Pyrrole-modified graphite-phase carbon nitride nanoparticles, preparation method, and application in biological imaging

Abstract

A pyrrole-modified graphite-phase carbon nitride nanoparticle, a preparation method, and its application in biological imaging belong to the technical field of fluorescence imaging of two-dimensional semiconductor materials. The present invention first obtains graphite-phase carbon nitride by a calcination method, then obtains a pyrrole-modified graphite-phase carbon nitride material by pyrrole exfoliation, and finally prepares PSMA-coated pyrrole-modified graphite-phase carbon nitride nanoparticles by a reprecipitation method. The nanoparticles of the present invention have excellent absorption and emission properties and good fluorescence scintillation characteristics. The scintillation characteristics provide potential possibilities for the application of the nanoparticles in super-resolution imaging; the nanoparticles have a stable chemical structure, excellent biosafety and biomaterial compatibility, and can further modify biomolecules to achieve targeted binding to specific biological structures; after the nanoparticles are used to mark cell structures, microscopic imaging is performed using a fluorescence imaging system to achieve related applications in biological imaging.

Description

Technical Field

[0001] The present invention belongs to the technical field of two-dimensional semiconductor material fluorescence imaging, and specifically relates to pyrrole-modified graphite-phase carbon nitride nanoparticles, a preparation method and applications thereof in biological imaging. Background Art

[0002] With the development of bioluminescence technology, researchers are increasingly in need of fluorescent probes with excellent biosafety, high stability, and high signal intensity. Conventional fluorescent materials are often limited by potential biotoxicity, easy photobleaching, low fluorescence intensity, and inability to be stably dispersed in biological systems. At the same time, these materials have limited support for new super-resolution imaging technologies. The new two-dimensional material system provides new opportunities for related applications. Its high chemical stability eliminates the possibility of biotoxicity and photochemical reactions. The unique electronic structure of two-dimensional materials gives them rich and adjustable optoelectronic properties. However, this type of material has always lacked material refinement methods, which makes the material itself large in size, making it difficult to be widely used in micro-nano biological systems.

[0003] Carbon nitride material is a widely used material among two-dimensional materials. This material has very excellent electronic structure properties and a wide band gap (2.7eV). In addition to being widely used in photocatalytic hydrogen and oxygen production systems, carbon nitride materials also have good fluorescence emission characteristics. However, this material is difficult to peel and refine due to its strong two-dimensional intra-plane covalent interaction, and the weak interaction between the surfaces further exacerbates the above problems. Although it has good fluorescence emission properties, it is not suitable for application in cell imaging due to its large size, so preparing small-sized carbon nitride is a feasible solution. In the past, the refinement and modification of carbon nitride materials were all methods of high-temperature treatment with strong oxidants or strong acids and strong bases. This method is not only dangerous and costly, but may also bring unnecessary toxic and side effects when used in biological related fields. At present, there are no reports on the synthesis and application of pyrrole-modified carbon nitride materials. Therefore, it is very meaningful that the present invention synthesizes a new modified carbon nitride material through a safe experimental process and biosafety method. Summary of the Invention

[0004] The present invention aims to provide pyrrole-modified graphite-phase carbon nitride nanoparticles, a preparation method, and applications thereof in bioimaging. The pyrrole-modified graphite-phase carbon nitride nanoparticles can be used as probes in conventional bioluminescence imaging and super-resolution bioimaging.

[0005] The present invention uses pyrrole as a stripping agent to strip the graphite phase carbon nitride material. After the stripping is completed, pyrrole is directly modified on the surface of the graphite phase carbon nitride as a stabilizer. The preparation method of the graphite phase carbon nitride is to adopt a high-temperature calcination method to obtain a graphite phase carbon nitride material with higher purity. The graphite phase carbon nitride material is stripped using a new room temperature treatment process. By using pyrrole as a stripping agent, the graphite phase carbon nitride material is refined, and the size of the obtained carbon nitride material can reach the nanometer level. The stripped graphite phase carbon nitride material will remove the unreacted pyrrole by vacuum distillation, and the collected product will then be dissolved in an organic solvent such as tetrahydrofuran for long-term storage. The material described in the invention can also be further treated by methods such as reprecipitation to form stable nanoparticles in water.

[0006] The pyrrole-modified graphite-phase carbon nitride nanoparticles of the present invention have multiple advantages: first, the preparation process of the material is simple. High-purity graphite-phase carbon nitride can be obtained by only one high-temperature treatment. Pyrrole exfoliation of the graphite-phase carbon nitride material does not require high temperature, high pressure or strong acid or strong base treatment. Secondly, the material obtained after this exfoliation has a unique affinity for organic solvents and can be dissolved in various organic solvents and prepared into a precursor solution for storage or further subjected to other synthesis and material composite operations. For example, through the reprecipitation method, it can be prepared into nanoparticles that have biological environmental solubility stability, biosafety and biomodifiability. Finally, the prepared nanoparticles have higher quantum efficiency in fluorescence properties than conventional carbon nitride materials. At the same time, the pyrrole-modified graphite-phase carbon nitride nanoparticles also have fluorescence scintillation characteristics. This scintillation characteristic gives it a unique application advantage in super-resolution imaging systems.

[0007] The pyrrole-modified graphite-phase carbon nitride nanoparticles prepared by the present invention can achieve the following functions:

[0008] 1) This material has excellent absorption and emission properties, emitting blue fluorescence under excitation by commonly used short-wavelength lasers. Compared to conventional unmodified carbon nitride materials, its fluorescence intensity is higher. Furthermore, these pyrrole-modified graphite-phase carbon nitride nanoparticles also exhibit excellent fluorescence scintillation properties, which offer potential applications for super-resolution imaging.

[0009] 2) These nanoparticles possess a stable chemical structure, excellent biosafety, and excellent biomaterial compatibility. They can also be further modified with biomolecules to achieve targeted binding to specific biological structures. By labeling cellular structures with these nanoparticles, bioimaging applications can be realized through microscopic imaging using a fluorescence imaging system.

[0010] The method for preparing pyrrole-modified graphite-phase carbon nitride nanoparticles of the present invention comprises the following steps:

[0011] 1) Obtain graphite phase carbon nitride by calcination

[0012] First, 5-8 g of nitrogen-rich carbon source is placed in a quartz crucible and placed in a muffle furnace. The temperature is raised to 400-600° C. at a rate of 5-10° C. per minute and maintained for 2-8 hours. The crucible is then naturally cooled to room temperature and taken out. The crucible is then washed repeatedly with deionized water for 1-3 minutes, and then centrifuged at 6000-12000 rpm for 8-15 minutes. The precipitate is collected to obtain graphite phase carbon nitride.

[0013] 2) Refining graphite-phase carbon nitride materials by pyrrole exfoliation

[0014] 5-10 mg of the graphite phase carbon nitride obtained in step 1) is mixed with 5-20 mL of pyrrole, and then reacted at 40-90° C. in an anhydrous and oxygen-free environment and protected from light for 12-72 hours. During the reaction, 10-20 mL of ethanol and / or 20-50 mL of tetrahydrofuran are optionally added to fully dissolve the reactants and products. 10-30 mg of a Lewis base can also be optionally added to promote the reaction. The obtained product is subjected to reduced pressure distillation to remove the reaction solvent; deionized water is then added and the mixture is distilled under reduced pressure again, the solid residue is collected, washed with water, and centrifuged at 6000-12000 rpm for 8-15 minutes. The resulting precipitate is a pure pyrrole-modified graphite phase carbon nitride material;

[0015] 3) Preparation of graphite phase carbon nitride nanoparticles by reprecipitation method

[0016] A tetrahydrofuran solution of a mixture of pyrrole-modified graphite-phase carbon nitride material and PSMA is prepared, wherein the total mass concentration of the pyrrole-modified graphite-phase carbon nitride material and PSMA is 2 ppm, and the mass concentration ratio of the two components is 1:0.25-4; 1-5 mL of the tetrahydrofuran solution is injected into 10-20 mL of deionized water and ultrasonically treated, and then the obtained solution is heated and purged with nitrogen to remove tetrahydrofuran, and then concentrated to a total mass concentration of the pyrrole-modified graphite-phase carbon nitride material and PSMA of 8-15 ppm to obtain PSMA-coated pyrrole-modified graphite-phase carbon nitride nanoparticles.

[0017] The nitrogen-rich carbon source described in the present invention includes but is not limited to cyanuric acid, melamine, urea, etc.; the Lewis base includes but is not limited to tert-butyl sodium, tert-butyl potassium, potassium thiocyanate, etc.; the hydroxyl activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and the protein antibody used is an α-tubulin monoclonal antibody.

[0018] In the above preparation process, we used the amphiphilic polymer poly(styrene-maleic anhydride copolymer) (PSMA, blocked isopropylbenzene, average degree of polymerization 1700, styrene content 68%) as a stabilizer when the pyrrole-modified graphite carbon nitride phase was transferred to the aqueous solution to form nanoparticles. Its structural formula is shown below:

[0019]

[0020] n and m are positive integers, indicating the number of repeating units.

[0021] The pyrrole-modified graphite-phase carbon nitride nanoparticles of the present invention are prepared by the above-mentioned preparation method.

[0022] The pyrrole-modified graphite-phase carbon nitride nanoparticles of the present invention can be used in biological imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Absorption spectrum of pyrrole-modified graphite-phase carbon nitride nanoparticles in Example 1 ( Figure 1 a) and emission spectra ( Figure 1 b);

[0024] Figure 2 : Transmission electron micrograph of pyrrole-modified graphite-phase carbon nitride nanoparticles in Example 1;

[0025] Figure 3 : Infrared spectrum of pyrrole-modified graphite-phase carbon nitride nanoparticles in Example 1;

[0026] Figure 4 : C1S photoelectron spectrum of pyrrole-modified graphite-phase carbon nitride nanoparticles in Example 1 ( Figure 4 a) and N1S photoelectron spectrum ( Figure 4 b);

[0027] Figure 5 : Single particle imaging photograph of pyrrole-modified graphite-phase carbon nitride nanoparticles in Example 2;

[0028] Figure 6 : Fluorescence signal blinking curve of pyrrole-modified graphite-phase carbon nitride nanoparticles in Example 2;

[0029] Figure 7 : A bar graph showing cell survival rates in the biotoxicity test of pyrrole-modified graphitic carbon nitride nanoparticles in Example 3;

[0030] Figure 8 : Fluorescence imaging photos of cells by pyrrole-modified graphite-phase carbon nitride nanoparticles in Example 3; wherein, Figure 8 a is a photo taken in bright field. Figure 8 b is a photo taken with fluorescence imaging;

[0031] Figure 9 : The super-resolution imaging photos of cell microtubule structure by pyrrole-modified graphite-phase carbon nitride nanoparticles in Example 4; wherein, Figure 9 a is a photo taken with conventional fluorescence imaging. Figure 9 b is the result of 4-order SOFI super-resolution fluorescence imaging processing, Figure 9 c is Figure 9 a and Figure 9 b Comparison chart of the two. DETAILED DESCRIPTION

[0032] The technical solutions of the present invention are described in more detail below with reference to specific embodiments, but the embodiments do not limit the present invention.

[0033] Example 1

[0034] 1) Synthesis of graphite-phase carbon nitride materials

[0035] First, 5 g of melamine was placed in a quartz crucible and placed in a muffle furnace. The temperature was raised at a rate of 5°C per minute to 400°C and maintained for 2 hours. After naturally cooling to room temperature, the sample was removed. The sample was then repeatedly washed with deionized water for 2 minutes, followed by centrifugation at 8000 rpm for 10 minutes. The precipitate was collected to obtain graphite phase carbon nitride, with a product mass of 2.3 g.

[0036] 2) Refining graphite-phase carbon nitride materials by pyrrole exfoliation

[0037] 5 mg of graphite-phase carbon nitride and 5 mL of pyrrole were mixed, and then the reaction was maintained at 40°C for 12 hours under anhydrous, oxygen-free and light-proof conditions. The product after the reaction was completed was distilled under reduced pressure to remove the reaction solvent, and then deionized water was added and distilled under reduced pressure again. The solid residue was collected, washed with water and centrifuged at 8000 rpm for 10 minutes. The resulting precipitate was pure pyrrole-modified graphite-phase carbon nitride material with a product mass of 3.4 mg.

[0038] 3) Preparation of pyrrole-modified graphite-phase carbon nitride nanoparticles

[0039] 5 mL of a tetrahydrofuran solution of a mixture of pyrrole-modified graphite-phase carbon nitride material and PSMA was prepared, wherein the total mass concentration of the pyrrole-modified graphite-phase carbon nitride material and PSMA was 2 ppm, and the mass concentration ratio of the pyrrole-modified graphite-phase carbon nitride material to PSMA was 4:1; 1 mL of the above solution was injected into 10 mL of deionized water and accompanied by ultrasonic treatment, and then the tetrahydrofuran was removed from the above solution by heating and nitrogen purging, and the volume was concentrated to 0.4 mL, thereby obtaining pyrrole-modified graphite-phase carbon nitride particles coated with PSMA with a total mass concentration of pyrrole-modified graphite-phase carbon nitride material and PSMA of 8 ppm.

[0040] Example 1 Performance Test

[0041] The fluorescence properties of the pyrrole-modified graphite-phase carbon nitride nanoparticles prepared by the present invention were tested. The absorption spectrum was measured using a UV-2600 ultraviolet-visible spectrometer, and the infrared spectrum was measured using a Shimadzu Fourier transform infrared spectrometer. The morphology and size of the nanoparticles were characterized using a TM-600 transmission electron microscope. XPS-related tests were performed using a Thermo Scientific K-Alpha XPS analyzer. The fluorescence spectrum was measured using an F4500 fluorescence spectrometer. The tests confirmed that the pyrrole-modified graphite-phase carbon nitride nanoparticles can emit blue-green fluorescence when excited by ultraviolet light. This fluorescence can be used as a signal for bioluminescence imaging to characterize cell structures.

[0042] like Figure 1 As shown: Figure 1 a is the absorption spectrum, Figure 1 b is the fluorescence spectrum. The abscissa of the spectrum is the absorption (or emission) wavelength, and the ordinate is the absorption intensity at the corresponding absorption (or emission) wavelength. The emission spectrum shows that the fluorescence emission position of pyrrole-modified graphitic carbon nitride nanoparticles is around 470 nm, which can be applied to cell imaging.

[0043] like Figure 2 As shown in the transmission electron microscope photos, it can be seen that the particle size distribution of pyrrole-modified graphite-phase carbon nitride nanoparticles is about 20 to 50 nm, and the morphology is close to lamellar;

[0044] like Figure 3 As shown in the figure: the horizontal axis is the absorption wave number, and the vertical axis is the absorption intensity of the corresponding wavelength. From the infrared spectrum, we can see the stretching vibration characteristic peak of the carbon-hydrogen bond on the carbon-carbon double bond in the pyrrole molecule (2900cm -1 ), indicating that the pyrrole group has been incorporated into the graphite phase carbon nitride nanoparticles;

[0045] like Figure 4As shown in the figure: the horizontal axis is the electron binding energy, and the vertical axis is the signal intensity of the corresponding binding energy photoelectron. The photoelectron energy spectrum shows the signal peaks of carbon (283.91eV and 284.18eV) and nitrogen (392.8eV and 394.9eV) in the graphitic carbon nitride nanoparticles. At the same time, there are also the signal peaks of carbon (284.24eV) and nitrogen (396.2eV) in the pyrrole molecules, indicating that the pyrrole molecules have successfully exfoliated the graphitic carbon nitride and attached to the graphitic carbon nitride surface.

[0046] Example 2

[0047] The preparation process of Example 2 for graphite phase carbon nitride, the steps for preparing pyrrole-modified graphite phase carbon nitride material and preparing pyrrole-modified graphite phase carbon nitride nanoparticles remain unchanged, except that the specific preparation parameters are adjusted.

[0048] 1) Synthesis of graphite-phase carbon nitride materials

[0049] First, 5g of melamine was placed in a quartz crucible and placed in a muffle furnace. The temperature was raised at a rate of 5°C per minute to 450°C and maintained for 4 hours. After naturally cooling to room temperature, the sample was removed. The sample was then repeatedly washed with deionized water for 2 minutes, followed by centrifugation at 6000rpm for 15 minutes. The precipitate was collected to obtain graphite phase carbon nitride, with a product mass of 2.1g.

[0050] 2) Refining graphite-phase carbon nitride materials by pyrrole exfoliation

[0051] 5 mg of graphite phase carbon nitride and 5 mL of pyrrole were mixed, and then 50 mL of tetrahydrofuran, 20 mL of ethanol, 10 mg of potassium thiocyanate and 20 mg of tert-butyl sodium were added under anhydrous, oxygen-free and light-proof conditions, maintaining the temperature at 90°C, and the reaction was carried out for 48 hours. After the reaction was completed, the product was distilled under reduced pressure to remove the reaction solvent, and then deionized water was added and distilled under reduced pressure again. The solid residue was collected, washed with water and centrifuged at 6000 rpm for 15 minutes. The resulting precipitate was pure pyrrole-modified carbon nitride material with a product mass of 3.6 mg.

[0052] 3) Preparation of pyrrole-modified graphite-phase carbon nitride nanoparticles

[0053] Prepare 5 mL of a tetrahydrofuran solution of pyrrole-modified graphite carbon nitride (GCN) and PSMA (PSMA) at a combined mass concentration of 2 ppm (1:1). Add 2 mL of this solution to 15 mL of deionized water and ultrasonicate. Then, heat the solution with nitrogen purge to remove the THF and concentrate it to a combined mass concentration of 15 ppm. This yields PSMA-coated GCN particles.

[0054] The pyrrole-modified graphite-phase carbon nitride nanoparticles obtained in Example 2 were studied using single-particle imaging. A small amount of the nanoparticle solution was placed on a quartz glass plate and allowed to stand for 15 minutes before the remaining liquid was removed. Single-particle imaging revealed that the nanoparticles produced in this example were bright and exhibited scintillating properties. They were well dispersed and free of agglomerates. Statistical results also indicate that these nanoparticles exhibited excellent scintillating properties.

[0055] like Figure 5 As shown: From the single particle imaging results of pyrrole-modified graphitic carbon nitride nanoparticles, it can be seen that the prepared nanoparticles are well dispersed and bright.

[0056] like Figure 6 As shown in the figure, the horizontal axis is time, and the vertical axis is the relative fluorescence intensity of the nanoparticles at the corresponding time. The statistical results of the fluorescence signal fluctuations of pyrrole-modified graphitic carbon nitride nanoparticles over time show that the prepared nanoparticles have obvious fluorescence blinking behavior.

[0057] Example 3

[0058] The steps for preparing graphite phase carbon nitride and pyrrole-modified graphite phase carbon nitride materials in the preparation process of Example 3 remain unchanged. The difference is the specific preparation conditions. The pyrrole-modified graphite phase carbon nitride nanoparticles subsequently obtained are applied to biological imaging.

[0059] 1) Synthesis of graphite phase carbon nitride materials:

[0060] First, 8 g of melamine was placed in a quartz crucible and placed in a muffle furnace. The temperature was raised at a rate of 10°C per minute to 550°C and maintained for 8 hours. After naturally cooling to room temperature, the sample was removed. The sample was then repeatedly washed with deionized water for 2 minutes, followed by centrifugation at 12,000 rpm for 8 minutes. The precipitate was collected to obtain graphite phase carbon nitride, with a product mass of 3.8 g.

[0061] 2) Refining graphite-phase carbon nitride materials by pyrrole exfoliation:

[0062] 10 mg of graphite phase carbon nitride and 20 mL of pyrrole were mixed, and then 20 mL of tetrahydrofuran, 10 mL of ethanol, and 10 mg of potassium thiocyanate were added under anhydrous, oxygen-free, and light-proof conditions, maintaining the temperature at 70°C, and the reaction was carried out for 48 hours. After the reaction was completed, the product was distilled under reduced pressure to remove the reaction solvent, and then deionized water was added and distilled under reduced pressure again. The solid residue was collected, washed with water, and centrifuged at 12000 rpm for 8 minutes. The resulting precipitate was pure pyrrole-modified carbon nitride material with a product mass of 6.7 mg.

[0063] 3) Preparation of pyrrole-modified graphite-phase carbon nitride nanoparticles:

[0064] A 5 mL tetrahydrofuran solution of a mixture of pyrrole-modified graphite-phase carbon nitride (GCN) and PSMA was prepared. The total mass concentration of the pyrrole-modified GCN and PSMA was 2 ppm, with a mass ratio of 2:1. This solution was then injected into 20 mL of deionized water and ultrasonicated. The solution was then heated with a nitrogen purge to remove the THF and concentrated to a total mass concentration of 10 ppm of the pyrrole-modified GCN and PSMA, yielding PSMA-coated GCN particles.

[0065] Bioimaging Application of Example 3

[0066] The pyrrole-modified graphite-phase carbon nitride nanoparticles prepared in Example 3 can be used for bioimaging applications. A 10 ppm solution of the nanoparticles was filtered through a 220 nm filter membrane and then used to stain cells for 2 hours before imaging. The imaging results confirmed that the nanoparticles had a good cell-labeling effect.

[0067] like Figure 7 As shown: the horizontal axis is the concentration of pyrrole-modified graphitic carbon nitride nanoparticles, and the vertical axis is the cell survival rate. The results of the biological toxicity test of pyrrole-modified graphitic carbon nitride nanoparticles show that these nanoparticles maintain stable biosafety even at higher concentrations.

[0068] like Figure 8 As shown: From the cell labeling effect of pyrrole-modified graphite-phase carbon nitride nanoparticles, it can be seen that the nanoparticles can be well absorbed by cells, thereby achieving accurate labeling of cells.

[0069] Example 4

[0070] The steps for preparing graphite phase carbon nitride and pyrrole-modified graphite phase carbon nitride materials in the preparation process of Example 4 are the same as those in Example 1, except that the specific preparation conditions are different. The pyrrole-modified graphite phase carbon nitride nanoparticles finally obtained are applied to super-resolution imaging of cell microtubules through conjugated antibodies.

[0071] 1) Synthesis of graphite phase carbon nitride materials:

[0072] First, 5 g of melamine was placed in a quartz crucible and placed in a muffle furnace. The temperature was raised at a rate of 10°C per minute to 600°C and maintained for 6 hours. After naturally cooling to room temperature, the sample was removed. The sample was then repeatedly washed with deionized water for 2 minutes, followed by centrifugation at 8000 rpm for 10 minutes. The precipitate was collected to obtain graphite phase carbon nitride, with a product mass of 2.1 g.

[0073] 2) Refining graphite-phase carbon nitride materials by pyrrole exfoliation:

[0074] 5 mg of graphite phase carbon nitride and 20 mL of pyrrole were mixed, and then 50 mL of tetrahydrofuran, 20 mL of ethanol, 5 mg of potassium thiocyanate, 5 mg of sodium tert-butoxide and 5 mg of potassium tert-butoxide were added under anhydrous, oxygen-free and light-proof conditions, maintaining the temperature at 90°C, and the reaction was carried out for 72 hours. After the reaction was completed, the product was distilled under reduced pressure to remove the reaction solvent, and then deionized water was added and distilled under reduced pressure again. The solid residue was collected, washed with water and centrifuged at 8000 rpm for 10 minutes. The resulting precipitate was pure pyrrole-modified graphite phase carbon nitride material with a product mass of 3.8 mg.

[0075] 3) Preparation of pyrrole-modified graphite-phase carbon nitride nanoparticles:

[0076] A 5 mL tetrahydrofuran solution of a mixture of pyrrole-modified graphite-phase carbon nitride (GCN) and PSMA was prepared. The total mass concentration of the pyrrole-modified GCN and PSMA was 2 ppm, and the mass ratio of the pyrrole-modified GCN to PSMA was 1:4. This solution was then injected into 15 mL of deionized water and ultrasonicated. The solution was then heated with a nitrogen purge to remove the THF and concentrated to a total mass concentration of 10 ppm of the pyrrole-modified GCN and PSMA, yielding PSMA-coated GCN particles.

[0077] Super-resolution imaging of the cell microtubule skeleton of Example 4

[0078] The nanoparticles prepared in Example 4 can mark the microtubule skeleton of the cell, and combined with SOFI super-resolution imaging technology, accurate imaging of the cell microtubule skeleton with a resolution of about 180nm can be achieved. First, PSMA coated with pyrrole-modified graphite phase carbon nitride nanoparticle colloids is activated by EDC, and then coupled with α-tubulin antibodies. After the coupling, 5ppm of antibody-modified nanoparticles are used to mark the cell microtubule skeleton, and then cell super-resolution imaging is performed using a 405nm laser as a light source. Imaging results show that this kind of nanoparticles has a good cell labeling effect, and combined with its scintillation characteristics, high-resolution super-resolution imaging can be achieved.

[0079] like Figure 9 As shown: From the super-resolution imaging results of cell microtubules by pyrrole-modified graphitic carbon nitride nanoparticles, it can be seen that the nanoparticles prepared by this material have good super-resolution imaging performance.

Claims

1. A method for preparing pyrrole-modified graphite-phase carbon nitride nanoparticles, comprising the following steps: 1) Obtain graphite phase carbon nitride by calcination First, 5-8 g of nitrogen-rich carbon source was placed in a quartz crucible and placed in a muffle furnace. The temperature was raised to 400-600 °C at a rate of 5-10 °C per minute and maintained for 2-8 hours. After cooling naturally to room temperature, the crucible was taken out and repeatedly washed with deionized water. The crucible was then centrifuged at 6000-12000 rpm for 8-15 minutes. The precipitate was collected to obtain graphite carbon nitride. 2) Refining graphite-phase carbon nitride materials by pyrrole exfoliation 5-10 mg of the graphite-phase carbon nitride obtained in step 1) is mixed with 5-20 mL of pyrrole, and then reacted at 40-90°C in an anhydrous and oxygen-free environment and protected from light for 12-72 hours. The resulting product is subjected to reduced pressure distillation to remove the reaction solvent; deionized water is then added and the product is distilled under reduced pressure again, and the solid residue is collected, washed with water, and centrifuged at 6000-12000 rpm for 8-15 minutes. The resulting precipitate is a pure pyrrole-modified graphite-phase carbon nitride material; 3) Preparation of graphite-phase carbon nitride nanoparticles by reprecipitation A tetrahydrofuran solution of a mixture of pyrrole-modified graphite-phase carbon nitride material and PSMA was prepared, with the total mass concentration of the pyrrole-modified graphite-phase carbon nitride material and PSMA being 2 ppm, and the mass concentration ratio of the two components being 1:0.25-4. 1-5 mL of the tetrahydrofuran solution was injected into 10-20 mL of deionized water and ultrasonically treated. The resulting solution was then heated and purged with nitrogen to remove the tetrahydrofuran, and then concentrated to a total mass concentration of the pyrrole-modified graphite-phase carbon nitride material and PSMA of 8-15 ppm, thereby obtaining PSMA-coated pyrrole-modified graphite-phase carbon nitride nanoparticles. PSMA is a polystyrene-maleic anhydride copolymer, end-capped cumene, with an average degree of polymerization of 1700 and a styrene content of 68%. Its structural formula is shown below: ； n and m are positive integers, indicating the number of repeating units.

2. The method for preparing pyrrole-modified graphite-phase carbon nitride nanoparticles according to claim 1, wherein: The nitrogen-rich carbon source in step 1) is one or more of cyanuric acid, melamine, and urea.

3. The method for preparing pyrrole-modified graphite-phase carbon nitride nanoparticles according to claim 1, wherein: During the reaction of step 2), 10-20 mL of ethanol and / or 20-50 mL of tetrahydrofuran are added to fully dissolve the reactants and products, and 10-30 mg of Lewis base is added to promote the reaction.

4. The method for preparing pyrrole-modified graphite-phase carbon nitride nanoparticles according to claim 3, wherein: The Lewis base is one or more of tert-butyl sodium, tert-butyl potassium, and potassium thiocyanate.

5. A pyrrole-modified graphite-phase carbon nitride nanoparticle, characterized in that: The method is prepared by the method according to any one of claims 1 to 4.

6. Use of the pyrrole-modified graphite-phase carbon nitride nanoparticles according to claim 5 in biological imaging.

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