Dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets, preparation method and application thereof

Abstract

The present application relates to a dual-targeted fluorescent carbon dot for endoplasmic reticulum and lipid droplets, and a preparation method and application thereof. The preparation method includes: dissolving o-phenylenediamine and phenylalanine in pure water to obtain a first solution, transferring the first solution to a reaction kettle lined with polytetrafluoroethylene for a first heating reaction to obtain a precipitate, subjecting the precipitate to column chromatography purification, rotary evaporation and drying in sequence to obtain a target solid; dissolving caffeic acid in dichloromethane and adding CDI for stirring to obtain a second solution; adding the target solid to the second solution for a second heating reaction to obtain the dual-targeted fluorescent carbon dot for endoplasmic reticulum and lipid droplets. The dual-targeted fluorescent carbon dot obtained by this preparation method can distinguish endoplasmic reticulum and lipid droplets, solving the problem of the lack of a lifetime-sensitive dual-targeted fluorescent carbon dot that can simultaneously localize to endoplasmic reticulum and lipid droplets and sensitively monitor the changes in their microenvironment.

Description

Technical Field

[0001] The present application relates to the field of fluorescent materials, and particularly to a dual-targeted fluorescent carbon dot for endoplasmic reticulum and lipid droplets, a preparation method thereof, and an 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 they were discovered in 2004, carbon dots still face the problems of lacking precise targeting of specific organelles, especially distinguishable targeting of two interrelated organelles and microenvironment response, which greatly limits the application of carbon dots in the dynamics of organelles 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 processes such as calcium ion storage and signal transduction. Lipid droplets are important organelles in cells for storing lipids and energy, consisting of a core of neutral lipids and a monolayer of phospholipids and specific proteins wrapping outside. Research has confirmed that the generation of lipid droplets originates from the endoplasmic reticulum, and there are frequent and extensive interactions between mature lipid droplets and the endoplasmic reticulum. Moreover, changes in the microenvironments of the endoplasmic reticulum and lipid droplets will affect their physiological functions. For example, excessive reactive oxygen species (ROS) will lead to endoplasmic reticulum stress, and changes in the viscosity of lipid droplets are also closely related to lipid metabolism. Therefore, real-time synchronous monitoring of the interaction between the two and the dynamic changes in the microenvironment is of great significance for deeply understanding their physiological and pathological mechanisms. At present, single-targeted fluorescent carbon dots often need to be combined with multiple commercial dyes (commercial dye combination) to achieve synchronous visualization of the two, and cannot simultaneously meet the multifunctional requirements of dual targeting and environmental sensitive response, making it difficult to comprehensively reflect complex pathological states and resulting in one-sided detection information.

[0004] Therefore, there is an urgent need to develop multifunctional fluorescent carbon dots with the dual-targeting ability of the endoplasmic reticulum and lipid droplets and sensitive response to changes in their microenvironments, so as to realize real-time imaging and functional regulation of the association between endoplasmic reticulum oxidative stress and lipid droplet viscosity changes, and provide a powerful tool for the early diagnosis, targeted treatment, and drug delivery of related diseases.

[0005] Regarding the current problem of the lack of lifetime-sensitive dual-targeted fluorescent carbon dots that can simultaneously localize to the endoplasmic reticulum and lipid droplets and sensitively monitor changes in their microenvironments, no effective solution has been proposed yet. Summary of the Invention

[0006] In the present invention, a dual-targeted fluorescent carbon dot for endoplasmic reticulum and lipid droplets, its preparation method and application are provided to solve the problem that there is currently a lack of dual-targeted fluorescent carbon dots that are sensitive to the microenvironmental changes of the endoplasmic reticulum and lipid droplets.

[0007] In a first aspect, a preparation method of a dual-targeted fluorescent carbon dot for endoplasmic reticulum and lipid droplets is provided in the present invention, including:

[0008] Dissolve o-phenylenediamine and phenylalanine in pure water to obtain a first solution, transfer the first solution to a reaction kettle lined with polytetrafluoroethylene for a first heating reaction to obtain a precipitate, and subject the precipitate to column chromatography purification, rotary evaporation and drying in sequence to obtain a target solid;

[0009] Dissolve caffeic acid in dichloromethane and add CDI for stirring to obtain a second solution;

[0010] Add the target solid to the second solution for a second heating reaction to obtain the dual-targeted fluorescent carbon dot for endoplasmic reticulum and lipid droplets.

[0011] In a second aspect, a dual-targeted fluorescent carbon dot for endoplasmic reticulum and lipid droplets is provided in the present invention, which is obtained by the preparation method of the dual-targeted fluorescent carbon dot for endoplasmic reticulum and lipid droplets described in the first aspect.

[0012] In a third aspect, the application of the dual-targeted fluorescent carbon dot for endoplasmic reticulum and lipid droplets described in the second aspect in cell monitoring is provided in the present invention.

[0013] Compared with the related technology, the present invention introduces phenylalanine into the reaction system to regulate the photoluminescence properties of CDs. On the one hand, it has a benzene ring itself; on the other hand, amino groups and carboxyl groups are easily cross-linked through amide bonds, both of which promote the formation of carbonaceous graphite nuclei and cause a red shift in the photoluminescence emission. In addition, caffeic acid can recognize superoxide anions, and caffeic acid can be connected to the carbon dots through the condensation reaction of amino groups and carboxyl groups. 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, can effectively distinguish the endoplasmic reticulum and lipid droplets, and solve the problem that there is currently a lack of lifetime-sensitive dual-targeted fluorescent carbon dots that can simultaneously localize to the endoplasmic reticulum and lipid droplets and sensitively monitor the microenvironmental changes thereof.

[0014] The details of one or more embodiments of the present application are set forth in the following drawings and description to make the other features, objects and advantages of the present application more concise and understandable. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a schematic diagram of the synthesis of fluorescent carbon dots in the embodiment;

[0016] Figure 2 Schematic diagram of the FT-IR results of the fluorescent carbon dots in the examples;

[0017] Figure 3 Schematic diagram of the UV-visible spectrum of the fluorescent carbon dots in the examples;

[0018] Figure 4 Schematic diagram of the fluorescence emission spectrum of the fluorescent carbon dots under different excitation lights in the examples;

[0019] Figure 5 Schematic diagram of the change in fluorescence intensity of the fluorescent carbon dots under different UV irradiation times in the examples;

[0020] Figure 6 Schematic diagram of the TEM image of the fluorescent carbon dots in the examples;

[0021] Figure 7 Schematic diagram of the size distribution results of the fluorescent carbon dots in the examples;

[0022] Figure 8 Schematic diagram of the XPS spectrum analysis of the fluorescent carbon dots in the examples; where: part (a) is the total XPS spectrum of the fluorescent carbon dots, part (b) is the XPS spectrum of the C element in the fluorescent carbon dots, part (c) is the XPS spectrum of the N element in the fluorescent carbon dots, and part (d) is the XPS spectrum of the O element in the fluorescent carbon dots;

[0023] Figure 9 Schematic diagram of the Log P diagram analysis of the fluorescent carbon dots in the examples;

[0024] Figure 10 Schematic diagram of the selectivity analysis of the fluorescent carbon dots for various analysis substrates contained in cells in the examples;

[0025] Figure 11 Schematic diagram of the fluorescence emission spectrum of the fluorescent carbon dots in response to superoxide anions in vitro in the examples;

[0026] Figure 12 Schematic diagram of the fluorescence emission spectrum of the fluorescent carbon dots in response to viscosity in vitro in the examples;

[0027] Figure 13 Schematic diagram of the selectivity analysis of the fluorescent carbon dots for pH in the examples;

[0028] Figure 14 Schematic diagram of the cytotoxicity analysis of the fluorescent carbon dots in the examples;

[0029] Figure 15 Schematic diagram of the real-time imaging of the fluorescent carbon dots entering cells in the examples;

[0030] Figure 16Schematic diagram of the co-localization imaging analysis of fluorescent carbon dots in cells in the examples;

[0031] Figure 17 Schematic diagram of the fluorescence lifetime imaging of fluorescent carbon dots in cells during endoplasmic reticulum oxidative stress in the examples;

[0032] Figure 18 Schematic diagram of the fluorescence lifetime imaging of fluorescent carbon dots in zebrafish in a liver injury model in the examples. Detailed implementation manners

[0033] To more clearly understand the purpose, technical solution and advantages of the present application, the present application will be described and illustrated below with reference to the accompanying drawings and examples.

[0034] Unless otherwise defined, the technical terms or scientific terms involved in the present application shall have the general meaning understood by those with ordinary skills in the technical field to which the present application belongs. In the present application, words such as "a", "one", "a kind", "the", "these" and the like do not indicate a limitation in quantity, and they can be singular or plural. The terms "including", "comprising", "having" and any variants thereof involved in the present application are intended to cover non-exclusive inclusion. The terms "first", "second", "third" and the like involved in the present application are only used to distinguish similar objects and do not represent a specific order for the objects.

[0035] In this example, a preparation method of dual-targeting fluorescent carbon dots for endoplasmic reticulum and lipid droplets is provided. Figure 1 Synthesis schematic diagram of the dual-targeting fluorescent carbon dots for endoplasmic reticulum and lipid droplets in this example. Refer to Figure 1 and Figure 2 , this process includes step S110, step S120 and step S130.

[0036] Step S110: Dissolve o-phenylenediamine and phenylalanine in pure water to obtain a first solution, transfer the first solution to a reaction kettle with a polytetrafluoroethylene liner for a first heating reaction to obtain a precipitate (take out the precipitate in the reaction kettle after the reaction ends), and perform column chromatography purification, rotary evaporation and drying on the precipitate in sequence to obtain a target solid, and the target solid is a black-brown solid.

[0037] Among them, o-phenylenediamine and phenylalanine can be dissolved in pure water by ultrasound. The mass ratio of o-phenylenediamine, phenylalanine and pure water is 1:2:190 - 1:2:210, such as 1:2:200, and the pure water can account for 3:5 of the volume of the reaction kettle; the temperature of the first heating reaction can be 160 - 200 °C and the time can be 6 - 10 h. Among them, the preferred temperature of the first heating reaction is 180 °C and the time is 8 h; the eluent for column chromatography purification is a mixed solution of dichloromethane and methanol. In the mixed solution of dichloromethane and methanol, the volume ratio of dichloromethane and methanol can be 30:1.

[0038] Step S120: Dissolve caffeic acid in dichloromethane and add CDI (1,1'-carbonyldiimidazole) and stir to obtain a second solution.

[0039] Among them, the stirring is ice bath stirring and the time is 30 - 60 min, and the mass ratio of CDI to caffeic acid is 1:1 - 1:2.

[0040] Step S130: Add the target solid to the second solution for a second heating reaction to obtain dual-targeted fluorescent carbon dots for the endoplasmic reticulum and lipid droplets.

[0041] Among them, 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 from the reaction is the dual-targeted fluorescent carbon dots.

[0042] Through the above preparation method of dual-targeted fluorescent carbon dots for the endoplasmic reticulum and lipid droplets, dual-targeted fluorescent carbon dots for the endoplasmic reticulum and lipid droplets can be prepared.

[0043] In the above method for preparing dual-targeted fluorescent carbon dots for the endoplasmic reticulum and lipid droplets, o-phenylenediamine is a well-known material for condensation, polymerization, and carbonization into carbon dots. Phenylalanine is introduced into the reaction system to regulate the photoluminescence properties of the CDs. On the one hand, it has a benzene ring itself; on the other hand, the amino group and carboxyl group are easily cross-linked through amide bonds, both of which promote the formation of a carbonaceous graphite core, causing a red shift in the photoluminescence emission. Caffeic acid can recognize superoxide anions and can be attached to the carbon dots through the condensation reaction of the amino group and 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. In the presence of superoxide anions, the fluorescence intensity and fluorescence lifetime of the carbon dots decrease significantly. Therefore, it is possible to determine whether cells are stressed or damaged by the fluorescence intensity and lifetime of the fluorescent carbon dots (specifically, refer to the subsequent experiments). At the same time, the dual-targeted fluorescent carbon dots prepared by this method are sensitive to the cell microenvironment and can effectively distinguish the endoplasmic reticulum and lipid droplets through fluorescence lifetime imaging (specifically, refer to the subsequent experiments), solving the problem of the lack of lifetime-sensitive dual-targeted fluorescent carbon dots that can simultaneously localize to the endoplasmic reticulum and lipid droplets and sensitively monitor changes in their microenvironments.

[0044] In this embodiment, a dual-targeted fluorescent carbon dot for the endoplasmic reticulum and lipid droplets is also provided, which is obtained by the method for preparing dual-targeted fluorescent carbon dots for the endoplasmic reticulum and lipid droplets in this embodiment.

[0045] As can be seen from the above preparation method, the dual-targeted fluorescent carbon dots are obtained by using o-phenylenediamine and phenylalanine as carbon sources, pure water as a solvent, and reacting in a reaction kettle by a solvothermal method to obtain carbon dots, followed by column chromatography purification and then attaching caffeic acid groups to the carbon dots.

[0046] The dual-targeted fluorescent carbon dots are a kind of photoluminescent fluorescent carbon dots with a high fluorescence quantum yield, which can be used as a fluorescent probe 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 different fluorescence lifetimes, and the probe shows responses to superoxide anions and viscosity. The association between endoplasmic reticulum oxidative stress and lipid metabolism can be studied through changes in fluorescence intensity and fluorescence lifetime. The optimal excitation wavelength of the dual-targeted fluorescent carbon dots is 390 nm, the maximum emission wavelength is 570 nm, and the particle size distribution is in the range of 2.0 - 4.5 nm. The mass contents of C, N, and O in the dual-targeted fluorescent carbon dots are 88% - 89%, 6% - 7%, and 9% - 10% respectively. The dual-targeted fluorescent carbon dots are relatively small in size, with an average particle size of 3.3 nm.

[0047] The oil-water partition coefficient (LogP value) of the dual-targeted fluorescent carbon dots is between 1.4 and 1.5, with good liposolubility. It has dual-target specificity for the endoplasmic reticulum and lipid droplets, is sensitive to the microenvironment, can distinguish the endoplasmic reticulum and lipid droplet parts through fluorescence lifetime, can sensitively detect the content of superoxide anions and the change of lipid droplet viscosity, can monitor the degree of endoplasmic reticulum oxidative stress and the change of lipid droplet viscosity in real time, has strong anti-photobleaching performance, and has little toxicity to cells. The dual-targeted fluorescent carbon dots have the characteristics of high quantum yield, good anti-photobleaching performance, and good biocompatibility. Therefore, the dual-targeted fluorescent carbon dots in this example are particularly suitable for live cell real-time imaging and observing the association between endoplasmic reticulum oxidative stress and lipid metabolism.

[0048] As follows, the characteristics of the dual-targeted fluorescent carbon dots (Y-CDs) in this example are verified through experimental data. The specific preparation steps of the verified dual-targeted fluorescent carbon dots are as follows:

[0049] Add o-phenylenediamine and phenylalanine into pure water, ultrasonicate for 10 minutes, transfer it to a reaction kettle with a PTFE liner with a volume of 50 mL after o-phenylenediamine and phenylalanine are completely dissolved, react at 180 °C for 8 h, and then naturally cool to room temperature. Purify the precipitate in the reaction kettle by column chromatography, and the eluent is a mixed solution of dichloromethane and methanol (V dichloromethane:V methanol = 30:1). Rotate and evaporate, and dry to obtain a black-brown solid. Dissolve caffeic acid in dichloromethane, add CDI, and stir in an ice bath for 30 min, where the mass ratio of CDI to caffeic acid is 1:2. Add the purified black-brown solid in the above reaction kettle to the dichloromethane solution of caffeic acid, and react at 25 °C by magnetic stirring for 12 h. Among them, the mass ratio of caffeic acid to the purified black-brown solid is 1:1.2.

[0050] The characterization of the dual-targeted fluorescent carbon dots in this example is as Figure 2 shown. The surface functional groups of the synthesized Y-CDs are determined by FTIR spectroscopy. The absorption band of Y-CDs near 3155 cm -1 is attributed to -OH / -NH on its surface, and the absorption band at 2924 - 2851 cm -1 is attributed to the C-H bond. The absorption band corresponding to C=O is observed at 1723 - 1634 cm -1 , and the absorption band at 1300 - 1000 cm -1 is attributed to the C-O bond. The optical properties of the synthesized Y-CDs are confirmed by UV-Vis absorption spectroscopy and fluorescence spectroscopy.

[0051] Please refer to 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 exhibit a characteristic peak at 323 nm, further proving that the preparation method of the dual-targeted fluorescent carbon dots provided in this embodiment successfully incorporates caffeic acid into the carbon dots.

[0052] Please refer to Figure 4 and Figure 5 , in Figure 4 , as the excitation wavelength increases from 390 nm to 570 nm, the emission wavelength of the synthesized Y-CDs hardly changes. From Figure 4 the photograph depicted in the inset, the pale yellow methanol solution exhibits orange-yellow fluorescence under the irradiation of a (365 nm) ultraviolet lamp. Figure 5 Figure

[0053] Please refer to Figure 6 and Figure 7 , from Figure 6 and 7 it can be seen that the synthesized Y-CDs are approximately spherical in shape. 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, and the average diameter is about 3.3 nm, presenting a uniformly dispersed state when imaged in the solvent.

[0054] Please refer to Figure 8 , it can be known that: there are mainly three elements, C, N, and O, in the Y-CDs sample, and their contents are 88% - 89%, 6% - 7%, and 9% - 10% (atomic ratio), respectively. The high-resolution XPS spectrum of C1s can be fitted with three characteristic peaks at 284.8 eV (C-C), 285.9 eV (C-O-C), and 288.5 eV (C=O). The high-resolution XPS spectrum of N1s indicates that N mainly exists in the forms of pyrrole-N and graphitic-N. The high-resolution XPS spectrum of O1s can be fitted with three characteristic peaks at 531.7 eV (C=O), 532.1 eV (C-O-C), and 533.5 eV (O-H).

[0055] Please refer to Figure 9 , it can be known that: the oil-water partition coefficient (LogP value) of Y-CDs is between 1.4 and 1.5, indicating good liposolubility.

[0056] 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.

[0057] Investigation of the selectivity of fluorescent carbon dots:

[0058] To test the selectivity of the fluorescent carbon dots for biomolecules contained in cells, such as inorganic salts, amino acids, and other proteins or enzymes, a fluorescent probe (10 μg / mL) was added to a PBS buffer solution (0.01 M, pH 7.4), and then common biomolecules in cells, 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), liposome (Lipsome), H2O2, ClO - 、ONOO - 、 1 O2、O2 - were added respectively, and the fluorescence changes after their reaction with the fluorescent carbon dots were measured. Please refer to Figure 10 and it can be seen from Figure 10 that the fluorescent carbon dots do not respond to other biomolecules contained in cells, but respond to superoxide anions, glycerol, and liposomes. The responses to proteins and liposomes are mainly due to viscosity.

[0059] Study on the response of fluorescent carbon dots to superoxide anions:

[0060] To test the response of the fluorescent carbon dots to superoxide anions, potassium peroxide DMSO solutions with different concentrations were prepared. A fluorescent probe (10 μg / mL) was added to a PBS buffer solution (0.01 M, pH 7.4), and then potassium peroxide solutions with different concentrations were added respectively. The fluorescence spectra were measured under an excitation light of 470 nm to observe the sensitivity of Y-CDs to detect superoxide anions. Please refer to Figure 11 and it can be seen from Figure 11 that as the concentration of superoxide anions increases, the fluorescence intensity decreases significantly, indicating that Y-CDs are sensitive to superoxide anions.

[0061] Study on the response of fluorescent carbon dots to viscosity:

[0062] To test the response of the fluorescent carbon dots to viscosity, different viscosity solutions were first prepared by different ratios of glycerol and water. A fluorescent probe (10 μg / mL) was added to the solutions with different viscosities, shaken well, and the changes in fluorescence intensity were studied. Please refer to Figure 12 and it can be seen from Figure 12It can be seen that with the increase in the viscosity of the solution, the fluorescence intensity of the fluorescent carbon dots changes significantly, indicating that the fluorescent carbon dots are sensitive to viscosity.

[0063] Changes in the fluorescence intensity of fluorescent carbon dots at different pH values:

[0064] For the study of the change in 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 a concentration of 10 μg / mL, and the change in their fluorescence intensity was studied. Please refer to Figure 13 , from Figure 13 it can be seen that the change in pH does not affect the fluorescence intensity of the probe, indicating that the probe remains stable in strong alkaline and strong acidic environments.

[0065] Cytotoxicity analysis:

[0066] Human hepatoma cells (HepG2) were seeded into a 96-well plate at a density of 1×104 cells per well. The 96-well plate was placed in a cell incubator with the culture conditions of 37 °C, 5% CO2, and saturated humidity for 24 h to allow the cells to adhere completely. Then, the fresh culture medium was replaced, and 10 μL of fluorescent carbon dot dispersion with different concentrations was added. After culturing for 24 h, MTT solution (5 mg / mL) was added to each well and incubated for another 4 h. Then, 100 μL of DMSO was added to each well. The 96-well plate was placed on a horizontal shaking incubator and shaken for 10 min. The absorbance (OD value) of the solution in each well of the 96-well plate was measured at a wavelength of 492 nm on an enzyme-linked immunosorbent assay (ELISA) reader. The cell survival rate was calculated according to the following formula: Cell survival rate = (OD of the test group - OD of the blank group) / (OD of the cell group - OD of the blank group) × 100%. Please refer to Figure 14 , from Figure 14 it can be seen that the fluorescent carbon dots have almost no cytotoxicity in the range of 1 - 30 μg / mL.

[0067] Real-time imaging of the process of fluorescent carbon dots entering cells:

[0068] Hepatoma cells (HepG2) were seeded into a glass-bottom culture dish and cultured in an environment of 37 °C for 2 days. Subsequently, the probe (10 μg / mL) was added to the cells and incubated at 37 °C for 20 min, and then imaged with a laser confocal microscope. The results are as Figure 15 shown. The fluorescent carbon dots can enter the cells within 5 minutes, and the fluorescence intensity reaches the highest after 20 minutes.

[0069] Intracellular co-localization imaging:

[0070] In the co-localization experiment, first, hepatoma cells (HepG2) were co-incubated with fluorescent carbon dots (10 μg / mL) for 20 min, and then incubated with 1 μM endoplasmic reticulum commercial staining reagent for 10 min. Finally, the cells were washed twice with PBS solution (pH 7.4) and imaged with a laser confocal microscope. The results are as Figure 16 shown. The fluorescence carbon dot Y-CDs channel perfectly overlaps with the endoplasmic reticulum commercial staining channel, demonstrating its localization in the endoplasmic reticulum organelle. The fluorescence carbon dot Y-CDs channel perfectly overlaps with the lipid droplet commercial staining channel, demonstrating its localization in lipid droplets. Therefore, this probe is a carbon dot that localizes in the endoplasmic reticulum and lipid droplets.

[0071] Real-time imaging of endoplasmic reticulum stress in cells:

[0072] In the experiment of endoplasmic reticulum stress in cells, first, hepatoma cells (HepG2) were co-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 endoplasmic reticulum stress in the cells and the endoplasmic reticulum stress was observed with a fluorescence lifetime imaging system. The results are as Figure 17 shown. The Bar value was uniformly set to 0 - 15. As the endoplasmic reticulum oxidative stress gradually increased, the fluorescence lifetime of the endoplasmic reticulum decreased (from 13.34 ns to 11.09 ns), while the fluorescence lifetime of lipid droplets increased (from 8.96 ns to 14.38 ns). The change in fluorescence lifetime was obvious, so this probe is sensitive to the response of superoxide anions and viscosity.

[0073] In vivo imaging of zebrafish liver injury model:

[0074] In the experiment of zebrafish liver injury model, first, a zebrafish liver injury model was established: Zebrafish embryos were maintained in a 6-well plate containing fish medium (3.0 ml methylene 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 of fish medium was added to each well. The zebrafish embryos were divided into 2 groups, named blank control group (CON group) and model group (thioacetamide TAA group 0.4 mg / ml) respectively. All incubations were carried out under a 14-hour light / 10-hour dark cycle and a constant temperature of 28 °C. During the chemical treatment, the incubation solution of zebrafish larvae was renewed daily. From 6 dpf to 9 dpf, the larvae were fed with microcapsule feed for zebrafish larvae. Zebrafish in the blank control group and the model group were taken out and incubated in a medium containing 60 μg / mL fluorescent carbon dots for 3 - 4 h, and then the fish fry were washed three times with normal medium. The zebrafish were anesthetized by hypothermic anesthesia, placed on a glass slide and imaged in vivo with a fluorescence lifetime imaging system. The results are as Figure 18As shown, the Bar value is uniformly set. In zebrafish nine days after fertilization, the fluorescence lifetime of the liver tissue in the liver tissue imaging of the blank control group with fluorescent carbon dots is 14.254 ns, while the lifetime of the liver tissue of zebrafish in the liver tissue injury model group is 8.961 ns. In the liver injury model, the fluorescence lifetime of the fluorescent carbon dots decreases significantly. Therefore, this probe has an indicative effect on the liver tissue injury situation.

[0075] In summary, the fluorescent carbon dots prepared in this example can be used in the field of in vivo bioimaging, and it has a high quantum yield, above 22.05%. At the same time, it can be selectively localized in the endoplasmic reticulum and lipid droplets of cells and can be distinguished by fluorescence lifetime imaging, and it can sensitively detect the content of superoxide anions in the endoplasmic reticulum and the viscosity change of lipid droplets when oxidative stress occurs in the endoplasmic reticulum. It can be used for live cell imaging and real-time monitoring of the association between endoplasmic reticulum stress and lipid metabolism, and can be used for the monitoring of zebrafish liver tissue injury, greatly improving its application in the biological field and not causing radiation damage to biological samples. In addition, the fluorescent carbon dots prepared in this example have high photostability and good biocompatibility, overcoming the problem of the lack of lifetime-sensitive dual-target fluorescent carbon dots that can simultaneously localize to the endoplasmic reticulum and lipid droplets and sensitively monitor the changes in their microenvironment.

[0076] From the above description, it can be seen that the dual-target fluorescent carbon dots prepared by the preparation method of the dual-target fluorescent carbon dots in this example can be used for imaging and monitoring of the endoplasmic reticulum and lipid droplets. Therefore, in this example, the application of the above dual-target fluorescent carbon dots for the endoplasmic reticulum and lipid droplets in cell monitoring is also provided, and this application includes real-time imaging of endoplasmic reticulum oxidative stress and lipid metabolism in cells.

[0077] It should be understood that the specific embodiments described here are only used to explain this application, rather than to limit it. According to the embodiments provided in this application, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the protection scope of this application.

[0078] Obviously, the drawings are only some examples or embodiments of this application. For those of ordinary skill in the art, this application can also be applied to other similar situations based on these drawings without creative work. In addition, it can be understood that although the work done during the development process here may be complex and time-consuming, for those of ordinary skill in the art, some design, manufacturing or production changes based on the technical content disclosed in this application are only conventional technical means and should not be regarded as insufficient disclosure of 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 performing column chromatography purification, rotary evaporation and drying on the precipitate to obtain a target solid, wherein the mass ratio of the o-phenylenediamine, the phenylalanine and the pure water is 1:2:190-1:2:210, the pure water accounts for 3:5 of the volume of the reactor, and the temperature of the first heating reaction is 160-200° C. and the time is 6-10 hours; Dissolving caffeic acid in dichloromethane and adding CDI to the mixture for stirring to obtain a second solution, wherein the mass ratio of the CDI to the caffeic acid is 1:1-1:2; The target solid is added to the second dissolving solution for a second heating reaction to obtain the dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets. 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.

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:

200.

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 was 180° C. and the time was 8 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 carried out in an ice bath and the stirring time is 30-60 min.

6. 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 5.

7. The dual-targeting fluorescent carbon dots for endoplasmic reticulum and lipid droplets according to claim 6, 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.

8. Use of the dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets according to claim 6 or 7 in cell monitoring, wherein the application is non-disease diagnosis or treatment.

9. The use of dual-targeted fluorescent carbon dots for endoplasmic reticulum and lipid droplets in cell monitoring according to claim 8, characterized in that: Includes real-time imaging of cellular endoplasmic reticulum oxidative stress and lipid metabolism.

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