Dual-targeting fluorescent carbon dots for endoplasmic reticulum and lipid droplets as well as preparation method and application of dual-targeting fluorescent carbon dots

Abstract

The invention relates to a dual-targeting fluorescent carbon dot for endoplasmic reticulum and lipid droplets and a preparation method and application thereof.The preparation method comprises the steps that o-phenylenediamine and phenylalanine are dissolved in pure water to obtain a first dissolved solution; transferring the first dissolving solution into a poly-tetrafluoroethylene lined reaction kettle, carrying out a first heating reaction to obtain a precipitate, and sequentially carrying out column chromatography purification, rotary evaporation and drying on the precipitate to obtain a target solid; dissolving caffeic acid in dichloromethane, adding CDI, and stirring to obtain a second dissolved solution; and adding the target solid into the second dissolving solution, and carrying out a second heating reaction to obtain the dual-targeting fluorescent carbon dots for the endoplasmic reticulum and the lipid droplets. The dual-targeting fluorescent carbon dots obtained by the preparation method can distinguish endoplasmic reticulum and lipid droplets, and solve the problem of lack of life-sensitive dual-targeting fluorescent carbon dots capable of simultaneously positioning the endoplasmic reticulum and lipid droplets and sensitively monitoring microenvironment changes of the endoplasmic reticulum and lipid droplets.

Description

Technical Field

[0001] The present application relates to the field of fluorescent materials, and in particular to a dual-targeted fluorescent carbon dot for endoplasmic reticulum and lipid droplets, and a preparation method and application thereof. Background Art

[0002] Fluorescent carbon dots (CDs) have shown great potential in the fields of biosensing, cell imaging and disease diagnosis due to their excellent optical properties, low cytotoxicity, good biocompatibility and easy functional modification. Although the synthesis of carbon dots has developed rapidly since its discovery in 2004, the current carbon dots still lack precise targeting of specific organelles, especially the distinguishable targeting and microenvironmental response of interconnected dual organelles, which greatly limits the application of carbon dots in organelle dynamics in living cells.

[0003] The endoplasmic reticulum is the largest and most variable closed membrane organelle in eukaryotic cells. It is the synthesis base of proteins, lipids, and carbohydrates, and also plays an important role in calcium ion storage and signal transduction. Lipid droplets are important organelles for storing lipids and energy in cells. They are composed of an inner core composed of neutral lipids and a monolayer of phospholipids and specific proteins wrapped around it. Studies have confirmed that the generation of lipid droplets originates from the endoplasmic reticulum, and mature lipid droplets also have frequent and extensive interactions with the endoplasmic reticulum. Moreover, changes in the microenvironment of the endoplasmic reticulum and lipid droplets can affect their physiological functions. For example, excessive reactive oxygen species (ROS) can cause endoplasmic reticulum stress, and changes in lipid droplet viscosity are also closely related to lipid metabolism. Therefore, real-time and synchronous monitoring of the interaction between the two and the dynamic changes of the microenvironment is of great significance for a deep understanding of the physiological and pathological mechanisms of the two. At present, single-target fluorescent carbon dots often need to be combined with multiple commercial dyes (commercial dye combination) to achieve simultaneous visualization of the two, and cannot simultaneously meet the multifunctional requirements of dual targeting and environmental sensitive response, making it difficult to fully reflect complex pathological states, resulting in one-sided detection information.

[0004] Therefore, there is an urgent need to develop multifunctional fluorescent carbon dots that have dual targeting capabilities for the ER and lipid droplets and that can sensitively respond to changes in their microenvironment, so as to achieve real-time imaging and functional regulation of the association between ER oxidative stress and lipid droplet viscosity changes, and provide powerful tools for early diagnosis, targeted treatment, and drug delivery of related diseases.

[0005] There is currently a lack of lifespan-sensitive dual-targeted fluorescent carbon dots that can simultaneously localize to the endoplasmic reticulum and lipid droplets and sensitively monitor changes in their microenvironment. No effective solution has been proposed so far. Summary of the invention

[0006] The present invention provides a dual-targeted fluorescent carbon dot for endoplasmic reticulum and lipid droplets, and a preparation method and application thereof, to solve the current problem of lack of dual-targeted fluorescent carbon dots that are sensitive to microenvironmental changes of the endoplasmic reticulum and lipid droplets.

[0007] In a first aspect, the present invention provides a method for preparing dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets, comprising: Dissolving o-phenylenediamine and phenylalanine in pure water to obtain a first solution, transferring the first solution to a polytetrafluoroethylene-lined reactor for a first heating reaction to obtain a precipitate, and sequentially subjecting the precipitate to column chromatography purification, rotary evaporation, and drying to obtain a target solid; Dissolving caffeic acid in dichloromethane and adding CDI and stirring to obtain a second solution; The target solid is added into the second dissolving solution to carry out a second heating reaction to obtain the dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets.

[0008] In a second aspect, the present invention provides dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets, which are obtained by the preparation method of dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets described in the first aspect.

[0009] In a third aspect, the present invention provides the use of the dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets described in the second aspect in cell monitoring.

[0010] Compared with the related art, the present invention introduces phenylalanine into the reaction system to adjust the photoluminescence properties of CDs. On the one hand, it has a benzene ring itself; on the other hand, the amino group and the carboxyl group are easily cross-linked through an amide bond, both of which promote the formation of carbonaceous graphite cores, causing the red shift of photoluminescence emission. In addition, caffeic acid can recognize superoxide anions, and can be connected to the carbon dots through the condensation reaction of the amino group and the carboxyl group. When superoxide anions are present, the caffeic acid residues on the carbon dots can be converted from the electron donor catechol to the electron acceptor benzoquinone. The dual-targeted fluorescent carbon dots obtained by this preparation method are sensitive to the cell microenvironment and can effectively distinguish the endoplasmic reticulum and lipid droplets, solving the current problem of lack of life-sensitive dual-targeted fluorescent carbon dots that can simultaneously locate the endoplasmic reticulum and lipid droplets and sensitively monitor their microenvironmental changes.

[0011] Details of one or more embodiments of the present application are set forth in the following drawings and description to make other features, objects, and advantages of the present application more readily apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a schematic diagram of the synthesis of fluorescent carbon dots in the embodiment; Figure 2 is a schematic diagram of the FT-IR results of the fluorescent carbon dots in the embodiment; Figure 3 is a schematic diagram of the UV-visible spectrum of the fluorescent carbon dots in the embodiment; Figure 4 Schematic diagram of fluorescence emission spectra of fluorescent carbon dots under different excitation lights in the embodiment; Figure 5 is a schematic diagram of the change in fluorescence intensity of the fluorescent carbon dots under different UV irradiation times in the embodiment; Figure 6 is a schematic diagram of a TEM image of fluorescent carbon dots in the embodiment; Figure 7 is a schematic diagram of the size distribution results of the fluorescent carbon dots in the embodiment; Figure 8 Schematic diagram of XPS spectrum analysis of fluorescent carbon dots in the embodiment; wherein: (a) is the total XPS spectrum of fluorescent carbon dots, (b) is the XPS spectrum of C element in fluorescent carbon dots, (c) is the XPS spectrum of N element in fluorescent carbon dots, and (d) is the XPS spectrum of O element in fluorescent carbon dots; Fig. 9 Schematic diagram of Log P analysis of fluorescent carbon dots in the embodiment; Fig.10 is a schematic diagram of the selective analysis of various analytical substrates contained in cells by fluorescent carbon dots in the embodiment; Fig.11 is a schematic diagram of the fluorescence emission spectrum of the fluorescent carbon dots in vitro in response to superoxide anions in the embodiment; Fig.12 is a schematic diagram of the fluorescence emission spectrum of the fluorescent carbon dots in vitro in response to viscosity in the embodiment; Fig.13 Schematic diagram of the selective analysis of pH by fluorescent carbon dots in the embodiment; Fig.14 is a schematic diagram of fluorescent carbon dot cytotoxicity analysis in the embodiment; Fig.15 Schematic diagram of real-time imaging of fluorescent carbon dots entering cells in the embodiment; Fig.16 is a schematic diagram of co-localization imaging analysis of fluorescent carbon dots in cells in the embodiment; Fig.17 Schematic diagram of fluorescence lifetime imaging of fluorescent carbon dots in the endoplasmic reticulum of cells under oxidative stress in the embodiment; Fig.18 This is a schematic diagram of fluorescence lifetime imaging of fluorescent carbon dots in a zebrafish liver injury model in the embodiment. DETAILED DESCRIPTION

[0013] In order to more clearly understand the purpose, technical solutions and advantages of the present application, the present application is described and illustrated below in conjunction with the accompanying drawings and embodiments.

[0014] Unless otherwise defined, the technical terms or scientific terms involved in this application should have the general meaning understood by people with ordinary skills in the technical field to which this application belongs. The words such as "one", "a", "the", "these" and the like in this application do not represent quantitative restrictions, and they can be singular or plural. The terms "include", "comprise", "have" and any variants thereof involved in this application are intended to cover non-exclusive inclusions. The terms "first", "second", "third", etc. involved in this application are only used to distinguish similar objects and do not represent a specific ordering of objects.

[0015] In this embodiment, a method for preparing dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets is provided. Figure 1 Schematic diagram of the synthesis of dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets in this embodiment. Figure 1 and Figure 2 , the process includes step S110, step S120 and step S130.

[0016] Step S110, dissolving o-phenylenediamine and phenylalanine in pure water to obtain a first solution, transferring the first solution to a polytetrafluoroethylene-lined reactor for a first heating reaction to obtain a precipitate (after the reaction is completed, taking out the precipitate from the reactor), and sequentially performing column chromatography purification, rotary evaporation and drying on the precipitate to obtain a target solid, which is a dark brown solid.

[0017] Among them, ultrasound can be used to dissolve o-phenylenediamine and phenylalanine in pure water. The mass ratio of o-phenylenediamine, phenylalanine and pure water is 1:2:190-1:2:210, such as 1:2:200, and pure water can account for 3:5 of the volume of the reactor; the temperature of the first heating reaction can be 160-200°C and the time can be 6-10 hours, wherein the preferred temperature of the first heating reaction is 180°C and the time is 8 hours; the eluate purified by column chromatography is a mixture of dichloromethane and methanol, and in the mixture of dichloromethane and methanol, the volume ratio of dichloromethane to methanol can be 30:1.

[0018] Step S120, dissolving caffeic acid in dichloromethane and adding CDI (1,1'-carbonyldiimidazole) and stirring to obtain a second solution.

[0019] The stirring is performed in an ice bath for 30-60 min, and the mass ratio of CDI to caffeic acid is 1:1-1:2.

[0020] Step S130, adding the target solid into the second dissolving solution to perform a second heating reaction to obtain dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets.

[0021] The temperature of the second heating reaction is 25°C and the time is 12-24 h, and the mass ratio of caffeic acid to the target solid is 1:1-1:1.2. The precipitate obtained by the reaction is a dual-targeted fluorescent carbon dot.

[0022] By using the above-mentioned preparation method of dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets, dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets can be prepared.

[0023] In the above-mentioned preparation method of dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets, o-phenylenediamine is a known good material for condensation, polymerization, and carbonization into carbon dots, and phenylalanine is introduced into the reaction system to adjust the photoluminescence properties of CDs. On the one hand, it has a benzene ring itself; on the other hand, the amino and carboxyl groups are easily cross-linked through amide bonds, both of which promote the formation of carbonaceous graphite cores and red-shift the photoluminescence emission. Caffeic acid can recognize superoxide anions and can be connected to carbon dots through the condensation reaction of amino and carboxyl groups. When superoxide anions are present, the caffeic acid residues on the carbon dots can be converted from electron donor catechol to electron acceptor benzoquinone. In the presence of superoxide anions, the fluorescence intensity and fluorescence lifetime of the carbon dots decrease significantly. Therefore, the fluorescence intensity and lifetime of the fluorescent carbon dots can be used to judge whether the cells are stressed or damaged (for details, please refer to subsequent experiments). At the same time, the dual-targeted fluorescent carbon dots prepared by this method are sensitive to the cellular microenvironment and can effectively distinguish between the endoplasmic reticulum and lipid droplets through fluorescence lifetime imaging (see subsequent experiments for details), which solves the current problem of the lack of lifetime-sensitive dual-targeted fluorescent carbon dots that can simultaneously locate the endoplasmic reticulum and lipid droplets and sensitively monitor changes in their microenvironment.

[0024] In this embodiment, a dual-targeted fluorescent carbon dot for endoplasmic reticulum and lipid droplets is also provided, which is obtained by the preparation method of the dual-targeted fluorescent carbon dot for endoplasmic reticulum and lipid droplets in this embodiment.

[0025] Through the above preparation method, it can be known that the dual-targeted fluorescent carbon dots use o-phenylenediamine and phenylalanine as carbon sources and pure water as solvent. The carbon dots are obtained by solvothermal reaction in a reactor. After purification by column chromatography, caffeic acid groups are connected to the carbon dots.

[0026] Dual-targeted fluorescent carbon dots are photoluminescent carbon dots with high fluorescence quantum yield. They can be used as fluorescent probes for real-time imaging of the association between endoplasmic reticulum oxidative stress and lipid metabolism in living cells. The endoplasmic reticulum and lipid droplet parts can be distinguished by the difference in fluorescence lifetime. The probe responds to superoxide anions and viscosity. The association between endoplasmic reticulum oxidative stress and lipid metabolism can be studied by changes in fluorescence intensity and fluorescence lifetime. The optimal excitation wavelength of dual-targeted fluorescent carbon dots is 390nm, the maximum emission wavelength is 570nm, and the particle size distribution is 2.0-4.5nm. The mass contents of C, N, and O in dual-targeted fluorescent carbon dots are 88%-89%, 6%-7%, and 9%-10%, respectively. The size of dual-targeted fluorescent carbon dots is small, with an average particle size of 3.3nm.

[0027] The oil-water partition coefficient (LogP value) of the dual-targeted fluorescent carbon dots is between 1.4 and 1.5, has good lipid solubility, and has dual targeting specificity for the endoplasmic reticulum and lipid droplets. It is sensitive to the microenvironment, can distinguish the endoplasmic reticulum and lipid droplet parts by fluorescence lifetime, can sensitively detect the content of superoxide anions and changes in lipid droplet viscosity, can monitor the degree of endoplasmic reticulum oxidative stress and changes in lipid droplet viscosity in real time, has strong resistance to photobleaching, and is basically non-toxic to cells. The dual-targeted fluorescent carbon dots have high quantum yield, good resistance to photobleaching, and good biocompatibility. Therefore, the dual-targeted fluorescent carbon dots of this embodiment are particularly suitable for real-time imaging of living cells and observing the association between endoplasmic reticulum oxidative stress and lipid metabolism.

[0028] As follows, the characteristics of the dual-targeted fluorescent carbon dots (Y-CDs) in this embodiment are verified through experimental data. The specific preparation steps of the verified dual-targeted fluorescent carbon dots are: Add o-phenylenediamine and phenylalanine to pure water, ultrasonicate for 10 minutes, transfer to a 50 mL polytetrafluoroethylene-lined reactor after o-phenylenediamine and phenylalanine are completely dissolved, react at 180°C for 8 hours, and then cool naturally to room temperature. The precipitate in the reactor is purified by column chromatography, and the eluate is a mixture of dichloromethane and methanol (V dichloromethane: V methanol = 30:1), which is rotary evaporated and dried to obtain a dark brown solid. Caffeic acid is dissolved in dichloromethane, CDI is added, and stirred in an ice bath for 30 minutes, wherein the mass ratio of CDI to caffeic acid is 1:2. The purified dark brown solid in the above reactor is added to the dichloromethane solution of caffeic acid, and reacted for 12 hours at 25°C by magnetic stirring. Among them, the mass ratio of caffeic acid to the purified dark brown solid is 1:1.2.

[0029] The characterization of the dual-targeted fluorescent carbon dots in this example is as follows Figure 2 As shown, the surface functional groups of the synthesized Y-CDs were determined by FTIR spectroscopy. -1The absorption bands nearby are attributed to -OH / -NH on the surface, 2924-2851 cm -1 The absorption band attributed to C-H bonds was observed at 1723–1634 cm -1 Corresponding to the C=O absorption band, 1300-1000 cm -1 The absorption band of Y-CDs was attributed to CO bonds. The optical properties of the synthesized Y-CDs were confirmed by UV-Vis absorption and fluorescence spectra.

[0030] See also Figure 3 The ultraviolet absorption peak at 323 nm is the characteristic absorption peak of caffeic acid. By externally connecting caffeic acid, it can be observed that Y-CDs also has a characteristic peak at 323 nm, which further proves that the preparation method of dual-targeted fluorescent carbon dots provided in this embodiment successfully connects caffeic acid to carbon dots.

[0031] See also Figure 4 and Figure 5 ,exist Figure 4 In the figure, as the excitation wavelength increases from 390 nm to 570 nm, the emission wavelength of the synthesized Y-CDs hardly changes. Figure 4 In the photograph depicted in the inset, the pale yellow methanol solution exhibits orange-yellow fluorescence under irradiation with (365 nm) ultraviolet light. Figure 5 The fluorescence intensity of the carbon dots after continuous irradiation with ultraviolet light (365nm) for 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, 95 min and 100 min respectively showed no obvious decrease, which indicated that Y-CDs had excellent photostability.

[0032] See also Figure 6 and Figure 7 ,from Figure 6 and 7 It can be seen that the shape of the synthesized Y-CDs is approximately spherical, and the size distribution results of Y-CDs show that the diameter of Y-CDs is distributed in the range of 2.0-4.5 nm, with an average diameter of about 3.3 nm, and the imaging is uniformly dispersed in the solvent.

[0033] See also Figure 8It can be seen that the Y-CDs sample mainly contains three elements: C, N and O, with contents of 88%-89%, 6%-7% and 9%-10% (atomic ratio), respectively. The high-resolution XPS spectrum of C1s can fit three characteristic peaks of 284.8 eV (CC), 285.9 eV (COC) and 288.5 eV (C=O). The high-resolution XPS spectrum of N1s shows that N mainly exists in the form of pyrrole-N and graphite-N. The high-resolution XPS spectrum of O1s can fit three characteristic peaks of 531.7 eV (C=O), 532.1 eV (COC) and 533.5 eV (OH).

[0034] See also Fig. 9 It can be seen that the oil-water partition coefficient (LogP value) in Y-CDs is between 1.4 and 1.5, and the fat solubility is good.

[0035] As follows, the cell imaging application effect of the dual-targeted fluorescent carbon dots (Y-CDs) in this embodiment is further explored and verified through experimental data.

[0036] Selective exploration of fluorescent carbon dots: To test the selectivity of fluorescent carbon dots for intracellular biomolecules such as inorganic salts, amino acids, and other proteins or enzymes, fluorescent probes (10 μg / mL) were added to PBS buffer solution (0.01 M, pH 7.4), and then common intracellular biomolecules such as potassium ions, calcium ions, copper ions, iron ions, sodium ions, glutathione (GSH), cysteine ​​(Cys), leucine (Leu), arginine (Arg), threonine (Thr), glycine (Gly), methionine (Met), bovine serum albumin (BSA), glycerol (TG), liposomes (Lipsome), H2O2, ClO - 、ONOO - , 1 O2, O2 - , and measure the fluorescence changes after reacting with fluorescent carbon dots. Fig.10 ,Depend on Fig.10 It can be seen that the fluorescent carbon dots have no response to other biological molecules contained in the cells, but respond to superoxide anions, glycerol, and liposomes. The response to proteins and liposomes is mainly due to viscosity.

[0037] Study on the response of fluorescent carbon dots to superoxide anions: To test the response of fluorescent carbon dots to superoxide anions, different concentrations of potassium peroxide DMSO solutions were prepared, and the fluorescent probe (10 μg / mL) was added to the PBS buffer solution (0.01 M, pH 7.4). Then, different concentrations of potassium peroxide solutions were added, and the sensitivity of Y-CDs to detect superoxide anions was observed by measuring the fluorescence spectrum under 470 nm excitation light. Fig.11 ,Depend on Fig.11 It can be seen that with the increase of superoxide anion concentration, the fluorescence intensity decreases significantly, indicating that Y-CDs responds sensitively to superoxide anions.

[0038] Study on the response of fluorescent carbon dots to viscosity: To test the response of fluorescent carbon dots to viscosity, different viscosity solutions were prepared by varying the ratio of glycerol to water. Fluorescent probes (10 μg / mL) were added to the solutions with different viscosities, shaken and mixed, and the changes in fluorescence intensity were studied. Fig.12 ,Depend on Fig.12 It can be seen that with the increase of solution viscosity, the fluorescence intensity of the fluorescent carbon dots changes significantly, indicating that the fluorescent carbon dots respond sensitively to viscosity.

[0039] Changes in fluorescence intensity of fluorescent carbon dots at different pH values: To test the change of fluorescence intensity of fluorescent carbon dots under different pH conditions, fluorescent carbon dots were added to 1 ml of PBS (0.01 M) with different pH values ​​to make the concentration of 10 μg / mL, and the change of fluorescence intensity was studied. Fig.13 ,Depend on Fig.13 It can be seen that changes in pH will not affect the fluorescence intensity of the probe, indicating that the probe remains stable in strong alkaline and strong acid environments.

[0040] Cytotoxicity assay: Human hepatoma cells (HepG2) were seeded into 96-well plates at 1×104 cells per well, and the 96-well plates were placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 24 hours to allow them to completely adhere to the wall. After that, fresh culture medium was replaced, and 10 μL of fluorescent carbon dot dispersions of different concentrations were added. After 24 hours of culture, MTT solution (5 mg / mL) was added to each well and incubated for another 4 hours. Then, 100 μL of DMSO was added to each well, and the 96-well plate was placed on a horizontal shaking shaker for 10 minutes. The wavelength was set to 492 nm on the microplate reader, and the absorbance (OD value) of the solution in each well of the 96-well plate was measured. The cell survival rate was calculated according to the following formula: Cell survival rate = (OD test group-OD blank group) / (OD cell group-OD blank group) × 100%. Please refer to Fig.14 ,from Fig.14As can be seen from the figure, the fluorescent carbon dots have almost no cytotoxicity in the range of 1-30 μg / mL.

[0041] Real-time imaging of fluorescent carbon dots entering cells: Hepatocellular carcinoma cells (HepG2) were seeded into glass-bottomed culture dishes and cultured at 37 °C for 2 days. Subsequently, the probe (10 μg / mL) was added to the cells, incubated at 37 °C for 20 min, and imaged using a laser confocal microscope. Fig.15 As shown, the fluorescent carbon dots can enter the cells within 5 minutes, and the fluorescence intensity reaches the highest after 20 minutes.

[0042] Intracellular co-localization imaging: In the colocalization experiment, liver cancer cells (HepG2) were first incubated with fluorescent carbon dots (10 μg / mL) for 20 min, and then incubated with 1 μM endoplasmic reticulum staining reagent for 10 min. Finally, the cells were washed twice with PBS solution (pH 7.4) and imaged using a laser confocal microscope. The results are shown in Figure 2. Fig.16 As shown in the figure, the Y-CDs channel of the fluorescent carbon dots perfectly overlaps with the endoplasmic reticulum staining channel, proving that it is localized to the endoplasmic reticulum organelle. The Y-CDs channel of the fluorescent carbon dots perfectly overlaps with the lipid droplet staining channel, proving that it is localized to the lipid droplets. Therefore, this probe is a carbon dot localized to the endoplasmic reticulum and lipid droplets.

[0043] Real-time imaging of ER stress in cells: In the ER stress experiment, hepatoma cells (HepG2) were first incubated with fluorescent carbon dots (10 μg / mL) for 20 min, then the cells were washed twice with PBS solution (pH 7.4), and finally thioacetamide (TAA, 0.4 mg / mL) was added to induce ER stress in the cells and the ER stress was observed using a fluorescence lifetime imaging system. Fig.17 As shown in the figure, the Bar value is uniformly set to 0-15. As the oxidative stress of the endoplasmic reticulum gradually increases, the fluorescence lifetime of the endoplasmic reticulum decreases (from 13.34 ns to 11.09 ns), while the fluorescence lifetime of lipid droplets increases (from 8.96 ns to 14.38 ns). The fluorescence lifetime changes significantly, so the probe is sensitive to superoxide anions and viscosity responses.

[0044] In vivo imaging of zebrafish liver injury model: In the zebrafish liver injury model experiment, the zebrafish liver injury model was first established: zebrafish embryos were maintained in a 6-well plate containing fish culture medium (3.0 ml methyl blue, 0.8 g sodium bicarbonate, 2.66 g sea salt and water to prepare 8 L). Zebrafish embryos two days after fertilization were placed in a 6-well plate, and 6 ml fish culture medium was added to each well. Zebrafish embryos were divided into 2 groups, named blank control group (CON group) and model group (thioacetamide TAA group 0.4 mg / ml). All incubations were carried out under a 14-h light / 10-h dark cycle and a constant temperature of 28 °C. During the chemical treatment period, the incubation solution of zebrafish larvae was renewed every day. From 6 dpf to 9 dpf, the larvae were fed with microcapsule feed for zebrafish larvae. Zebrafish from the blank control group and the model group were extracted and incubated in a culture medium containing 60 μg / mL fluorescent carbon dots for 3-4 h, and then the fry were washed three times with normal culture medium. The zebrafish were anesthetized by hypothermia, placed on a glass slide, and imaged in vivo using a fluorescence lifetime imaging system. Fig.18 As shown in the figure, the Bar value is uniformly set. In zebrafish nine days after fertilization, the fluorescence lifetime of the liver tissue in the blank control group is 14.254ns, while the lifetime of the liver tissue in the liver tissue injury model group is 8.961ns. In the liver injury model, the fluorescence lifetime of the fluorescent carbon dots decreases significantly. Therefore, the probe has an indicative effect on liver tissue damage.

[0045] In summary, the fluorescent carbon dots prepared in this embodiment can be used in the field of biological in vivo imaging, and it has a high quantum yield of more than 22.05%. At the same time, it can be selectively positioned in the endoplasmic reticulum and fat droplets of cells and can be distinguished by fluorescence lifetime imaging, and the content of superoxide anions in the endoplasmic reticulum and the viscosity change of fat droplets when the endoplasmic reticulum undergoes oxidative stress can be sensitively detected, which can be used for living cell imaging and real-time monitoring of the association between endoplasmic reticulum stress and lipid metabolism, and can be used for monitoring of zebrafish liver tissue damage, greatly improving its application in the biological field, and will not cause radiation damage to biological samples. In addition, the fluorescent carbon dots prepared in this embodiment have high photostability and good biocompatibility, which overcomes the problem of lacking life-span sensitive dual-targeted fluorescent carbon dots that can simultaneously locate the endoplasmic reticulum and fat droplets and sensitively monitor changes in their microenvironment.

[0046] It can be seen from the above description that the dual-targeted fluorescent carbon dots prepared by the preparation method of the dual-targeted fluorescent carbon dots in this embodiment can be used for imaging monitoring of endoplasmic reticulum and lipid droplets. Therefore, this embodiment also provides the application of the dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets in cell monitoring, which includes real-time imaging of cellular endoplasmic reticulum oxidative stress and lipid metabolism.

[0047] It should be understood that the specific embodiments described herein are only used to explain the application, rather than to limit it. Based on the embodiments provided in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the protection scope of this application.

[0048] Obviously, the drawings are only some examples or embodiments of the present application. For ordinary technicians in the field, the present application can also be applied to other similar situations based on these drawings without creative work. In addition, it is understandable that although the work done in this development process may be complicated and lengthy, for ordinary technicians in the field, certain changes in design, manufacturing or production based on the technical content disclosed in this application are only conventional technical means and should not be regarded as insufficient content disclosed in this application.

Claims

1. A method for preparing dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets, characterized in that: include: Dissolving o-phenylenediamine and phenylalanine in pure water to obtain a first solution, transferring the first solution to a polytetrafluoroethylene-lined reactor for a first heating reaction to obtain a precipitate, and sequentially subjecting the precipitate to column chromatography purification, rotary evaporation, and drying to obtain a target solid; Dissolving caffeic acid in dichloromethane and adding CDI and stirring to obtain a second solution; The target solid is added into the second dissolving solution to carry out a second heating reaction to obtain the dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets.

2. The method for preparing dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets according to claim 1, characterized in that: The mass ratio of the o-phenylenediamine, the phenylalanine and the pure water is 1:2:190-1:2:210, and the pure water accounts for 3:5 of the volume of the reactor.

3. The method for preparing dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets according to claim 1, characterized in that: The temperature of the first heating reaction is 160-200° C. and the time is 6-10 h.

4. The method for preparing dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets according to claim 1, characterized in that: The eluate of the column chromatography purification is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol in the mixture of dichloromethane and methanol is 30:

1.

5. The method for preparing dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets according to claim 1, characterized in that: The stirring is ice bath stirring for 30-60 min, and the mass ratio of the CDI to the caffeic acid is 1:1-1:

2.

6. The method for preparing dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets according to claim 1, characterized in that: The temperature of the second heating reaction is 25° C. and the time is 12-24 h. The mass ratio of the caffeic acid to the target solid is 1:1-1:1.

2.

7. A dual-targeted fluorescent carbon dot for endoplasmic reticulum and lipid droplets, characterized in that: The method for preparing dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets is used as described in any one of claims 1 to 6.

8. The dual-targeting fluorescent carbon dots for endoplasmic reticulum and lipid droplets according to claim 7, characterized in that: The optimal excitation wavelength is 390 nm, the maximum emission wavelength is 570 nm, and the particle size distribution is 2.0-4.5 nm with an average particle size of 3.3 nm.

9. Use of the dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets according to claim 7 or 8 in cell monitoring.

10. The use of dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets in cell monitoring according to claim 9, characterized in that: Includes real-time imaging of cellular endoplasmic reticulum oxidative stress and lipid metabolism.

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