A fluorine-nitrogen co-doped carbon dot, its preparation method and application

Abstract

This invention discloses a fluorine-nitrogen co-doped carbon dot, its preparation method, and its applications. The fluorine-nitrogen co-doped carbon dot is a nanoparticle constructed using a one-step hydrothermal synthesis method with fluorine-containing and nitrogen-containing precursors. The nanoparticle is spherical with a particle size of 5 nm, and its surface is rich in carboxyl, amino, and hydroxyl functional groups. Its fluorescence quantum yield is not less than 20%, and its emission wavelength is in the 500-550 nm range. It has the characteristic of targeting the H3K36me2 histone modification in tumor cell nuclei, and can achieve specific labeling of tumor cells within 5 minutes. The enrichment intensity in tumor tissue is more than 2.72 times that in normal tissue. The fluorine-nitrogen co-doped carbon dot of this invention is expected to serve as a novel fluorescent labeling probe for tumor identification and imaging, contributing to the early and accurate diagnosis of malignant tumors. It has multiple advantages such as speed, accuracy, safety, and visualization, and has broad application prospects in tumor pathology identification and intraoperative navigation.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials and biomedical imaging technology, specifically relating to a fluorine-nitrogen co-doped carbon dot (FNCDs) and its preparation method and application. Background Technology

[0002] Malignant tumors are one of the leading causes of death worldwide. According to the latest data from the International Agency for Research on Cancer (IARC), a branch of the World Health Organization, approximately 20 million new cases of malignant tumors were diagnosed globally in 2022, with nearly 9.7 million deaths, posing a serious threat to human life and health.

[0003] Studies have shown that early diagnosis is crucial for improving treatment success rates and prolonging patient survival. For example, the five-year survival rate for lung cancer patients diagnosed early can exceed 90%, while it is less than 10% for those in the middle and late stages. However, because early-stage cancerous tissues differ only slightly in morphology and structure from normal tissues, traditional imaging techniques (such as magnetic resonance imaging and X-ray computed tomography) still have limitations in resolution, sensitivity, and safety, including high radiation exposure, high imaging costs, and limited resolution. Therefore, there is an urgent need to develop novel, low-toxicity, efficient, and high-resolution diagnostic methods to achieve early and accurate identification of malignant tumors.

[0004] In recent years, the development of nanotechnology has provided new ideas for the diagnosis and treatment of tumors. Among them, carbon dots (CDs), as a novel class of fluorescent nanomaterials, have attracted widespread attention due to their advantages such as good optical properties, biocompatibility, and ease of preparation. CDs mainly include carbon quantum dots (CQDs), carbon nanodots (CNDs), graphene quantum dots (GQDs), and carbonized polymer dots (CPDs), and are widely used in various fields such as bioimaging, drug delivery, disease diagnosis, and optoelectronic devices. CDs exhibit excellent photoluminescence properties in bioimaging, and some carbon dots possess excitation wavelength dependence, good resistance to photobleaching, and high fluorescence stability, making them suitable for visualization labeling at the in vivo or cellular level. However, traditional carbon dots suffer from problems such as short excitation wavelengths (e.g., 405 nm), low quantum yield (QY), and insufficient water solubility, which limit their application in in vivo imaging and clinical diagnosis.

[0005] In the field of tumor identification and treatment, CDs-based targeted fluorescent probes have been widely used for tumor cell identification and tissue localization. Traditional strategies often rely on the binding of targeting molecules such as peptides, proteins, antibodies, or aptamers to receptors on the tumor surface. However, due to the lack of specific biomarkers in the early stages of some tumor types, the sensitivity and specificity of identification are often insufficient to meet clinical requirements. Therefore, the development of novel carbon dot probes with active targeting capabilities, long-wavelength excitation, and high fluorescence efficiency is of great significance. These materials are expected to serve as highly sensitive, high-resolution bioimaging tools, widely used in the early identification of malignant tumors, intraoperative assessment, and post-treatment monitoring, demonstrating significant clinical translational potential. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a fluorine-nitrogen co-doped carbon dot, its preparation method, and its application. This fluorine-nitrogen co-doped carbon dot possesses excellent optical properties, biocompatibility, and specific tumor targeting capabilities, and is expected to serve as a novel nanoprobe for diagnosing malignant tumors, thereby solving the problems of inaccurate positioning, cumbersome operation, or high toxicity of traditional imaging agents in the early identification of tumors.

[0007] To solve the above-mentioned technical problems and achieve the above-mentioned technical effects, the present invention is implemented through the following technical solution: A method for preparing fluorine-nitrogen co-doped carbon dots, comprising a one-step hydrothermal synthesis using fluorine-containing and nitrogen-containing precursors, including the following steps: Step 1) Glycine, 2,4-difluorobenzoic acid, ethanol and ultrapure water are added sequentially to a stainless steel autoclave lined with polytetrafluoroethylene in a molar ratio of 2.65:1:5.44:220.16 to form a reaction solution. Step 2) After sealing the autoclave, place it in a heating device and heat it for a period of time to ensure that the reaction solution reacts completely; Step 3) After the reaction is complete, allow the reaction solution to cool naturally, and then dialyze the reaction solution to remove small molecule byproducts and unreacted raw materials. Step 4) The dialyzed reaction solution is freeze-dried to obtain a dry orange powder, namely nitrogen co-doped carbon dots (FNCDs).

[0008] Furthermore, in step 2, the heating temperature is 160℃ and the heating time is 4 hours.

[0009] Furthermore, in step 3, after the reaction solution is naturally cooled to 25°C, the reaction solution is transferred to a dialysis bag with a molecular weight cutoff of 500 Da for dialysis.

[0010] Furthermore, in step 4, the freeze-drying conditions are -60°C and 10 Pa.

[0011] A fluorine-nitrogen co-doped carbon dot (FNCD) is prepared using the above-mentioned method for preparing fluorine-nitrogen co-doped carbon dots.

[0012] The fluorine-nitrogen co-doped carbon dots (FNCDs) of the present invention have the following characteristics: 1. Hydrophilicity and biodispersibility: The FNCDs nanoparticles are spherical with a particle size of about 5 nm. Their surface is rich in functional groups such as carboxyl, amino, and hydroxyl groups, which gives them good hydrophilicity and biodispersibility.

[0013] 2. Epigenetic target colocalization capability: The FNCDs have the characteristic of targeting H3K36me2 histone modification in the nucleus of tumor cells. That is, the FNCDs can colocalize with the tumor-related epigenetic modification marker H3K36me2, and the colocalization intensity is significantly higher in tumor cells than in normal cells, which has the potential to detect epigenetic abnormalities.

[0014] 3. Optical properties: The fluorescence quantum yield of the fluorine-nitrogen co-doped carbon dots (FNCDs) is not less than 20%, and the emission wavelength is in the range of 500-550nm. In particular, it emits bright and stable green fluorescence at an excitation wavelength of 480nm. The longer excitation wavelength can effectively avoid interference from the autofluorescence of biological tissues and reduce phototoxicity, making it suitable for in vivo imaging.

[0015] 4. Tumor cell recognition characteristics: The FNCDs can complete the specific labeling of tumor cells within 5 minutes. That is, the FNCDs can selectively enter the nucleus of tumor cells and accumulate in the nucleolar region within 5 minutes. In normal cells, they are only distributed in the cytoplasm, showing significant cell-specific recognition ability, and do not affect normal cell proliferation.

[0016] 5. Cell cycle stability: The FNCDs maintain stable fluorescence throughout the entire cell division process and can continuously emit light in the nuclei of daughter cells, making them suitable for dynamic tracking of live cells.

[0017] 6. In vivo targeting ability of in situ tumors in mice: The enrichment intensity of the fluorine-nitrogen co-doped carbon dots (FNCDs) in tumor tissue was more than 2.72 times that in normal tissue. Animal experiments showed that the FNCDs could enter the bloodstream via intravenous injection and specifically target in situ breast tumor tissue. 24 hours after injection, fluorescence imaging of frozen sections showed that the fluorescence intensity of the FNCDs in the tumor region (48.18±2.21) was significantly higher than that in the surrounding normal tissue (17.73±2.20), and the fluorescence signal in the tumor tissue was 2.72 times that of the surrounding tissue, indicating a significant tumor-to-normal tissue ratio and clearly delineating the tumor boundary, demonstrating its potential in in vivo tumor imaging and boundary determination.

[0018] The aforementioned fluorine-nitrogen co-doped carbon dots (FNCDs) can be used as fluorescently labeled probes for the preparation of rapid diagnostic reagents for malignant tumors.

[0019] Furthermore, the malignant tumors include thyroid malignant tumors.

[0020] Furthermore, the rapid diagnostic reagent for malignant tumors is a rapid staining diagnostic reagent suitable for fine needle biopsy specimens.

[0021] In clinical sample studies, the FNCDs were used for rapid staining of thyroid fine-needle aspiration (FNA) samples. Significant green fluorescence was observed in the tumor tissue after only 5 minutes of incubation. Pathological examination confirmed the tumor as papillary carcinoma (PTC). Non-malignant samples showed only weak peripheral fluorescence. These results validate that the FNCDs possess excellent tumor recognition capabilities in human tissues, providing a basis for clinical application.

[0022] The aforementioned fluorine-nitrogen co-doped carbon dots (FNCDs) can also be used to prepare tumor boundary delimitation reagents. By using the FNCDs for in vivo fluorescent labeling, tumor boundaries can be delimited.

[0023] The aforementioned fluorine-nitrogen co-doped carbon dots (FNCDs) can also be used to prepare rapid intraoperative pathological detection reagents for tumors. By using the FNCDs to rapidly stain fresh tissue specimens, rapid intraoperative pathological detection of tumors can be achieved.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: The fluorine-nitrogen co-doped carbon dots (FNCDs) of this invention have small particle size, abundant surface functional groups, and good water solubility and cell membrane penetration ability.

[0025] The fluorine-nitrogen co-doped carbon dots of this invention can rapidly identify various types of tumor cells, especially showing targeted enrichment of the nuclear H3K36me2 high-expression region, with high recognition efficiency.

[0026] The fluorine-nitrogen co-doped carbon dots of this invention have no obvious toxicity, no effect on cell proliferation, and strong biosafety.

[0027] The fluorine-nitrogen co-doped carbon dots of this invention can be imaged at high resolution in both tissues and living organisms, with clear tumor boundaries and high imaging resolution.

[0028] The fluorine-nitrogen co-doped carbon dots of this invention can be applied to the rapid identification and interpretation of basic research, animal experiments, and clinical puncture samples, and have the feasibility of being translated from the laboratory to the clinical setting, with wide applicability.

[0029] In summary, the fluorine-nitrogen co-doped carbon dots of this invention possess excellent optical properties, biocompatibility, and specific tumor targeting capabilities. They are expected to be used as novel fluorescently labeled probes in tumor identification and imaging, contributing to the early and accurate diagnosis of malignant tumors. In particular, they offer multiple advantages such as speed, accuracy, safety, and visualization in the cellular level identification of malignant tumors, in vivo targeted imaging, and rapid interpretation of clinical puncture samples. They solve the problems of inaccurate positioning, cumbersome operation, or high toxicity of traditional imaging agents in early tumor identification, and have broad application prospects in tumor pathology identification and intraoperative navigation.

[0030] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0031] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings: Figure 1 This is a characterization result diagram of FNCDs in an embodiment of the present invention, wherein... Figure 1 In the diagram, 'a' represents the particle size distribution of FNCDs. Figure 1 In this context, 'b' represents the photoluminescence spectrum of FNCDs at different excitation wavelengths (400-750 nm). Figure 1 In this context, 'c' represents the Fourier transform infrared (FTIR) spectrum of NCDs. Figure 1 In this context, d represents the transmission electron microscope (TEM) image of the FNCDs.

[0032] Figure 2 This is a diagram showing the EdU cell proliferation experiment results of FNCDs in an embodiment of the present invention.

[0033] Figure 3 This is an H&E staining image of the major organs after systemic administration of FNCDs in an embodiment of the present invention.

[0034] Figure 4 This is a graph showing the serum biochemical index detection results in an embodiment of the present invention, wherein... Figure 4 The 'a' indicates the activity level of aspartate aminotransferase (AST). Figure 4 In this context, 'b' represents the creatine kinase (CK) activity level. Figure 4 In this context, 'c' represents the creatinine (CRE) concentration level.

[0035] Figure 5This is a diagram showing the results of a live cell staining experiment with FNCDs in an embodiment of the present invention. Figure 5 In the graph, 'a' represents a line graph showing the quantitative analysis of the average fluorescence intensity of cell nuclei during incubation for different cell lines. Figure 5 In this image, b represents the bright-field and fluorescence images of 293T (normal) and HeLa (tumor) cells co-cultured after FNCD treatment. Figure 5 In the image, 'c' represents fluorescence images of four cancer cell lines (HeLa, SCC15, 4T1, T24) and two normal cell lines (293T, MN9D) co-incubated with FNCDs for 5, 20, and 30 min.

[0036] Figure 6 This is a diagram showing the results of FNCDs immunofluorescence staining experiments in an embodiment of the present invention.

[0037] Figure 7 This is a diagram showing the co-localization analysis results of FNCDs and H3K36me2 in an embodiment of the present invention. Figure 7 In this context, 'a' represents the Pearson correlation coefficient between FNCDs and histone H3. Figure 7 In this context, 'b' represents the Pearson correlation coefficient between FNCDs and H3K36me1. Figure 7 In this context, 'c' represents the Pearson correlation coefficient between FNCDs and H3K36me2. Figure 7 In this context, d represents the Pearson correlation coefficient between FNCDs and H3K27me3.

[0038] Figure 8 These are fluorescence images from animal experiments in embodiments of the present invention.

[0039] Figure 9 This is a fluorescence image of thyroid fine-needle aspiration tissue after FNCDs treatment in an embodiment of the present invention, wherein... Figure 9 In this image, 'a' represents a fluorescence image of normal thyroid tissue. Figure 9 In the image, 'b' represents a fluorescence image of papillary thyroid carcinoma. Detailed Implementation

[0040] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings to provide a clearer understanding of the invention's purpose, features, and advantages. It should be understood that the embodiments shown in the drawings are not intended to limit the scope of the invention, but are merely illustrative of the essential spirit of the invention's technical solution.

[0041] In the following description, certain specific details are set forth for the purpose of illustrating various disclosed embodiments in order to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the art will recognize that embodiments may be practiced without one or more of these specific details. In other instances, well-known apparatuses, structures, and techniques associated with this application may not have been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0042] Unless the context requires otherwise, throughout the specification and claims, the word “comprising” and its variations, such as “including” and “having”, shall be understood to have an open, inclusive meaning, that is, to be interpreted as “including, but not limited to”.

[0043] Throughout this specification, references to "an embodiment" or "an embodiment" indicate that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Therefore, the appearance of "in an embodiment" or "an embodiment" in various places throughout the specification does not necessarily refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in any manner in one or more embodiments.

[0044] The singular forms “a” and “the” used in this specification and the appended claims include plural references unless otherwise expressly stated herein. It should be noted that the term “or” is generally used to mean “and / or” unless otherwise expressly stated herein.

[0045] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0046] To improve the performance of carbon dots, this invention attempts to optically modulate CDs through a heteroatom doping strategy. Nitrogen (N) doping can effectively adjust the emission wavelength and enhance fluorescence intensity by forming lone pairs of electrons and surface defects. Fluorine (F), due to its extremely high electronegativity, can form hydrogen bonds with the carbon dot surface, further enhancing fluorescence stability and imaging resolution. Fluorine / nitrogen co-doped carbon dots (FNCDs) are thus developed. Related experiments show that FNCDs not only possess high quantum yield (QY up to 57%), excellent solution stability, and resistance to photobleaching, but also long-wavelength excitation characteristics (e.g., λex = 488 nm), making them suitable for long-term, low-damage live-cell imaging and in vivo detection. Simultaneously, the positively charged surface of FNCDs allows them to effectively bind to negatively charged biomolecules (such as DNA and RNA), enabling active targeting of the cell nucleus or nucleolus. In super-resolution fluorescence microscopy (STED), FNCDs exhibit good resolution (up to 22.1 nm) and imaging stability, making them suitable for dynamic monitoring in complex biological environments. More importantly, FNCDs can achieve long-term fluorescence signal acquisition without affecting cell viability, demonstrating significant biosafety and clinical application potential.

[0047] The embodiments of the present invention and their related experimental results are described in detail below.

[0048] 1. Preparation of fluorine-nitrogen co-doped carbon dots (FNCDs): FNCDs were constructed using a one-step hydrothermal synthesis method with fluorine-containing and nitrogen-containing precursors, including the following steps: Step 1) Add 16.7 mmol glycine, 6.3 mmol 2,4-difluorobenzoic acid, 2 mL ethanol and 25 mL ultrapure water sequentially to a stainless steel autoclave lined with polytetrafluoroethylene in a molar ratio of 2.65:1:5.44:220.16 to form a reaction solution. It should be noted that since the density of ethanol is approximately 0.789 g / mL and the density of ultrapure water is 1 g / mL, when the amount of glycine is 16.7 mmol and the amount of 2,4-difluorobenzoic acid is 6.3 mmol, the volume of ethanol is 2 mL and the volume of ultrapure water is 25 mL.

[0049] Step 2) After sealing the autoclave, place it in a heating device, raise the temperature to 160°C and maintain it for 4 hours to ensure that the reaction solution reacts completely; Step 3) After the reaction is complete, allow the system temperature of the reaction solution to cool naturally to 25°C, and then transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 500 Da for dialysis to remove small molecule byproducts and unreacted raw materials. Step 4) After dialysis is completed, the dialysis reaction solution is freeze-dried at -60°C and 10 Pa to obtain a dry orange powder, namely nitrogen co-doped carbon dots (FNCDs).

[0050] Finally, the FNCDs were redissolved in ultrapure water for subsequent quantitative analysis and related characterization experiments.

[0051] 2. Characterization of FNCDs: 2.1 The characterization methods for FNCDs are as follows: The morphology of FNCDs was characterized using a transmission electron microscope (TEM) (model: FEI Tecnai G2 F20 TWIN). The particle size distribution of FNCDs was determined using a dynamic light scattering instrument (Malvern Zetasizer Nano ZS, model: ZEN 3690). Fourier transform infrared spectroscopy (FTIR) was performed using a Bruker Hyperion infrared spectrometer.

[0052] 2.2 Characterization results of FNCDs: See Figure 1 As shown in d, transmission electron microscopy (TEM) characterization shows that the FNCDs prepared in this embodiment have a uniformly dispersed spherical morphology.

[0053] See Figure 1 As shown in Figure a, the particle size distribution of the FNCDs prepared in this embodiment was characterized by dynamic light scattering (DLS) technology, and the results showed that the average particle size was 5 nm.

[0054] See Figure 1 As shown in b, optical performance tests show that the FNCDs prepared in this embodiment have fluorescence characteristics that are independent of the excitation wavelength, exhibiting the strongest fluorescence emission in the 480nm excitation range.

[0055] See Figure 1 As shown in c, Fourier transform infrared spectroscopy (FTIR) analysis shows that the surface of the FNCDs prepared in this embodiment is rich in hydroxyl (-OH), carbonyl (C=O) and nitrogen-containing functional groups (CN).

[0056] Characterization results of FNCDs show that the FNCDs prepared in this embodiment have small particle size, abundant surface functional groups, good hydrophilicity and biodispersibility, good water solubility, strong cell membrane penetration ability, and emit bright and stable green fluorescence at an excitation wavelength of 480 nm. The longer excitation wavelength can effectively avoid interference from the autofluorescence of biological tissues and reduce phototoxicity, making it suitable for in vivo imaging.

[0057] 3. EdU cell proliferation experiment: 3.1 The reagents used in the EdU cell proliferation experiment are as follows: (1) Washing solution (20 mL), containing 20 mL PBS buffer and 0.6 g BSA; (2) Permeation solution (20 mL), containing 600 μL 10% Triton and 19.4 mL PBS buffer; (3) Click Additive Solution (1.3 mL), containing 1 tube of Click Additive and 1.3 mL of deionized water.

[0058] (4) Click reaction solution (4 mL, to be used within 15 min after preparation), containing 3.44 mL Click Reaction Buffer, 160 μL CuSO4, 8 μL Azide 647 and 400 μL Click Additive Solution.

[0059] 3.2 The EdU cell proliferation assay method is as follows: (1) Cell seeding: The experiment included a control group and an FNCDs treatment group, with 3 replicates per group. HeLa cells in good growth condition were digested, centrifuged, and resuspended. 1×10⁶ cells were seeded into each well of a 6-well plate. 5 Observe the adhesion status of individual cells, and proceed to the next step when the growth density reaches 80%. (2) Cell treatment: FNCDs working solution (final concentration: 25µg / mL) was added to the FNCDs treatment group, and the cells were cultured in an incubator for 20 min. The culture medium was then discarded and the cells were washed three times with PBS. (3) EdU-647 labeling: Add 2 mL of preheated EdU working solution (concentration: 10 μM) at 37 °C to each well and incubate for 2 h in an incubator. Discard the culture medium, wash 3 times with PBS, digest the cells with trypsin, and centrifuge (1000 rpm, 5 min). (4) Fixation and permeabilization: Add 1 mL of 4% paraformaldehyde to each well and fix at room temperature for 15 min, then centrifuge (1500 rpm, 5 min). Wash cells with 1 mL of washing buffer (PBS containing 3% BSA) and centrifuge (1500 rpm, 5 min, repeated 3 times). Add 500 μL of permeabilization buffer and permeabilize for 15 min. Discard the permeabilization buffer, wash cells once with washing buffer and centrifuge (1500 rpm, 5 min); (5) Add Click reaction solution: Add 500 μL of Click reaction solution to each well and incubate at room temperature in the dark for 30 min. Centrifuge to remove the reaction solution, wash with washing buffer, centrifuge again, discard the washing buffer, resuspend the cells in 500 μL of PBS, and store at 4°C in the dark; (6) Data acquisition: Unlabeled cell samples were used as negative controls, and an appropriate voltage was selected. The cell suspensions obtained in the above experimental steps were analyzed using flow cytometry.

[0060] 3.3 Effects of FNCDs on cell proliferation: To assess the effect of FNCDs on cell proliferation, this study used a 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay combined with flow cytometry (FC) to detect the effects on HeLa cells.

[0061] See Figure 2 As shown, the experimental results indicated that, compared with the control group (negative control, NC), there was no significant statistical difference in the proportion of EdU weakly positive cells (non-proliferating cells) and EdU strongly positive cells (proliferating cells) after 20 min of FNCD treatment (P>0.05).

[0062] EdU cell proliferation experiments showed that FNCDs had no obvious toxicity, and treatment with FNCDs did not affect the normal proliferation capacity of HeLa cells, demonstrating strong biosafety.

[0063] 4. In vivo safety studies: 4.1 H&E staining experiment: Mice sacrificed 24 hours after intravenous injection of FNCDs (5 µg / g body weight) or an equal volume of physiological saline (control group) were subjected to histopathological analysis of representative hematoxylin-eosin (H&E) stained sections of the heart, liver, spleen, lung, and kidney. See also Figure 3As shown, H&E staining images of major organs in two groups of mice after systemic administration of FNCDs are displayed (images acquired from the cropped area of ​​a whole-section scan, full image scale bar: 1000µm, display area scale bar: 100µm). Compared with the control group injected with saline, no obvious histopathological changes, including tissue necrosis, hemorrhage, edema or acute inflammatory infiltration, were observed in any of the examined organs in the FNCDs-treated group.

[0064] 4.2 Serum biochemical marker detection: Five mice in each group were intravenously injected with either FNCDs (5 µg / g body weight) or an equal volume of physiological saline (control group). Serum was collected 24 hours after injection. Serum biochemical markers related to systemic toxicity following intravenous injection of FNCDs were assessed. See [link to relevant documentation] Figure 4 As shown in the ac data, no significant abnormalities were found in the activity levels of aspartate aminotransferase (AST), creatine kinase (CK), and creatinine (CRE). All data are expressed as mean ± standard error. Unpaired t-tests were used for statistical comparisons between groups (ns: no significant difference). 4.3 Results of in vivo safety experiments: In vivo safety assessments showed that FNCDs did not cause significant pathological damage to major organs such as the heart, liver, spleen, lungs, or kidneys. Serum biochemical indicators (aspartate aminotransferase AST, creatinine CRE, and creatine kinase CK) also showed no significant abnormalities. These results confirm the good biocompatibility and in vivo safety of FNCDs, providing a safe basis for their subsequent translational applications.

[0065] 5. FNCDs live cell staining experiment: 5.1 The method for FNCDs cell staining experiment is as follows: This study selected two non-tumor cell types (293T, MN9D) and four tumor cell types (HeLa, SCC15, 4T1, T24) for experimental validation. After the cells were cultured to adherence, they were treated with FNCDs (25µg / mL) and co-incubated at 37℃ for specific times (5min / 20min / 30min). The cells were then washed three times with PBS, and the fluorescence signal of the cells was observed and captured under an inverted fluorescence microscope (excitation wavelength: 488nm).

[0066] 5.2 Results of FNCDs live cell staining experiment: See Figure 5As shown in Figure a, quantitative analysis of the mean fluorescence intensity of cell nuclei during incubation for different cell lines is presented (data are expressed as mean ± standard deviation, n=30 cells per group). Quantitative analysis showed that strong green fluorescence was observed in the nucleoli of all tumor cell lines 5 min after incubation, while only weak fluorescence was detected in normal cell lines at this time. The fluorescence intensity of tumor cells increased over time, reaching optimal imaging conditions at 20 min, and showed no significant change at 30 min. Furthermore, throughout the entire incubation period, the mean fluorescence intensity of tumor cells was consistently higher than that of normal cells, indicating that cancer cells rapidly and efficiently uptake FNCDs.

[0067] See Figure 5 As shown in b, bright-field and fluorescence images (scale bar: 10 μm) of 293T (normal) and HeLa (tumor) cells co-cultured after FNCD treatment are displayed. Bright-field imaging shows no morphological changes before and after FNCD treatment, while fluorescence imaging shows that FNCDs are significantly aggregated in the nuclei of HeLa cells within 5 min, while the signal in 293T cells is much weaker.

[0068] See Figure 5 Figure c shows fluorescence images (excitation wavelength: 488 nm, scale bar = 20 μm) of four cancer cell lines (HeLa, SCC15, 4T1, T24) and two normal cell lines (293T, MN9D) after co-incubation with FNCDs for 5, 20, and 30 min. The results show that tumor cells exhibited significant green fluorescence within the nucleolus after 5 min of co-incubation, while non-tumor cells showed only enhanced green fluorescence around the cell membrane at the same time point, with no significant green fluorescence signal in the cytoplasm, suggesting that FNCDs were mainly distributed in the cytoplasm and had not yet entered the nucleus. After 20 min of staining, significant green fluorescence was visible in the nuclei of all six cell types, indicating that FNCDs had completely entered the nucleus.

[0069] FNCDs live cell staining results showed that FNCDs can rapidly and selectively accumulate in the nuclei of tumor cells in various cell types, have the ability to rapidly enter the tumor cell nucleus, and produce significant fluorescent signals in the nucleolus, suggesting that they have good application potential in the rapid identification of tumor cells, and provide a rapid and reliable strategy for distinguishing cancer cells from normal cells in vitro.

[0070] 6. FNCDs immunofluorescence staining experiment: 6.1 The reagents used in the FNCDs immunofluorescence staining experiment are as follows: (1) 0.1% PBST (50 mL) is prepared by mixing 50 mL of PBS buffer and 50 μL of Tween-20. (2) 5% BSA (10mL) is prepared from 10mL PBST and 0.5g BSA.

[0071] 6.2 The method for FNCDs immunofluorescence staining is as follows: (1) Cell culture and plating: After trypsin digestion, the cells were centrifuged (1000 rpm, 5 min), resuspended, and seeded into 24-well plates (1×10⁶ cells / well). 4 / well (containing sterile climbing sheet), incubate at 37℃ and 5% CO2 until adherent; (2) FNCDs treatment: Add FNCDs (25µg / mL) and incubate for 20 min. Discard the culture medium and wash 3 times with PBS. (3) Fixation: Fix with 4% paraformaldehyde for 15 min, wash with PBS 3 times (5 min / time); (4) Blocking: Block with 5% BSA for 30 min; (5) Primary antibody incubation: Add primary antibody (5% BSA diluted, 300 μL / well) and incubate overnight at 4°C; (6) Secondary antibody incubation: After washing 3 times with PBST, add secondary antibody (300 μL, protected from light, 2 h), and wash again; (7) Covering: DAPI cover, nail polish to fix the edges, store at 4℃ away from light.

[0072] 6.3 Results of FNCDs immunofluorescence staining experiment: H3K36me2 refers to the dimethylation modification of lysine (Lys36, K36) at position 36 of histone H3. It plays a key role in gene transcription regulation, DNA damage repair, and the formation of other histone modifications. Abnormal changes in its expression level can affect cell proliferation and differentiation and are closely related to tumorigenesis.

[0073] To investigate the distribution of fibrinogen-free nuclear deposits (FNCDs) in the cell nucleus and their relationship with histone modification, this study analyzed the co-localization of FNCDs with histone H3 and its methylation sites (H3K36me1, H3K36me2, H3K27me3) in different cell types. Two tumor cell lines (HeLa and SCC15) and one non-tumor cell line (MN9D) were selected for the experiment. Cells were stained with FNCDs (25 µg / mL) for 20 min, fixed, and then subjected to immunofluorescence staining.

[0074] Confocal laser scanning microscopy (CLSM) was used to observe the distribution of FNCDs in the cell nucleus and analyze their co-localization with histone H3 and related methylation modifications. DAPI (blue) was used for nuclear labeling, and the immunofluorescence signals of histone H3, H3K36me1, H3K36me2, and H3K27me3 are presented in red. See also Figure 6The image shown in the figure illustrates a three-color immunofluorescence image of cells captured by CLSM. (DAPI excitation wavelength: 350 nm; FNCDs excitation wavelength: 488 nm; histone H3, H3K36me1, H3K36me2, H3K27me3 excitation wavelengths: 640 nm; scale bar = 10 μm). Note: Merged in the figure is a merged image of the green and red channels, and orange represents colocalization signals.

[0075] See Figure 6 As shown in the image, FNCDs (green) and DAPI (blue) highly overlap, indicating that FNCDs can specifically perform fluorescence imaging of cell nuclei.

[0076] See Figure 6 As shown in c, after further merging the green (FNCDs) and red (antibody) channels, the colocalization of fluorescence signals was analyzed. The results showed that the colocalization signals of FNCDs and H3K36me2 differed significantly between tumor cells and non-tumor cells. Specifically: (1) the expression level of H3K36me2 was higher in tumor cells (HeLa, SCC15); (2) colocalization signals of H3K36me2 and FNCDs (orange) were observed in tumor cells, while no obvious colocalization was observed in non-tumor cells (MN9D).

[0077] To further quantify the colocalization of FNCDs with histone H3 and its methylation modifications, colocalization analysis was performed using a colocalization tool to analyze the correlation between FNCDs (green channel) and H3, H3K36me1, H3K36me2, and H3K27me3 (red channel) along the Z-axis. Ten consecutive slices were selected for the experiment, with a step size of 0.2 μm, and Pearson's correlation coefficient (PCC) was used to assess the correlation between fluorescence signals.

[0078] See Figure 7 As shown in the figure, statistical analysis results indicate that H3K36me2 and FNCDs showed moderate to high correlation in HeLa cells (0.525±0.012) and SCC15 cells (0.492±0.012), while the correlation was weaker in MN9D cells (0.201±0.014). Furthermore, in tumor cells, FNCDs exhibited higher signal overlap coefficients and colocalization rates with H3K36me2, suggesting that FNCDs can more effectively target H3K36me2 within tumor cells.

[0079] 7. Animal experiments: 7.1 The reagents used in animal experiments are as follows: FNCDs injection solution is prepared by mixing 100 μL of FNCDs working solution with 900 μL of PBS buffer.

[0080] 7.2. Construction of a mouse subcutaneous tumor model: (1) Cell culture: Digest mouse breast cancer cells (4T1), centrifuge and resuspend (5×10⁻⁶ cells / year). 6 / 100μL DPBS); (2) Skin preparation: The mice were prepared (the 4th and 5th mammary gland areas were treated with hair removal cream), anesthetized, fixed and disinfected; (3) Breast injection: Insert the needle through the 5th nipple, and inject the cell suspension (100μL) subcutaneously to the 4th nipple on the same side. (4) Tumor monitoring: When the tumor volume reaches 50-100 mm 3 (0.5×L×W) 2 Then continue the experiment; (5) FNCDs injection: Anesthetize tumor-bearing mice and inject 200 μL of FNCDs injection solution into the tail vein. After observing their condition, feed them for 24 h. (6) Tissue collection: The tumor and its surrounding tissues are collected after euthanasia.

[0081] 7.3 Imaging of frozen tissue sections: (1) Tissue collection: Tumors and surrounding tissues were collected after euthanizing mice; (2) Quick freezing: The tissue was loaded into EP tubes, quick frozen in liquid nitrogen for 10-20 seconds, and then stored at -80℃; (3) Pre-cooling slicer: Set the machine to -20℃ and operate it after the temperature stabilizes; (4) Embedding: After the tissue is rewarmed, it is fixed in the sample holder, embedded by OCT, and frozen into a block at -20℃; (5) Sectioning: Adjust the blade angle and anti-roll plate, trim the section to the maximum cut surface and then section (20μm), flatten with a brush, and attach the glass slide; (6) Mounting: Add DAPI mounting solution, place a coverslip at 45° to avoid air bubbles; (7) Observation and photography: Transfer to a laser confocal microscope for photography.

[0082] 7.4 Animal Experiment Results: Cellular experiments have demonstrated that FNCDs can rapidly and accurately identify H3K36me2 in tumor cells through mitochondrial membrane potential mediation. Furthermore, the longer excitation wavelength of FNCDs effectively reduces phototoxicity to organisms and avoids interference from tissue autofluorescence, showcasing their potential in in vivo imaging.

[0083] To clarify the targeting ability of FNCDs on tumors in vivo, this study selected 4-6 week old female BALB / c mice as the experimental target to construct an orthotopic mammary tumor model. The tumor was targeted until it grew to 50-100 mm. 3 Around 24 hours later, tumor-bearing mice were injected with a tail vein injection solution (5µg / g) and observed for 24 hours before being dissected.

[0084] To more clearly demonstrate whether FNCDs have the potential to differentiate tumor tissue, we sampled the tumor and surrounding tissues, prepared frozen sections, and observed the tissue morphology and fluorescence intensity under a confocal microscope. (See attached image.) Figure 8 As shown, the fluorescence of tumor tissue and surrounding tissue captured by CLSM (FNCDs excitation wavelength: 488nm; scale bar = 100μm) is displayed. Here, a, b, and c represent three independent experiments, N represents normal tissue, and T represents tumor tissue. It can be seen that the fluorescence of the tumor site is significantly higher than that of the surrounding tissue.

[0085] Fluorescence intensity analysis of the tumor and surrounding tissues was performed using the Histogram tool in LAS X Office. CLSM images showed that 24 hours after injection, the fluorescence intensity of FNCDs in the tumor tissue (48.18±2.21) was 2.72 times that in the surrounding tissue (17.73±2.20), indicating that after entering the bloodstream, FNCDs can specifically target the tumor tissue, exhibiting a significant tumor-to-normal ratio (TNR).

[0086] Furthermore, the fluorescence images clearly delineated the boundary between the tumor and normal tissue, highlighting the potential of intravenous FNCDs for the diagnosis and treatment of malignant tumors, particularly for small lesions and tumors in situ.

[0087] 8. Thyroid fine-needle aspiration specimen staining experiment: 8.1 The method for staining thyroid fine-needle aspiration specimens is as follows: (1) Fresh thyroid puncture tissue was obtained. All tissue samples were collected with the patient's informed consent and an informed consent form was signed. The tissues were preserved in DMEM medium without serum using confocal culture dishes. (2) Add FNCDs to the culture medium, incubate for 5 min and then take fluorescence images using a confocal microscope; (3) Compare the acquired fluorescence images with the histopathological report.

[0088] 8.2 Results of staining experiment on thyroid fine needle aspiration specimen: To further evaluate the potential of FNCDs in the rapid interpretation of thyroid malignancies, this study explored their role in the visualization of thyroid malignancies. We collected fresh fine-needle aspiration (FNA) samples from patients with thyroid nodules and stored them in culture medium. Subsequently, FNCDs (2.5 µg / mL) were added to the samples and incubated for 5 min. Fluorescence imaging was performed using laser confocal microscopy, and histological verification was performed using pathological reports.

[0089] Results of fine-needle aspiration staining of thyroid specimens show, see [link / reference needed]. Figure 9 As shown in b (FNCDs excitation wavelength: 488 nm; scale bar = 50 μm), some FNA samples exhibited significant green fluorescence after FNCD labeling, and pathological analysis confirmed them as papillary thyroid carcinoma (PTC). For comparison, see [link to relevant documentation]. Figure 9 As shown in Figure a (FNCDs excitation wavelength: 488 nm; scale bar = 50 μm), other FNA samples showed only weak green fluorescence at cell edges and were pathologically identified as non-malignant tumor tissue. These results demonstrate that FNCDs have the potential to rapidly and effectively identify malignant tumors in human tissues and are expected to be used for rapid in vitro imaging of human thyroid biopsy tissue, providing strong support for their application in clinical diagnosis.

[0090] The fluorine-nitrogen co-doped carbon dots (FNCDs) of the present invention have the following characteristics: 1. Hydrophilicity and biodispersibility: The FNCDs nanoparticles are spherical with a small particle size of about 5 nm. The surface is rich in functional groups such as carboxyl, amino, and hydroxyl groups, which gives them good hydrophilicity and biodispersibility, good water solubility, and strong cell membrane penetration.

[0091] 2. Epigenetic target colocalization capability: The FNCDs have the characteristic of targeting H3K36me2 histone modification in the nucleus of tumor cells. That is, the FNCDs can colocalize with the tumor-related epigenetic modification marker H3K36me2, and the colocalization intensity is significantly higher in tumor cells than in normal cells, which has the potential to detect epigenetic abnormalities.

[0092] 3. Optical properties: The fluorescence quantum yield of the fluorine-nitrogen co-doped carbon dots (FNCDs) is not less than 20%, and the emission wavelength is in the range of 500-550nm. In particular, it emits bright and stable green fluorescence at an excitation wavelength of 480nm. The longer excitation wavelength can effectively avoid interference from the autofluorescence of biological tissues and reduce phototoxicity, making it suitable for in vivo imaging.

[0093] 4. Tumor cell recognition characteristics: The FNCDs can complete the specific labeling of tumor cells within 5 minutes. That is, the FNCDs can selectively enter the nucleus of tumor cells and accumulate in the nucleolar region within 5 minutes. In normal cells, they are only distributed in the cytoplasm, showing significant cell-specific recognition ability, and do not affect normal cell proliferation.

[0094] 5. Cell cycle stability: The FNCDs maintain stable fluorescence throughout the entire cell division process and can continuously emit light in the nuclei of daughter cells, making them suitable for dynamic tracking of live cells.

[0095] 6. In vivo targeting ability of in situ tumors in mice: The enrichment intensity of the fluorine-nitrogen co-doped carbon dots (FNCDs) in tumor tissue was more than 2.72 times that in normal tissue. Animal experiments showed that the FNCDs could enter the bloodstream via intravenous injection and specifically target in situ breast tumor tissue. 24 hours after injection, fluorescence imaging of frozen sections showed that the fluorescence intensity of the FNCDs in the tumor region (48.18±2.21) was significantly higher than that in the surrounding normal tissue (17.73±2.20), and the fluorescence signal in the tumor tissue was 2.72 times that of the surrounding tissue, indicating a significant tumor-to-normal tissue ratio and clearly delineating the tumor boundary, demonstrating its potential in in vivo tumor imaging and boundary determination.

[0096] Based on the above characteristics, the fluorine-nitrogen co-doped carbon dots (FNCDs) of the present invention can be used as fluorescent labeling probes for the preparation of rapid diagnostic reagents for malignant tumors.

[0097] Furthermore, the malignant tumors include thyroid malignant tumors.

[0098] Furthermore, the rapid diagnostic reagent for malignant tumors is a rapid staining diagnostic reagent suitable for fine needle biopsy specimens.

[0099] In clinical sample studies, the FNCDs were used for rapid staining of thyroid fine-needle aspiration (FNA) samples. Significant green fluorescence was observed in the tumor tissue after only 5 minutes of incubation. Pathological examination confirmed the tumor as papillary carcinoma (PTC). Non-malignant samples showed only weak peripheral fluorescence. These results validate that the FNCDs possess excellent tumor recognition capabilities in human tissues, providing a basis for clinical application.

[0100] The aforementioned fluorine-nitrogen co-doped carbon dots (FNCDs) can also be used to prepare tumor boundary delimitation reagents. By using the FNCDs for in vivo fluorescent labeling, tumor boundaries can be delimited.

[0101] The aforementioned fluorine-nitrogen co-doped carbon dots (FNCDs) can also be used to prepare rapid intraoperative pathological detection reagents for tumors. By using the FNCDs to rapidly stain fresh tissue specimens, rapid intraoperative pathological detection of tumors can be achieved.

[0102] The concentrations of the FNCDs used in the above experiments are as follows: (1) Cell staining FNCDs concentration: 25µg / mL; (2) Injection solution via tail vein in mice: 5µg / g; (3) Thyroid biopsy tissue staining concentration: 2.5µg / mL.

[0103] The reagents and instruments used in the above experiments are as follows: Table 1. Reagents, kits, and manufacturers used in the experiment. Table 2 Information on antibodies used in experiments Table 3 Experimental Equipment Information The experimental cells are as follows: The cells were grown in an incubator at 37°C, 5% CO2 concentration, and 90% humidity.

[0104] Table 4. Cell information and culture conditions used in the experiment The experimental animals are as follows: The experimental mice used in the above experiments were 4-6 week old female BALB / c mice.

[0105] The human thyroid gland is structured as follows: Fresh thyroid tissue was obtained via fine-needle aspiration biopsy. Informed consent was obtained from all patients, and informed consent forms were signed. Tissues were preserved in confocal culture dishes in serum-free DMEM medium.

[0106] The data analysis results are as follows: Data analysis was performed using GraphPad Prism 9.0 statistical software. All experiments were performed with at least three independent biological replicates. Data are presented as mean ± standard deviation (Mean ± SD). Before performing the two-tailed Student's t-test, the Shapiro-Wilk test was used to confirm normality (P > 0.05), and the F-test was used to verify homogeneity of variance between groups (P > 0.05). The statistical significance threshold was set at P < 0.05, and Bonferroni correction was used for comparisons among multiple groups.

[0107] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing fluorine-nitrogen co-doped carbon dots, characterized in that, The method involves a one-step hydrothermal synthesis using fluorine-containing and nitrogen-containing precursors, including the following steps: Step 1) Glycine, 2,4-difluorobenzoic acid, ethanol and ultrapure water are added sequentially to a stainless steel autoclave lined with polytetrafluoroethylene in a molar ratio of 2.65:1:5.44:220.16 to form a reaction solution. Step 2) After sealing the autoclave, place it in a heating device and heat it for a period of time to ensure that the reaction solution reacts completely; Step 3) After the reaction is complete, allow the reaction solution to cool naturally, and then dialyze the reaction solution to remove small molecule byproducts and unreacted raw materials. Step 4) The dialyzed reaction solution is freeze-dried to obtain a dry orange powder, namely nitrogen co-doped carbon dots.

2. The method for preparing fluorine-nitrogen co-doped carbon dots according to claim 1, characterized in that: In step 2, the heating temperature is 160℃ and the heating time is 4 hours.

3. The method for preparing fluorine-nitrogen co-doped carbon dots according to claim 1, characterized in that: In step 3, after the reaction solution is naturally cooled to 25°C, it is then transferred to a dialysis bag with a molecular weight cutoff of 500 Da for dialysis.

4. The method for preparing fluorine-nitrogen co-doped carbon dots according to claim 1, characterized in that: In step 4, the freeze-drying conditions are −60℃ and 10Pa.

5. A fluorine-nitrogen co-doped carbon dot, characterized in that: The carbon dots are prepared by the method described in any one of claims 1-4. The carbon dots are spherical nanoparticles with a particle size of 5 nm. The surface is rich in functional groups of carboxyl, amino and hydroxyl groups, and they have the characteristic of targeting the modification of H3K36me2 histone in the nucleus of tumor cells.

6. The application of fluorine-nitrogen co-doped carbon dots prepared by the method of any one of claims 1-4 and / or the fluorine-nitrogen co-doped carbon dots of claim 5 as fluorescently labeled probes in the preparation of diagnostic reagents for malignant tumors.

7. The application according to claim 6, characterized in that: The malignant tumors include thyroid malignant tumors.

8. The application according to claim 6, characterized in that: The malignant tumor diagnostic reagent is a rapid staining diagnostic reagent suitable for fine needle biopsy specimens.

9. The application of fluorine-carbon co-doped carbon dots prepared by the method of any one of claims 1-4 and / or the fluorine-nitrogen co-doped carbon dots of claim 5 in the preparation of tumor boundary delimiting reagents.

10. The application of fluorocarbon co-doped carbon dots prepared by the method of any one of claims 1-4 and / or the fluorocarbon co-doped carbon dots of claim 5 in the preparation of rapid pathological detection reagents for tumor surgery.

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