloromethane was slowly added dropwise in the dark. The solution was stirred at room temperature. 1 mmol of 2,3-dichloro-5,6-dicyanoquinone was added, and the stirring was continued for 4 h. 5 mL of triethylamine (TEA) was slowly added dropwise under ice bath, and the stirring was continued for 30 min. 5 mL of boron trifluoride etherate (BF3·Et2O) solution was added dropwise, and the stirring was continued for 10 h. The reaction mixture was concentrated and purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the fluorescent dye (PTZ-BDP-C18).

[0054] The chemical reaction formula involved in Example 3 is as follows:

[0055] Example 4 Step i, 1 mmol of phenothiazine (A0), 1.5 mmol of 2-(2-(2-methoxyethoxy)ethoxy)ethyl p-toluenesulfonate, and 3 mmol of KOH were stirred in 50 mL of dry dimethyl sulfoxide, and the temperature was raised to 60 °C. The reaction was monitored by TLC until the phenothiazine was completely consumed. The solution was cooled to room temperature, poured into ice water, extracted with dichloromethane, washed with water, and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the first intermediate (D1).

[0056] Step ii, 10 mmol of phosphorus oxychloride was cooled to 0 °C under argon, 50 mmol of DMF was added dropwise, and the mixture was stirred for 30 min until the reaction system turned light yellow. 1 mmol of the first intermediate in DMF was added dropwise, and the temperature was slowly raised to 90 °C. The reaction was stirred until the first intermediate was completely reacted, which was monitored by TLC. The mixture was quickly poured into ice water, the pH was adjusted to 7.5 with a saturated sodium carbonate solution, and dichloromethane was used for extraction, washed with water and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the second intermediate (D2).

[0057] Step iii, 1 mmol of the second intermediate was dissolved in dry dichloromethane, 3 drops of trifluoroacetic acid (TFA) were added, 2.1 mmol of 2,4-dimethylpyrrole in dichloromethane was slowly added dropwise in the dark, and the solution was stirred at room temperature. 1 mmol of 2,3-dichloro-5,6-dicyanoquinone was added and the stirring was continued for 4 hours. 5 mL of triethylamine (TEA) was slowly added dropwise under ice bath, and the stirring was continued for 30 min. 5 mL of boron trifluoride etherate (BF3·Et2O) solution was added dropwise, and the stirring was continued for 10 hours. The reaction mixture was concentrated and purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the fluorescent dye (PTZ-BDP-Ether).

[0058] The chemical reaction formula involved in Example 4 is as follows:

[0059] Example 5 Step i, 1 mmol of phenothiazine (A0), 1.5 mmol of 1,3-propane sultone and 3 mmol of KOH were stirred in 50 mL of dry dimethyl sulfoxide, and the temperature was raised to 60 °C. The reaction was monitored by TLC until the phenothiazine was completely consumed. The temperature was cooled to room temperature, and the mixture was poured into ice water. Dichloromethane was used for extraction, washed with water and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the first intermediate (E1).

[0060] Step ii, 10 mmol of phosphorus oxychloride was cooled to 0 °C under argon, 50 mmol of DMF was added slowly dropwise, and the mixture was stirred for 30 min until the reaction system turned light yellow. 1 mmol of the first intermediate was added slowly dropwise, and the temperature was slowly increased to 90 °C. The reaction was stirred until the first intermediate was completely consumed, as monitored by TLC. The mixture was quickly poured into ice water, and the pH was adjusted to 7.5 with a saturated sodium carbonate solution. After extraction with dichloromethane, the organic phase was washed with water and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the second intermediate (E2).

[0061] Step iii, 1 mmol of the second intermediate was dissolved in dry dichloromethane, 3 drops of trifluoroacetic acid (TFA) were added, and 2.1 mmol of 2,4-dimethylpyrrole in dichloromethane was slowly added dropwise in the dark. The solution was stirred at room temperature. 1 mmol of 2,3-dichloro-5,6-dicyano- benzoquinone was added, and the stirring was continued for 4 h. 5 mL of triethylamine (TEA) was slowly added dropwise under ice bath, and the stirring was continued for 30 min. 5 mL of boron trifluoride etherate (BF3·Et2O) solution was slowly added dropwise, and the stirring was continued for 10 h. The reaction mixture was concentrated and purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the fluorescent dye (PTZ-BDP-SO3).

[0062] In Example 5, the chemical reaction formula is as follows:

[0063] Example 6 Step i, 1 mmol of phenothiazine (A0), 3 mmol of KOH, and 1.5 mmol of 1,3-propane sultone (3-Bromo-1-propanol) were stirred in 50 mL of dry dimethyl sulfoxide, and the temperature was increased to 60 °C. The reaction was monitored by TLC until the phenothiazine was completely consumed. The solution was cooled to room temperature, poured into ice water, extracted with dichloromethane, washed with water, and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the third intermediate (F1).

[0064] Step ii, 1 mmol of the first intermediate and 1 mL of pyridine were dissolved in 50 mL of dry chloroform and cooled to -15 °C. A mixture of 4.5 mL of phosphorus oxychloride and 40 mL of chloroform was slowly added dropwise within 30 min. The stirring was continued at -15 °C for 2 h, and the reaction system was slowly warmed to room temperature. The stirring was continued for 1.5 h. The chloroform was removed by distillation under reduced pressure, and the crude product was washed with toluene to obtain the fourth intermediate as a green oil (F2).

[0065] Step iii, the remaining crude product of step ii was added into 50 mL deionized water, the solution was stirred at room temperature, the crude product was extracted with ethyl acetate, washed with water, dried with anhydrous sodium sulfate, and the ethyl acetate was removed by distillation under reduced pressure to obtain the first intermediate (F3) as a white solid.

[0066] Step iv, 10 mmol of phosphorus oxychloride was cooled to 0°C in an ice bath under argon, 50 mmol of DMF was added slowly dropwise, and the mixture was stirred for 30 minutes until the reaction system turned light yellow. 1 mmol of the fourth intermediate was added in DMF dropwise, and the temperature was slowly raised to 90°C. The reaction was stirred until the first intermediate was completely reacted, which was monitored by TLC. The mixture was quickly poured into ice water, the pH was adjusted to 8 with saturated sodium carbonate solution, extracted with dichloromethane, washed with water, and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the second intermediate (F4).

[0067] Step v, 1 mmol of the second intermediate was dissolved in dry dichloromethane under argon, 3 drops of trifluoroacetic acid (TFA) were added dropwise, 2.1 mmol of 2,4-dimethylpyrrole in dichloromethane was slowly added dropwise in the dark, and the solution was stirred at room temperature. 1 mmol of 2,3-dichloro-5,6-dicyanoquinone was added and the stirring was continued for 4 hours. 5 mL of triethylamine (TEA) was slowly added dropwise in an ice bath, and the stirring was continued for 30 minutes. 5 mL of boron trifluoride etherate (BF3·Et2O) solution was added dropwise, and the stirring was continued for 10 hours. The reaction mixture was concentrated and purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the fluorescent dye (PTZ-BDP-PO3).

[0068] The chemical reaction formula involved in Example 6 is as follows:

[0069] Example 7 Step i, 1 mmol of phenothiazine (A0), 1.5 mmol of bromo-PEG2000 (PEG 2000 Br), and 3 mmol of KOH were stirred in 50 mL of dry dimethyl sulfoxide, and the temperature was raised to 60°C. The reaction was monitored by TLC until the phenothiazine was completely consumed. The solution was cooled to room temperature, poured into ice water, extracted with dichloromethane, washed with water, and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether in a volume ratio of 1:2) to obtain the first intermediate (G1).

[0070] Step ii, under argon, 10 mmol of phosphorus oxychloride was cooled to 0 °C in an ice bath, 50 mmol of DMF was added slowly dropwise, and the reaction was stirred for 30 minutes until the reaction mixture turned light yellow. 1 mmol of the first intermediate in DMF was added slowly dropwise, and the temperature was slowly increased to 90 °C. The reaction was stirred until the first intermediate was completely reacted, as monitored by TLC. The mixture was quickly poured into ice water, the pH was adjusted to 7.5 using a saturated sodium carbonate solution, and the mixture was extracted with dichloromethane, washed with water, and concentrated. The crude product was purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether at a volume ratio of 1:2) to obtain the second intermediate (G2).

[0071] Step iii, under argon, 1 mmol of the second intermediate was dissolved in dry dichloromethane, 3 drops of trifluoroacetic acid (TFA) were added, 2.1 mmol of 2,4-dimethylpyrrole in dichloromethane was slowly added dropwise in the dark, and the solution was stirred at room temperature. 1 mmol of 2,3-dichloro-5,6-dicyano-benzoquinone was added, and the stirring was continued for 4 hours. 5 mL of triethylamine (TEA) was slowly added dropwise in an ice bath, and the stirring was continued for 30 minutes. 5 mL of a boron trifluoride etherate (BF3·Et2O) solution was slowly added dropwise, and the stirring was continued for 10 hours. The reaction mixture was concentrated and purified by silica gel column chromatography (a mixture of dichloromethane and petroleum ether at a volume ratio of 1:2) to obtain the fluorescent dye (PTZ-BDP-PEG).

[0072] wherein Example 7 involves the following chemical reaction formula:

[0073] Example 8 The prepared fluorescent dyes PTZ-BDP-C1, PTZ-BDP-C12, PTZ-BDP-C18 were dissolved in DMSO to obtain 1 mM stock solution. The stock solution was further diluted with n-hexane, tetrahydrofuran, DMSO, methanol respectively to obtain 1 μM test solution. The absorption, emission spectrum and fluorescence lifetime of these solutions were tested. The absorption spectrum was measured by ultraviolet-visible spectrophotometer, and the fluorescence spectrum was measured by fluorescence spectrometer. Exemplarily, the ultraviolet-visible spectrophotometer can be Agilent Cary 4000 ultraviolet-visible spectrophotometer, and the fluorescence spectrum can be HORIBA FluoroMax+ fluorescence spectrometer. As shown in Table 1, the maximum absorption wavelength (λabs), the maximum emission wavelength (λem) and the fluorescence lifetime (τ) of the fluorescent dyes PTZ-BDP-C1, PTZ-BDP-C12, PTZ-BDP-C18 in different solvents are specifically listed in Table 1. From the data in Table 1, it can be seen that the maximum emission wavelength of the fluorescent dyes PTZ-BDP-C1, PTZ-BDP-C12, PTZ-BDP-C18 is between 500-600 nm, and the fluorescence lifetime has a response to different polar solvents and the fluorescence lifetime is positively correlated with the polarity of the solvent.

[0074] Table 1. Spectral properties of PTZ-BDP-C1, PTZ-BDP-C12, PTZ-BDP-C18

[0075] Example 9 The fluorescent dyes PTZ-BDP-C1, PTZ-BDP-C12, PTZ-BDP-C18 were dissolved in DMSO to obtain 1 mM stock solution. The stock solution was diluted according to different volume ratios of tetrahydrofuran-methanol mixed system (total volume 3 mL, concentration 10 μM, volume percentage of tetrahydrofuran in the mixed system was 0%, 20%, 40%, 60%, 80%, 100% respectively) to obtain test solution. The fluorescence spectrum and fluorescence lifetime decay spectrum of these test solutions were obtained by fluorescence spectrometer. Exemplarily, the fluorescence spectrometer can be HORIBA FluoroMax+ fluorescence spectrometer. As shown in Table 2 and Table 3, the fluorescence intensity and fluorescence lifetime change with the increase of the volume percentage of tetrahydrofuran. Figure 1 and Figure 2 As shown in Table 2 and Table 3, the fluorescence intensity and fluorescence lifetime change with the increase of the volume percentage of tetrahydrofuran. By utilizing the excellent power supply property of phenothiazine, the intramolecular charge transfer (ICT) of the fluorescent dye is effectively improved, and the response ability of the fluorescence lifetime of the fluorescent dye molecule to the microenvironment is effectively improved. Compared with the 2-3 ns of the conventional BODIPY dye, the fluorescence lifetime of the fluorescent dye of the present application is increased to 1-5 ns.

[0076] Example 10 The fluorescent dye PTZ-BDP-C1 was dissolved in DMSO to obtain a stock solution of 1 mM, and the stock solution was mixed with pure lipids cholesterol, triglyceride and phospholipid respectively according to the operation steps shown in Figure 3 , at a ratio of 1:50, incubated for 2 minutes, spotted on glass slides respectively, and mounted with neutral mounting medium, then imaged by a fluorescence lifetime imaging system, and the data were statistically analyzed according to 5 samples per group and 5 fields of view per sample, and the specific results are recorded in Table 2 and Figure 4 . At the same time, according to the steps shown in Figure 5 , the blood sample was centrifuged to obtain serum, and the serum sample was statistically analyzed according to the above processing steps of pure lipids, including sample preparation, imaging, analysis, etc. For the lifetime analysis of the serum sample, phasor plot analysis and classification were required. The specific results are recorded in Table 2. In addition, Nile Red was used as a control group, and the steps shown in Figure 3 and the steps shown in Figure 5 were followed to mix Nile Red and pure lipids to obtain corresponding data results, and to mix Nile Red and serum samples to obtain corresponding data results. The specific results are recorded in Table 2.

[0077] According to the results shown in Table 2 and Figure 4 , it can be seen that the fluorescent dye of the present application can solve the problems of low binding selectivity and overlapping lifetime fingerprint of traditional dyes in complex lipid systems, and achieve high-resolution identification of cholesterol ester (CE), triglyceride (TG), phospholipid (PL) and other lipid categories in serum environment; at the same time, it has very high signal-to-noise ratio and high reliability.

[0078] Table 2 Performance verification of dye

[0079] Example 11 According to the sample preparation process shown in Figure 5 , the serum of 10 normal people (Normal, N) and 10 patients with hyperlipidemia (Patient, P) was prepared, and PTZ-BDP-C1, PTZ-BDP-C12 and PTZ-BDP-C18 dyes were used for staining, and the FLIM imaging system was used to collect data and statistically analyze, as shown in Figure 6 , the FLIM image shows that the patient serum contains large size lipid aggregates (>5 μm); and the phasor plot analysis of the FLIM image shows that the CE enrichment area of the patient group increases by 35% (p<0.01), and the TG enrichment area increases by 42% (p<0.001), as shown in Figure 6 .

[0080] Furthermore, a third aspect of the embodiments of the present invention provides the use of fluorescent dyes for in situ detection and analysis of lipids in serum.

[0081] In an embodiment of the present invention, a fluorescent dye and a serum sample can be mixed, stained, and made into a mounting slide, which can be imaged using a fluorescence lifetime imaging system, wherein the volume ratio of the fluorescent dye solution to the serum sample is 1:50.

[0082] The specific operation process can be as follows: pre-treat the serum, dilute or centrifuge at low speed to remove particulate matter; add fluorescent dye and incubate for 2 to 45 minutes; apply the mixture evenly on a glass slide, seal it with neutral resin after drying, and obtain a seal; use pulsed laser excitation to collect photon time information to obtain 2D / 3D fluorescence lifetime data, and perform preliminary noise filtering and background subtraction. Due to the strong anti-serum interference ability of fluorescent dyes and high signal-to-noise ratio, lipid extraction is not required, and the test can be quick and simple, with the entire process taking no more than 1 hour; at the same time, it can also accurately distinguish subtypes such as CE / TG / PL with an accuracy rate of over 90%, providing new lipid subtype diagnostic indicators (such as CE / TG ratio) for cardiovascular disease, fatty liver, etc.; and the test has high stability, the fluorescence lifetime is not affected by concentration and / or photobleaching, and the RSD (relative standard deviation) is less than 5%; in addition, if Figure 6 As shown, it can also intuitively present the lipid distribution and lipoprotein structure.

[0083] In addition, it can be understood that in some embodiments, serum samples can be replaced by whole blood samples; in addition, fluorescent dyes can also be extended to lipid analysis of cells and tissues.

[0084] Furthermore, in some embodiments, the concentration and incubation conditions of the fluorescent dye can be optimized to investigate the optimal concentration range for fluorescent dye staining for different types of serum samples (normal, hyperlipidemia, disease-related, etc.). By conducting staining experiments across a range of concentration gradients and combining them with FLIM measurements, the optimal concentration that ensures sufficient signal intensity while avoiding dye aggregation and nonspecific binding can be identified. Furthermore, the effects of various incubation conditions, including temperature, time, and pH, on the binding efficiency of the fluorescent dye to lipids can be explored. By designing an experimental matrix, the optimal combination of incubation parameters can be determined to ensure sufficient binding of the dye to serum lipids within a short period of time and the stable generation of a characteristic fluorescence lifetime signal.

[0085] Furthermore, a fourth aspect of the present invention provides the use of fluorescent dyes for preparing lipid analysis and diagnostic products. Specifically, based on the characteristics of the fluorescent dyes, related products can be made based on the fluorescent dyes, and the related products can be used for lipid detection, analysis and diagnosis, thereby improving the practicality of the fluorescent dyes.

[0086] Further, the fifth aspect of the embodiments of the present application provides the application of the fluorescent dye in the preparation of the fluorescent probe for targeting lipids. Specifically, according to the characteristics of the fluorescent dye, the fluorescent probe for targeting lipids can also be prepared according to the fluorescent dye. The fluorescent probe can be used to monitor the lipids in the serum, play a role in rapid detection of lipids and prevention of diseases, and improve the practicability.

Claims

1. A fluorescent dye, characterized in that, The fluorescent dye has the following chemical structure: wherein R1, R2are each independently selected from H, Cl, Br and I; R3is selected from alkyl, , and wherein n is a positive integer.

2. The fluorescent dye according to claim 1, characterized in that, The alkyl group includes CH3 and C12-18 alkyl.

3. The method for preparing the fluorescent dye according to claim 1 or 2, wherein: Comprise: Step 1, phenothiazine and the first substance are added to an organic solvent for stirring; the first intermediate is obtained by column chromatography separation; the first substance is selected from any one of halogenated alkyl, 2-(2-(2-methoxyethoxy)ethoxy)ethyl p-toluenesulfonate, 1,3-propane sulfolane, brominated PEG2000 and a second substance; the second substance includes 3-bromo-1-propanol and phosphorus oxychloride; Step 2, under an inert atmosphere, the phosphorus oxychloride is cooled to-5~5℃ by ice bath, an equal amount of DMF is added for stirring for 30 minutes, then the DMF solution of the first intermediate is added, and the temperature is increased to 80~100℃, and the stirring is continued until the reaction of the first intermediate is completed; ice water is added to the mixture, and the pH is adjusted to 7~8; extraction, washing and concentration are performed; The second intermediate is obtained by column chromatography separation; Step 3, under an inert atmosphere, the second intermediate is dissolved in dichloromethane, trifluoroacetic acid is added dropwise, a dichloromethane solution of 2,4-dimethylpyrrole is added under light shielding condition, and the stirring solution is obtained; 2,3-dichloro-5,6-dicyanoquinone is added for stirring; under ice bath, triethylamine and boron trifluoride ether solution are added for stirring; after concentration, column chromatography separation is performed to obtain the fluorescent dye.

4. The production method according to claim 3, characterized by, When the first substance of step 1 is selected from any one of halogenated alkyl, 2-(2-(2-methoxyethoxy)ethoxy)ethyl p-toluenesulfonate, 1,3-propane sulfolane and brominated PEG2000, the step 1 comprises: The phenothiazine, the first substance and the KOH solution are placed in dimethyl sulfoxide, the temperature is increased to 55~65℃, and the stirring is continued until the phenothiazine is completely consumed; ice water is added, extraction, washing and concentration are performed; and the first intermediate is obtained by column chromatography purification.

5. The preparation method according to claim 3, characterized in that When the first substance of step 1 is 3-bromo-1-propanol and phosphorus oxychloride, the step 1 comprises: The phenothiazine, 3-bromo-1-propanol and the KOH solution are placed in dimethyl sulfoxide, the temperature is increased to 55~65℃, and the stirring is continued until the phenothiazine is completely consumed; ice water is added, extraction, washing and concentration are performed; and the third intermediate is obtained by column chromatography purification; The third intermediate and pyridine are dissolved in chloroform, and the temperature is cooled to-20~-10℃; a chloroform solution of phosphorus oxychloride is added, the stirring is continued, and the temperature is increased to 10~30℃; vacuum distillation is performed, and cleaning is performed to obtain the fourth intermediate; The fourth intermediate is added to deionized water, and the stirring solution is obtained; extraction, washing and drying are performed; and vacuum distillation is performed to obtain the first intermediate.

6. The production method according to claim 4 or 5, characterized by, Before the operation step of placing the phenothiazine, the first substance and the KOH solution in dimethyl sulfoxide in step 1, the method further comprises: drying the dimethyl sulfoxide by a drying agent or activated molecular sieve.

7. The preparation method according to claim 3, characterized in that Before the operation step of dissolving the second intermediate in dichloromethane under an inert atmosphere in step 3, the method further comprises: drying the dichloromethane by re-distillation.

8. The preparation method according to claim 3, characterized in that The step 2 adopts saturated sodium carbonate to adjust the pH value.

9. The fluorescent dye of claim 1 is used for in-situ detection and analysis of lipids in serum.

10. The fluorescent dye of claim 1 is used for preparing a fluorescent probe targeting lipids.

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