Preparation method of red fluorescent carbon dots with lysosome and nucleolus dual-targeting capability

Abstract

The invention discloses a preparation method of red fluorescent carbon dots with lysosome and nucleolus dual-targeting capability in the technical field of nano material synthesis and biological imaging analysis. According to the preparation method, o-phenylenediamine is taken as a carbon source, methanol is taken as a solvent in the presence of rare earth ions, and the red fluorescent carbon dots can be obtained through a solvothermal method reaction in a reaction kettle. The synthesis process is simple, the raw materials are cheap and easy to repeat, and the absolute quantum yield of the purified red fluorescent carbon dots is 11.45%, so that the luminous efficiency is high, and the technical problem that the absolute quantum yield of the red fluorescent carbon dots in the existing bioimaging field is low is solved. The prepared red fluorescent carbon dots have non-exciting light dependence, and the emission wavelength can be extended to 800 nm; the size is small, the particle size distribution is 2.5-5.5 nm, and the average particle size is about 3.75 nm; the oil-water partition coefficient (Log P value) is 0.3-0.4, the water solubility is good, the lysosome and nucleolus targeting specificity is achieved, the photobleaching resistance is high, the biocompatibility is good, and the cytotoxicity is low.

Description

Technical Field

[0001] The present application relates to the technical field of nanomaterial synthesis and biological imaging analysis, and specifically to a method for preparing red fluorescent carbon dots with dual targeting capabilities of lysosomes and nucleoli, red fluorescent carbon dots prepared by the preparation method, and an analyzer for co-localization imaging of the red fluorescent carbon dots in cells. Background Art

[0002] Carbon dots have shown great application prospects in cell imaging and targeted therapy due to their excellent optical properties, biocompatibility and low toxicity. Although the synthesis of carbon dots has been rapidly developed, the fluorescence emission of synthesized carbon dots is mainly concentrated in the blue and green spectral ranges, and there are few reports on infrared or near-infrared (NIR) emitting carbon dots with little photodamage to biological tissues and strong penetration. Moreover, compared with the high fluorescence quantum yield of carbon dots in the blue-green spectral region (which can reach more than 90%), the current synthesis of red light carbon dots still faces problems such as complex synthesis process, low yield, and low fluorescence quantum yield.

[0003] Lysosomes and nucleoli play an important role in the biological functions of cells. Lysosomes are involved in the degradation and recycling of substances in cells, while nucleoli are closely related to protein synthesis and RNA processing of cells. Real-time monitoring of the interaction and dynamic association between the two is of great significance for the early diagnosis, targeted treatment and drug delivery of related diseases. At present, the simultaneous visualization of the two requires the use of lysosomal commercial dyes and nucleolar dyes to mark the two separately. This multi-probe combination technology faces problems such as probe uptake efficiency, asynchronous biological localization, high cytotoxicity, and photobleaching. Therefore, it is urgent to develop high quantum yield red light carbon dots with dual targeting capabilities of lysosomes and nucleoli to achieve simultaneous imaging and functional regulation of the two, and provide powerful tools for the early diagnosis, targeted treatment and drug delivery of related diseases. Summary of the invention

[0004] In order to solve the technical problem of low quantum yield of red fluorescent carbon dots in the existing biological imaging field, the present invention provides a method for preparing red fluorescent carbon dots with lysosome and nucleolus dual targeting capabilities, red fluorescent carbon dots prepared by the preparation method, and an analyzer for co-localization imaging in cells using the red fluorescent carbon dots.

[0005] The present invention is implemented by the following technical scheme: a method for preparing red fluorescent carbon dots with dual targeting capabilities of lysosomes and nucleoli, using o-phenylenediamine as a carbon source, in the presence of rare earth ions, and methanol as a solvent, to obtain the red fluorescent carbon dots through a solvothermal reaction in a reactor.

[0006] As a further improvement of the above scheme, o-phenylenediamine and hexahydrate rare earth chloride solid are added to methanol, ultrasonically dissolved and transferred to a polytetrafluoroethylene lined reactor, a high temperature and high pressure reaction is carried out in the reactor, and the supernatant in the reactor is taken out after the reaction is completed;

[0007] The supernatant is purified, rotary evaporated, and dried to obtain a solid, namely the red fluorescent carbon dots.

[0008] As a further improvement of the above scheme, the molar ratio of o-phenylenediamine to hexahydrated rare earth chloride solid is 5:1.

[0009] As a further improvement of the above scheme, the amount of methanol used is 1 / 2-2 / 3 of the volume of the reactor.

[0010] As a further improvement of the above solution, the reactor is a polytetrafluoroethylene stainless steel reactor.

[0011] As a further improvement of the above scheme, the hexahydrated rare earth chloride solid includes GdCl 3 6H 2 O,EuCl 3 6H 2 O, ErCl 3 6H 2 At least one of O.

[0012] As a further improvement of the above scheme, the reaction temperature adopted in the solvothermal method is 200°C and the reaction time is 5-12h.

[0013] Furthermore, the supernatant is purified by column chromatography.

[0014] Furthermore, the eluent during column chromatography purification is a mixture of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 =80:1).

[0015] The present invention also provides a red fluorescent carbon dot with lysosome and nucleolus dual targeting capability, which is prepared by using any of the above-mentioned methods for preparing red fluorescent carbon dots with lysosome and nucleolus dual targeting capability.

[0016] As a further improvement of the above scheme, the red fluorescent carbon dots have non-excitation light-dependent fluorescence emission, with optimal excitation wavelengths at 580 and 630 nm, a maximum emission wavelength at 660 nm, and an emission wavelength extending to 800 nm.

[0017] As a further improvement of the above scheme, the particle size distribution of the red fluorescent carbon dots is 2.5-5.5 nm, and the average particle size is about 3.75 nm.

[0018] The present invention also provides an analyzer for co-localization imaging in cells, which uses any of the above-mentioned red fluorescent carbon dots with dual targeting capabilities of lysosomes and nucleoli to synchronously label lysosomes and nucleoli of living cells to achieve real-time imaging of lysosomes and nucleoli.

[0019] Compared with the prior art, the present invention has the following technical effects:

[0020] (1) The preparation method of the red fluorescent carbon dots with dual targeting capabilities of lysosomes and nucleoli of the present invention uses o-phenylenediamine as a carbon source, in the presence of rare earth ions, and methanol as a solvent, and the red fluorescent carbon dots can be obtained by solvothermal reaction in a reactor. The synthesis process is simple, the raw materials are cheap, and it is easy to repeat. Moreover, the absolute quantum yield of the purified red fluorescent carbon dots is 11.45%, so the red light carbon dots have high luminous efficiency. Therefore, the absolute quantum yield of the red fluorescent carbon dots synthesized by the present invention is greatly improved, which conveniently solves the technical problem of low absolute quantum yield of red fluorescent carbon dots in the existing biological imaging field.

[0021] (2) The red fluorescent carbon dots prepared by this method can also be independent of excitation light, with the best excitation wavelengths being 580 and 630 nm, the maximum emission wavelength being 660 nm, and the emission wavelength being extendable to 800 nm, thus having the characteristics of long-wavelength emission.

[0022] (3) The particle size distribution of the red fluorescent carbon dots prepared by this method is 2.5-5.5 nm, and the average particle size is about 3.75 nm, which is relatively small.

[0023] (4) The red fluorescent carbon dots prepared by this method have an oil-water partition coefficient (Log P value) between 0.3 and 0.4, good water solubility, lysosome and nucleolus targeting specificity, strong anti-photobleaching performance, good biocompatibility, and low cytotoxicity.

[0024] (5) The red fluorescent carbon dots prepared by the preparation method of the present invention have lysosome and nucleolus targeting specificity, which overcomes the problems of photobleaching of current dye probes and cytotoxicity of multiple probes, and are more suitable for synchronous real-time imaging of lysosomes and nucleoli in living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the synthesis of red fluorescent carbon dots provided in Example 1 of the present invention.

[0026] Figure 2 It is a schematic diagram of the FT-IR results of red fluorescent carbon dots prepared by the preparation method provided in Example 2 of the present invention.

[0027] Figure 3It is a schematic diagram of the UV-visible spectrum and excitation emission spectrum of the red fluorescent carbon dots prepared by the preparation method provided in Example 2 of the present invention.

[0028] Figure 4 It is a schematic diagram of the fluorescence emission spectrum of red fluorescent carbon dots prepared by the preparation method provided in Example 2 of the present invention under different excitation lights.

[0029] Figure 5 It is a schematic diagram of the change in fluorescence intensity of red fluorescent carbon dots prepared by the preparation method provided in Example 2 of the present invention under different ultraviolet irradiation times.

[0030] Figure 6 This is a schematic diagram of a TEM image of red fluorescent carbon dots prepared by the preparation method provided in Example 2 of the present invention.

[0031] Figure 7 It is a schematic diagram of the size distribution results of red fluorescent carbon dots prepared by the preparation method provided in Example 2 of the present invention.

[0032] Figure 8 It is a schematic diagram of XPS spectrum analysis of red fluorescent carbon dots prepared by the preparation method provided in Example 2 of the present invention.

[0033] Fig. 9 It is a schematic diagram of the Log P diagram analysis of red fluorescent carbon dots prepared by the preparation method provided in Example 2 of the present invention.

[0034] Fig.10 It is a schematic diagram of the selective analysis of various analytical substrates contained in cells by the red fluorescent carbon dots prepared by the preparation method provided in Example 2 of the present invention.

[0035] Fig.11 Schematic diagram of the selective analysis of pH by the red fluorescent carbon dots prepared in Examples 1 and 2.

[0036] Fig.12 Schematic diagram of cytotoxicity analysis of red fluorescent carbon dots prepared in Examples 1 and 2.

[0037] Fig.13 Schematic diagram of real-time imaging of red fluorescent carbon dots prepared in Examples 1 and 2 entering cells.

[0038] Fig.14 Schematic diagram of co-localization imaging analysis of red fluorescent carbon dots prepared in Examples 1 and 2 in cells. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] The red fluorescent carbon dots prepared by the preparation method of the red fluorescent carbon dots of the present invention have the dual targeting capability of lysosome and nucleolus. The preparation method of the red fluorescent carbon dots with the dual targeting capability of lysosome and nucleolus is to use o-phenylenediamine as a carbon source, methanol as a solvent in the presence of rare earth ions, and obtain the red fluorescent carbon dots by solvothermal reaction in a reactor. For example, o-phenylenediamine and hexahydrated rare earth chloride solid can be added to methanol, ultrasonically dissolved and then transferred to a polytetrafluoroethylene lined reactor, a high temperature and high pressure reaction is carried out in the reactor, and the supernatant in the reactor is taken out after the reaction is completed; the supernatant is purified, rotary evaporated, and dried to obtain a solid, namely the red fluorescent carbon dots.

[0041] The red fluorescent carbon dots can be obtained by solvothermal reaction in a reactor using o-phenylenediamine as a carbon source, in the presence of rare earth ions and methanol as a solvent. The synthesis process is simple, the raw materials are cheap, and it is easy to repeat. Moreover, the absolute quantum yield of the purified red fluorescent carbon dots is 11.45%, so the red light carbon dots have high luminous efficiency. Therefore, the absolute quantum yield of the red fluorescent carbon dots synthesized by the present invention is greatly improved, which solves the technical problem of low absolute quantum yield of red fluorescent carbon dots in the existing biological imaging field.

[0042] Example 1

[0043] This example is one example of the preparation method of the present invention. Figure 1 , Figure 1 The schematic diagram of the synthesis of red fluorescent carbon dots (R-CDs) is shown in FIG. The preparation method of R-CDs comprises the following steps:

[0044] (1) o-phenylenediamine and gadolinium chloride hexahydrate are added to methanol, dissolved by ultrasonication, and then transferred to a polytetrafluoroethylene-lined reactor, where a high-temperature and high-pressure reaction is carried out. After the reaction is completed, the supernatant in the reactor is taken out.

[0045] The molar ratio of o-phenylenediamine to gadolinium chloride hexahydrate is 5:1, and the amount of methanol used is 1 / 2-1 / 3 of the solvent in the reactor. When the high temperature and high pressure reaction is carried out in the reactor, the reaction temperature is preferably 200° C., and the reaction time is preferably 12 hours.

[0046] (2) The supernatant obtained in step (1) is purified by column chromatography, rotary evaporated, and dried to obtain a blue solid powder, i.e., the red fluorescent probe.

[0047] The eluent during column chromatography purification is a mixture of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 =80:1).

[0048] O-phenylenediamine is a known material that can be condensed, polymerized, and carbonized into carbon dots, while gadolinium chloride is used as a catalyst to expand the effective aromatic sp 2 The conjugated structure is favorable for red emission.

[0049] The red fluorescent carbon dots (R-CDs) prepared by the preparation method of the present invention are non-excitation light-dependent red fluorescent carbon dots with high fluorescence quantum yield, which can be used as fluorescent probes for real-time imaging of lysosomes and nucleoli in living cells. The particle size of the red fluorescent carbon dots is distributed in the range of 2.5-5.5 nm, and the mass contents of C, N, and O in the red fluorescent carbon dots are 87%-88%, 3%-4%, and 9%-10%, respectively. The size of the red fluorescent carbon dots is relatively small, with an average particle size of 3.75 nm.

[0050] The red fluorescent carbon dots (R-CDs) are prepared by solvothermal reaction in a polytetrafluoroethylene stainless steel reactor using o-phenylenediamine and gadolinium chloride hexahydrate as raw materials and methanol as solvent. The specific preparation process is shown in Example 2.

[0051] The red fluorescent carbon dots (R-CDs) of the present invention have an oil-water partition coefficient (Log P value) between 0.3 and 0.4, good water solubility, lysosome and nucleolus targeting specificity, strong photobleaching resistance, and are substantially non-toxic to cells. The red fluorescent carbon dots (R-CDs) have the characteristics of high absolute quantum yield, good photobleaching resistance, and good biocompatibility. Therefore, the red fluorescent carbon dots of the present invention are particularly suitable for real-time imaging of living cells.

[0052] Example 2

[0053] This embodiment is another example of the preparation method of the present invention.

[0054] The preparation method of R-CDs is as follows: o-phenylenediamine and gadolinium chloride hexahydrate solid are added to methanol, and the mixture is ultrasonicated for 10 minutes. After the o-phenylenediamine and gadolinium chloride hexahydrate are completely dissolved, the mixture is transferred to a polytetrafluoroethylene-lined reactor with a volume of 50 mL, and the mixture is reacted at 200° C. for 12 hours, and then naturally cooled to room temperature. The supernatant in the reactor is purified by column chromatography, and the eluent is a mixture of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 =80:1), rotary evaporation, and drying to obtain a blue solid powder, namely the red fluorescent carbon dots.

[0055] The red fluorescent carbon dots prepared in this example are characterized as follows: Figure 2 As shown, the surface functional groups of the synthesized R-CDs were determined by FTIR spectroscopy. -1 The absorption bands nearby are attributed to -OH / -NH on its surface, 2921-2852 cm -1The absorption band attributed to C-H bonds was observed at 1635 cm -1 Corresponding to the C=O / C=N absorption band, 1300-1000cm -1 The absorption band of is attributed to CO bond. The optical properties of the synthesized R-CDs were confirmed by UV-Vis absorption and fluorescence spectra.

[0056] See also Figure 3 , Figure 3 The absorption band from 400 nm to 660 nm, as shown by the dashed line, can be attributed to sp 2 The n→π* transition produced by the C=O / C=N bond at the edge of the hybrid system; as shown by the solid line, when the optimal excitation wavelength is 630nm, the corresponding maximum emission peak is 660nm. Figure 3 In the photograph depicted in the inset, the blue methanol solution appears deep red under (365nm) ultraviolet light.

[0057] See also Figure 4 and Figure 5 ,exist Figure 4 In the figure, the emission wavelength of the synthesized R-CDs hardly changes as the excitation wavelength increases from 410 nm to 670 nm. Figure 5 The fluorescence intensity of the carbon dots after continuous irradiation with ultraviolet light (365nm) for 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min and 60min respectively showed no obvious decrease, which indicated that R-CDs had excellent photostability.

[0058] See also Figure 6 and Figure 7 ,from Figure 6 and 7 It can be seen that the shape of the synthesized R-CDs is approximately spherical, and the size distribution results of R-CDs show that the diameter of R-CDs is distributed in the range of 2.5-5.5nm, with an average diameter of about 3.75nm, and the imaging is uniformly dispersed in the solvent.

[0059] See also Figure 8It can be seen that the R-CDs sample mainly contains three elements: C, N and O, with contents of 87%-88%, 3%-4% and 9%-10% (atomic ratio), respectively. The high-resolution XPS spectrum of C1s can fit three characteristic peaks of 284.8eV (CC), 286.1eV (COC) and 288.2eV (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 two characteristic peaks of 532eV (C=O) and 533.4eV (COC).

[0060] See also Fig. 9 It can be seen that the oil-water partition coefficient (Log P value) of R-CDs is between 0.3 and 0.4, and the water solubility is good.

[0061] The present invention has conducted various investigations on the red fluorescent carbon dots of the above-mentioned embodiments.

[0062] 1. Selective study of red fluorescent carbon dots.

[0063] To test the selectivity of red fluorescent carbon dots to intracellular biomolecules such as inorganic salts, amino acids, and other proteins or enzymes, fluorescent probes (4 μg / mL) were added to PBS buffer solution (0.01 M, pH 7.4), and then common intracellular biomolecules such as ferrous ions, iron ions, magnesium ions, copper ions, zinc ions, potassium ions, calcium ions, carbonate ions, sodium chloride, glutathione (GSH), cysteine ​​(Cys), leucine (Leu), arginine (Arg), threonine (Thr), glycine (Gly), glutamic acid (Glu), aspartic acid (Asp), bovine serum albumin (BSA), human serum albumin (HSA), and pepsin (PG) were added, and the fluorescence changes after the reaction with red fluorescent carbon dots were measured. Please refer to Fig.10 ,Depend on Fig.10 It can be seen that the red fluorescent carbon dots do not respond to other biological molecules contained in the cells, but respond to glutamate.

[0064] 2. Changes in fluorescence intensity of red fluorescent carbon dots at different pH values.

[0065] To test the change of fluorescence intensity of red fluorescent carbon dots under different pH conditions, red fluorescent carbon dots were added to 1 ml PBS buffer (0.01M, pH 7.4) to make the concentration 10 μg / mL, and the change of fluorescence intensity was studied. Fig.11 ,Depend on Fig.11 It can be seen that as the pH decreases, the fluorescence intensity at 660nm gradually increases. When the pH is 4, the increase of the red fluorescent carbon dots can reach 10 times, indicating that the fluorescence intensity of the carbon dots is higher in an acidic environment.

[0066] 3. Cytotoxicity analysis.

[0067] Human cervical cancer cells (HeLa) were plated at 1×10 4 The cells were inoculated into a 96-well plate and placed in a cell culture incubator at 37°C and 5% CO 2 , saturated humidity, culture for 24 hours to allow complete attachment. After that, fresh culture medium was replaced, and 10 μL of red fluorescent carbon dot dispersion of different concentrations was added. After culturing for 24 hours, MTT solution (5 mg / mL) was added to each well, and incubation continued for 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 each well of the 96-well plate was measured. The cell survival rate was calculated according to the following formula: Cell survival rate = (OD 待测组 -OD 空白组 ) / (OD 细胞组 -OD 空白组 ) × 100%. Please refer to Fig.12 ,from Fig.12 As can be seen from the figure, the red fluorescent carbon dots have almost no cytotoxicity in the range of 1-20 μg / mL.

[0068] 4. Real-time imaging of the process of red fluorescent carbon dots entering cells.

[0069] Human cervical cancer cells (HeLa) were seeded into glass-bottomed culture dishes and cultured at 37°C for 2 days. Subsequently, the probe (5 μg / mL) was added to the cells and imaged using a laser confocal microscope at 37°C. Fig.13 As shown, the red fluorescent carbon dots began to enter the cells at 30 seconds, and the fluorescence intensity reached the highest after 20 minutes.

[0070] 5. Intracellular co-localization imaging.

[0071] An analyzer using red fluorescent carbon dots with dual targeting capabilities for lysosomes and nucleoli (i.e., an analyzer for co-localization imaging in cells) can be used to synchronously label lysosomes and nucleoli in living cells, thereby achieving real-time imaging of lysosomes and nucleoli.

[0072] In the colocalization experiment, human cervical cancer cells (HeLa) were first incubated with red fluorescent carbon dots (5 μg / mL) for 30 min, and then incubated with 1 μM lysosomal 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.14As shown in the figure, the colocalization coefficient of the red fluorescent carbon dot R-CDs channel and the lysosome commercial stain (LysoSensor) channel is 85%, proving that it is localized in the lysosome organelle; in addition, it can be clearly observed in the cell image that the red fluorescent carbon dot can also be localized to the nucleolus. Therefore, the probe is a dual-localization probe that is localized in the lysosome and the nucleolus.

[0073] In summary, the red fluorescent carbon dots prepared in this application can be used in the field of biological imaging, and they have a very high absolute quantum yield of more than 11.45%. At the same time, they can be selectively located in cell lysosomes and nucleoli, and can be used for living cell imaging, greatly improving their application in the biological field, and will not cause radiation damage to biological samples. In addition, the red fluorescent carbon dots prepared in this application have high photostability and good biocompatibility, overcoming the current problems of photobleaching of dye probes and cytotoxicity of multiple probes.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing red fluorescent carbon dots with dual targeting capabilities for lysosomes and nucleoli, characterized in that: The red fluorescent carbon dots are obtained by using o-phenylenediamine as a carbon source, methanol as a solvent and a solvothermal reaction in a reaction vessel in the presence of rare earth ions.

2. The method for preparing red fluorescent carbon dots with lysosome and nucleolus dual targeting capabilities according to claim 1, characterized in that: o-phenylenediamine and hexahydrate rare earth chloride solid are added to methanol, and after ultrasonic dissolution, they are transferred to a polytetrafluoroethylene lined reactor, and heated to react in the reactor. After the reaction is completed, the supernatant in the reactor is taken out; The supernatant is purified, rotary evaporated, and dried to obtain a solid, namely the red fluorescent carbon dots.

3. The method for preparing red fluorescent carbon dots with lysosome and nucleolus dual targeting capabilities according to claim 1, characterized in that: The molar ratio of o-phenylenediamine to hexahydrated rare earth chloride solid is 5:

1.

4. The method for preparing red fluorescent carbon dots with lysosome and nucleolus dual targeting capabilities according to claim 1, characterized in that: The amount of methanol used is 1 / 2-2 / 3 of the volume of the reactor.

5. The method for preparing red fluorescent carbon dots with lysosome and nucleolus dual targeting capabilities according to claim 1, characterized in that: The reactor is a polytetrafluoroethylene stainless steel reactor; And / or, the hexahydrated rare earth chloride solid includes at least one of GdCl3·6H2O, EuCl3·6H2O, and ErCl3·6H2O; And / or, the reaction temperature used in the solvothermal method is 200° C. and the reaction time is 5-12 h.

6. The method for preparing red fluorescent carbon dots with lysosome and nucleolus dual targeting capabilities according to claim 2, characterized in that: The supernatant was purified by column chromatography.

7. The method for preparing red fluorescent carbon dots with lysosome and nucleolus dual targeting capabilities according to claim 6, characterized in that: The eluent during column chromatography purification is a mixture of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 =80:1).

8. A red fluorescent carbon dot with dual targeting capabilities for lysosomes and nucleoli, characterized in that: The method is used to prepare the lysosome-nucleolus dual-targeted red fluorescent carbon dots as claimed in any one of claims 1 to 7.

9. The red fluorescent carbon dots with dual targeting capabilities to lysosomes and nucleoli according to claim 8, characterized in that: The red fluorescent carbon dots have fluorescence emission that is not dependent on excitation light, with the best excitation wavelengths at 580 and 630 nm, the maximum emission wavelength at 660 nm, and the emission wavelength extending to 800 nm; And / or, the particle size distribution of the red fluorescent carbon dots is 2.5-5.5 nm, and the average particle size is about 3.75 nm.

10. An analyzer for co-localization imaging in cells, characterized in that: It uses the red fluorescent carbon dots with dual targeting capabilities of lysosomes and nucleoli as described in claim 8 to synchronously label the lysosomes and nucleoli of living cells to achieve real-time imaging of lysosomes and nucleoli.

Images