A method for rapidly synthesizing red fluorescent carbon dots based on room temperature schiff base reaction

Abstract

The application discloses a method for rapidly synthesizing red fluorescent carbon dots based on room-temperature Schiff base reaction, and belongs to the technical field of fluorescent carbon dot materials. The method uses 1,4-dihydroxyanthraquinone as a carbon source and ethylenediamine as a nitrogen source, fully dissolves in an organic solvent, realizes molecular condensation through a Schiff base reaction at room temperature, and completes in-situ construction of red fluorescent carbon dots with mild oxidation. The application takes room-temperature Schiff base reaction as a core driving mechanism, does not need high-temperature and high-pressure conditions and complex energy input, has controllable reaction process, short cycle, low energy consumption and simple process, and has excellent repeatability and industrial amplification potential. The obtained red fluorescent carbon dots have a fluorescent emission peak center at about 650 nm, an emission tail peak extending to the near-infrared I region, a fluorescent quantum yield of 15%, excellent light stability and environmental tolerance, and are suitable for the field of biological imaging, and have wide application prospects.

Description

Technical Field

[0001] This invention relates to the field of fluorescent carbon dot materials technology, specifically a method for rapidly synthesizing red fluorescent carbon dots based on a room-temperature Schiff base reaction. The method of this invention does not require high temperature and high pressure and is suitable for bioimaging scenarios. Background Technology

[0002] Fluorescent carbon dots (CDs) are a novel class of zero-dimensional carbon nanomaterials that have shown broad application prospects in biomedicine, environmental monitoring, fluorescence sensing, and anti-counterfeiting due to their excellent optical properties, biocompatibility, low cytotoxicity, and chemical stability. Among them, red fluorescent carbon dots (emission wavelength 600nm~700nm), whose emission light is located in the red region of the visible light spectrum, have stronger biological tissue penetration capabilities compared to blue-green carbon dots. They can effectively reduce the absorption and scattering of light signals by biological tissues such as hemoglobin and water, while also reducing autofluorescence interference, showing significant advantages in in vivo fluorescence imaging, deep tissue detection, and related optical applications. With the development of bioimaging and optical detection technologies, higher requirements are being placed on the mildness of synthesis conditions, preparation efficiency, and optical performance stability of red fluorescent carbon dots.

[0003] Currently, the preparation methods for red fluorescent carbon dots have made some progress, but there are still areas for improvement. Existing mainstream methods are mainly hydrothermal and solvothermal methods, which typically require high-temperature conditions of 160℃~220℃ and rely on specialized equipment such as high-pressure reactors. These methods have high energy consumption, stringent equipment requirements, and certain safety risks, and are also not conducive to the large-scale scaling of the reaction system. Furthermore, the high-temperature synthesis process usually takes 8h~24h, a long reaction cycle, which can lead to excessive oxidation or uneven carbonization of functional groups on the surface of the red fluorescent carbon dots, thus affecting the stability of their fluorescence performance and even causing emission wavelength drift.

[0004] To overcome the shortcomings of high-temperature synthesis methods, studies have attempted to prepare red fluorescent carbon dots at room temperature, such as ultrasound-assisted and microwave-assisted methods. Although these methods avoid high-temperature and high-pressure conditions, the fluorescence quantum yield of some red fluorescent carbon dots synthesized at room temperature is still relatively low, and their stability and environmental adaptability need to be improved, making it difficult to meet the requirements of high-sensitivity imaging and complex system applications. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to overcome the core problems of "harsh reaction conditions, long cycles, and poor fluorescence performance of room-temperature synthesized products" in current red fluorescent carbon dot synthesis techniques. It provides a method for the rapid synthesis of red fluorescent carbon dots based on a room-temperature Schiff base reaction. Through innovative raw material systems and a mild synthesis strategy, it achieves efficient and controllable preparation of red fluorescent carbon dots. The resulting red fluorescent carbon dots exhibit stable long-wavelength fluorescence emission characteristics, specifically suited to the application needs of the bioimaging field, providing high-performance and easily prepared fluorescent probe materials for biomedical imaging.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution: This invention protects a method for the rapid synthesis of red fluorescent carbon dots based on a room-temperature Schiff base reaction, comprising the following steps: Using 1,4-dihydroxyanthraquinone, a hydroxyanthraquinone derivative, as the carbon source and ethylenediamine, a primary amine, as the nitrogen source, the nitrogen source was added dropwise to the carbon source solution and stirred until homogeneous to obtain a precursor solution. An oxidant was added to the precursor solution, and a Schiff base reaction and in-situ carbon dot construction were carried out at room temperature for 50 min to 1 h. If the reaction time was too short, red fluorescence could not be generated, resulting in a crude product system. In a solvent, molecular condensation was achieved through a room temperature Schiff base reaction, supplemented by mild oxidation (air), to complete the in-situ construction of red fluorescent carbon dots. After purification by antisolvent method, the target product was obtained. The technical route focuses on "mild, rapid, and high fluorescence performance," and is corely adapted to bioimaging applications.

[0007] The reaction principle is as follows: In air, hydroxyanthraquinone can be rapidly oxidized to generate an unstable quinone intermediate. This intermediate undergoes a Michael addition reaction with a primary amine, followed by dehydration condensation to form a Schiff base. This process is the key step in the formation of red fluorescent carbon dots. From a molecular structural perspective, the Schiff base derivative possesses a rigid aromatic π-conjugated structure. The hydroxyl group not only participates in the coordination process but also promotes electron transfer in the Schiff base reaction. The presence of the conjugated framework provides the structural basis for achieving long-wavelength fluorescence emission of approximately 650 nm after the formation of red fluorescent carbon dots. Using ethylenediamine as a nitrogen source allows for participation in the Schiff base reaction through the formation of an imine bond (C=N) between the amino group and the carbon source. Furthermore, N doping allows for precise control of the electronic structure of the red fluorescent carbon dots, significantly optimizing red fluorescence performance. This reaction system can proceed efficiently at room temperature without additional energy input, fully demonstrating the advantages of mild reaction conditions and environmental friendliness.

[0008] In some specific embodiments, the solvent in the carbon source solution is N,N-dimethylformamide (DMF), ethanol, or dimethyl sulfoxide (DMSO).

[0009] In some specific embodiments, the mass-to-volume ratio of 1,4-dihydroxyanthraquinone, ethylenediamine, and solvent in the precursor solution is 0.8 g to 1.2 g : 1.5 mL to 2.5 mL : 80 mL to 120 mL.

[0010] In some specific embodiments, after the reaction is complete, an antisolvent is added to the crude product system. The antisolvent is a 25mM PBS aqueous solution with a pH of 7.2-7.4, and the volume ratio of the antisolvent to the solvent in the precursor solution is 1-3:1. Subsequently, centrifugation is performed at 8000-12000 rpm for 5-10 minutes, and the precipitate (crude product with red fluorescent carbon dots) is collected. The precipitate is redissolved in DMF, and the above antisolvent-centrifugation step is repeated 1-2 times to further remove unreacted raw materials and small molecule impurities.

[0011] In some specific embodiments, the purified precipitate is freeze-dried at -40℃ to -60℃ and a vacuum of 10Pa to 30Pa for 12h to 24h to obtain solid powdered red fluorescent carbon dots, which are convenient for storage and application in bioimaging experiments.

[0012] This invention also protects the red fluorescent carbon dots, which are prepared using the above-described method; In some specific embodiments, the red fluorescent carbon dots are composed of four elements: C, H, O, and N. The average particle size of the red fluorescent carbon dots is 2.72 ± 0.64 nm, with a particle size range of 2.08 nm to 3.36 nm. The fluorescence emission peak of the red fluorescent carbon dots is centered at 650 nm, and the emission tail peak extends into the near-infrared I region. The fluorescence quantum yield of the red fluorescent carbon dots is 15%, and they exhibit excellent photostability and environmental tolerance.

[0013] This invention also protects the application of red fluorescent carbon dots in the preparation of biomedical imaging fluorescent probes.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. Addressing the core problems of existing red fluorescent carbon dot synthesis techniques, namely "harsh reaction conditions, long cycles, and poor fluorescence performance of synthesized products at room temperature," this invention aims to provide a method for the rapid synthesis of red fluorescent carbon dots based on a room-temperature Schiff base reaction. This method uses a hydroxyanthraquinone derivative as the carbon source and a primary amine compound as the nitrogen source. Under room-temperature conditions, the Schiff base reaction serves as the core driving mechanism, achieving in-situ molecular condensation and carbon dot construction. It eliminates the need for high-temperature, high-pressure equipment or complex energy input, offering a controllable reaction process, short cycle time, low energy consumption, and simple technology, while also possessing excellent reproducibility and industrial scale-up potential. This method significantly shortens the reaction time (less than 1 hour), reduces energy consumption and operational difficulty, improves preparation efficiency and process reproducibility, and yields red fluorescent carbon dots with an emission peak at approximately 650 nm, a tail peak extending into the near-infrared I region, and good stability. This meets the needs of bioimaging and fluorescence sensing applications and provides technical support for the large-scale preparation of red fluorescent carbon dots.

[0015] 2. The synthesis conditions of this invention are mild and break through the dependence on high temperature: with the Schiff base reaction at room temperature as the core, there is no need for special equipment such as high pressure reactors and high temperature furnaces. The synthesis can be completed at room temperature of 25°C and normal temperature and pressure. The operation is safe and the energy consumption is low, which solves the problems of "high temperature and high pressure, high energy consumption and high risk" of traditional hydrothermal / solvothermal methods.

[0016] 3. The red fluorescent carbon dots of this invention have high reaction efficiency and are suitable for large-scale production: the reaction cycle is less than 1 hour, which is much shorter than the traditional high-temperature synthesis method (4h~24h), and the process steps are simple (dissolution-reaction-antisolvent purification-drying), without the need for complex post-processing, with good repeatability and potential for industrial scale-up.

[0017] 4. The red fluorescent carbon dots of this invention have excellent fluorescence performance and are precisely suited for imaging applications: the emission peak of the obtained red fluorescent carbon dots is concentrated in the 650nm red band, and the tail peak extends to the near-infrared I region. The fluorescence quantum yield reaches 15%, and the photostability and environmental tolerance are excellent. It can effectively reduce background fluorescence interference in bioimaging and improve the penetration effect of deep tissues, thus solving the problems of low fluorescence quantum yield and poor stability of some room temperature synthesized red fluorescent carbon dots.

[0018] 5. The raw materials and processes for preparing the red fluorescent carbon dots of this invention are environmentally friendly and have high application safety: DMF, ethanol or DMSO are used as solvents, and air is used as a mild oxidant. There are no toxic or harmful reagent residues, and the product has good biocompatibility (suitable for bioimaging applications). Moreover, the raw material solvent, 1,4-dihydroxyanthraquinone and ethylenediamine are all common chemicals with wide availability and controllable costs, taking into account both environmental protection and practical application, and the reaction cost is extremely low. Attached Figure Description

[0019] Figure 1This is a transmission electron microscope (TEM) characterization image of the red fluorescent carbon dots in Example 1.

[0020] Figure 2 This is a super-resolution transmission electron microscope (HR-TEM) characterization image of the red fluorescent carbon dots in Example 1.

[0021] Figure 3 The Fourier transform infrared (FT-IR) spectrum of the red fluorescent carbon dot in Example 1 is shown.

[0022] Figure 4 The image shows the UV-Vis absorption spectrum of the red fluorescent carbon dot ethanol solution in Example 1.

[0023] Figure 5 The excitation and emission spectra of the red fluorescent carbon dot ethanol solution in Example 1 are shown.

[0024] Figure 6 The fluorescence emission spectra of the red fluorescent carbon dots in DMSO, DMF, pyridine, ethyl acetate, ethanol, and dichloromethane, as well as the fluorescence emission spectrum of the same concentration of Rhodamine B in ethanol solution, are shown in Example 1. These spectra are used to calculate the fluorescence quantum yield of the red fluorescent carbon dots.

[0025] Figure 7 The image shows a laser confocal image of HeLa cells after DAPI staining and incubation with red fluorescent carbon dots from Example 1. The left image is a blue fluorescent image of the cell nucleus stained with DAPI, the middle image is a red fluorescent image of the red fluorescent carbon dots from Example 1, and the right image is a merged image of the left and middle images.

[0026] Figure 8 Fluorescence imaging of various organs in mice 24 hours after oral administration of red fluorescent carbon dots from Example 1 (Red channel, 590nm excitation, HB630 red filter).

[0027] Figure 9 The fluorescence intensity of each organ was statistically analyzed and quantitatively shown as a percentage of fluorescence intensity in each organ 24 hours after oral administration of the red fluorescent carbon dots of Example 1 to mice. Detailed Implementation

[0028] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0029] This invention uses 1,4-dihydroxyanthraquinone as the carbon source and ethylenediamine as the nitrogen source to carry out a Schiff base reaction and in-situ carbon dot construction under an air atmosphere to obtain red fluorescent carbon dots. Through a mild and efficient synthesis strategy and a high-performance product design, this invention not only fills the technological gap in the large-scale preparation of high-performance red fluorescent carbon dots at room temperature, providing a new paradigm for the green synthesis of nanofluorescent materials, but also provides a novel probe with high adaptability and transformation potential for the field of biomedical imaging. It has important scientific significance and practical value for promoting the transformation of nanomaterials from basic research to clinical applications and expanding the application boundaries of red fluorescent carbon dots.

[0030] The technical solution of the present invention will be studied using the following embodiments. The specific research methods and results are shown below: Example 1 A method for rapid synthesis of red fluorescent carbon dots based on room temperature Schiff base reaction includes the following steps: S1. Raw material preparation: Select 1,4-dihydroxyanthraquinone (carbon source), ethylenediamine (nitrogen source), and N,N-dimethylformamide (DMF, solvent). The purity of 1,4-dihydroxyanthraquinone is ≥98%, the purity of ethylenediamine is ≥99%, and DMF is analytical grade.

[0031] S2. Preparation of precursor solution: In air, accurately weigh 1,4-dihydroxyanthraquinone: ethylenediamine: DMF in a mass-to-volume ratio of 1.0 g: 2.0 mL: 100 mL. Add 1.0 g of 1,4-dihydroxyanthraquinone to 100 mL of DMF, place on a magnetic stirrer, and stir at 400 rpm until the 1,4-dihydroxyanthraquinone is completely dissolved, resulting in a uniform and transparent carbon source solution. Then, add 2.0 mL of ethylenediamine dropwise to the carbon source solution, and continue stirring at 400 rpm until the mixture is homogeneous to form the precursor solution.

[0032] S3. Schiff base reaction and carbon dot construction at room temperature: The precursor solution was placed at room temperature of 25°C under natural ventilation (air as oxidant) and stirred at 400 rpm for 50 min. During this period, the solution color gradually changed from yellowish-brown to dark red, completing the in-situ construction of red fluorescent carbon dots and obtaining the crude product system.

[0033] S4. Purification by antisolvent method: Add 25mM PBS aqueous solution with pH 7.4 to the crude product system as antisolvent. The volume ratio of antisolvent to DMF is 2:1. Stir and mix at 400 rpm to allow the red fluorescent carbon dots to be fully precipitated. Then place the mixture in a high-speed centrifuge and centrifuge at 10,000 rpm for 5 min. Collect the dark red precipitate at the bottom (crude carbon dots).

[0034] S5. Secondary purification and drying: The crude carbon dots were redissolved in 5 mL of DMF, and the above antisolvent-centrifugation steps were repeated once to further remove unreacted raw materials and small molecule impurities. Finally, the purified precipitate was transferred to a freeze dryer and freeze-dried at -50℃ and 20 Pa vacuum for 18 h to obtain a deep red solid powder of red fluorescent carbon dots.

[0035] Example 2 A method for rapid synthesis of red fluorescent carbon dots based on room temperature Schiff base reaction includes the following steps: S1. Raw material preparation: Select 1,4-dihydroxyanthraquinone (carbon source), ethylenediamine (nitrogen source), and N,N-dimethylformamide (DMF, solvent). The purity of 1,4-dihydroxyanthraquinone is ≥98%, the purity of ethylenediamine is ≥99%, and DMF is analytical grade.

[0036] S2. Preparation of precursor solution: In air, accurately weigh 0.8g of 1,4-dihydroxyanthraquinone: ethylenediamine: DMF in a mass-to-volume ratio of 0.8g: 1.5mL: 80mL. Add 0.8g of 1,4-dihydroxyanthraquinone to 80mL of DMF and place the solution on a magnetic stirrer. Stir at 400rpm until the 1,4-dihydroxyanthraquinone is completely dissolved, resulting in a homogeneous and transparent carbon source solution. Then, add 1.5mL of ethylenediamine dropwise to the carbon source solution and continue stirring at 400rpm until the mixture is homogeneous, forming the precursor solution.

[0037] S3. Schiff base reaction and carbon dot construction at room temperature: The precursor solution was placed at room temperature of 25°C under natural ventilation (air as oxidant) and stirred at 400 rpm for 1 hour. During this period, the solution color gradually changed from yellowish-brown to dark red, completing the in-situ construction of red fluorescent carbon dots and obtaining the crude product system.

[0038] S4. Purification by antisolvent method: Add 25mM PBS aqueous solution with pH 7.3 to the crude product system as antisolvent. The volume ratio of antisolvent to DMF is 1:1. Stir and mix at 400 rpm to allow the red fluorescent carbon dots to be fully precipitated. Then place the mixture in a high-speed centrifuge and centrifuge at 10,000 rpm for 5 min. Collect the dark red precipitate at the bottom (crude carbon dots).

[0039] S5. Secondary purification and drying: The crude carbon dots were redissolved in 5 mL of DMF, and the above antisolvent-centrifugation steps were repeated once to further remove unreacted raw materials and small molecule impurities. Finally, the purified precipitate was transferred to a freeze dryer and freeze-dried at -50℃ and 20 Pa vacuum for 18 h to obtain a deep red solid powder of red fluorescent carbon dots.

[0040] Example 3 A method for rapid synthesis of red fluorescent carbon dots based on room temperature Schiff base reaction includes the following steps: S1. Raw material preparation: Select 1,4-dihydroxyanthraquinone (carbon source), ethylenediamine (nitrogen source), and N,N-dimethylformamide (DMF, solvent). The purity of 1,4-dihydroxyanthraquinone is ≥98%, the purity of ethylenediamine is ≥99%, and DMF is analytical grade.

[0041] S2. Preparation of precursor solution: In air, accurately weigh 1,4-dihydroxyanthraquinone: ethylenediamine: DMF in a mass-to-volume ratio of 1.2g: 2.5mL: 120mL. Add 1.2g of 1,4-dihydroxyanthraquinone to 120mL of DMF, place on a magnetic stirrer, and stir at 400rpm until the 1,4-dihydroxyanthraquinone is completely dissolved, resulting in a uniform and transparent carbon source solution. Then, add 2.5mL of ethylenediamine dropwise to the carbon source solution, and continue stirring at 400rpm until the mixture is homogeneous to form the precursor solution.

[0042] S3. Schiff base reaction and carbon dot construction at room temperature: The precursor solution was placed at room temperature of 25°C under natural ventilation (air as oxidant) and stirred at 400 rpm for 55 min. During this period, the solution color gradually changed from yellowish-brown to dark red, completing the in-situ construction of red fluorescent carbon dots and obtaining the crude product system.

[0043] S4. Purification by antisolvent method: Add 25mM PBS aqueous solution with pH 7.2 as antisolvent to the crude product system. The volume ratio of antisolvent to DMF is 3:1. Stir and mix at 400 rpm to allow the red fluorescent carbon dots to be fully precipitated. Then place the mixture in a high-speed centrifuge and centrifuge at 10,000 rpm for 5 min. Collect the dark red precipitate at the bottom (crude carbon dots).

[0044] S5. Secondary purification and drying: The crude carbon dots were redissolved in 5 mL of DMF, and the above antisolvent-centrifugation steps were repeated once to further remove unreacted raw materials and small molecule impurities. Finally, the purified precipitate was transferred to a freeze dryer and freeze-dried at -50℃ and 20 Pa vacuum for 18 h to obtain a deep red solid powder of red fluorescent carbon dots.

[0045] In Examples 1 to 3 of this invention, red fluorescent carbon dots with stable long-wavelength fluorescence emission characteristics were prepared. The red fluorescent carbon dots of Example 1 are used as an example for further study. The specific research methods and results are as follows: I. Structural characterization of red fluorescent carbon dots: 1. Characterization method for red fluorescent carbon dots: Morphology and crystal structure characterization: The morphology and dispersibility of red fluorescent carbon dots were observed using transmission electron microscopy (TEM) with an accelerating voltage of 200 kV. The red fluorescent carbon dot samples were dispersed in ethanol, dropped onto a copper grid, and tested after air drying. The crystal fringes of the red fluorescent carbon dots were observed using super-resolution transmission electron microscopy (HR-TEM) with an accelerating voltage of 200 kV. The sample preparation method was the same as that for TEM testing.

[0046] Functional group characterization: The functional group structure of the red fluorescent carbon dots was tested using Fourier transform infrared spectroscopy (FT-IR) with a test range of 4000 cm⁻¹. -1 ~400cm -1 Samples were prepared using the KBr tableting method.

[0047] Ultraviolet-visible absorption spectroscopy characterization: The ultraviolet absorption characteristics of the red fluorescent carbon dots were tested using an ultraviolet-visible spectrophotometer. The red fluorescent carbon dots were dissolved in ethanol to prepare a solution with a concentration of 50 μg / mL. The test wavelength range was 400 nm to 700 nm.

[0048] Fluorescence spectroscopy characterization: The excitation and emission spectra of the red fluorescent carbon dots were measured using a fluorescence spectrophotometer. The red fluorescent carbon dots were dissolved in ethanol to prepare a solution with a concentration of 50 μg / mL. The excitation wavelength scanning range was 400 nm to 620 nm, the emission wavelength scanning range was 600 nm to 800 nm, and the slit width was 5 nm.

[0049] Fluorescence quantum yield (QY) test: Using Rhodamine B (ethanol solution, QY=65%) as the reference, red fluorescent carbon dots were prepared in concentrations of 50 μg / mL in ethanol, DMSO, DMF, pyridine, ethyl acetate, and dichloromethane, as well as in an ethanol solution of the same concentration. The fluorescence emission spectra of each solution were measured at an excitation wavelength of 545 nm. The fluorescence quantum yield of the red fluorescent carbon dots was calculated using the formula: Among them, QY x QY represents the fluorescence quantum yield of red fluorescent carbon dots. std For the fluorescence quantum yield of Rhodamine B (65%), I x I represents the integral intensity of fluorescence emission from the red fluorescent carbon dot solution. std Let A be the integral intensity of the fluorescence emission of the Rhodamine B solution. x A represents the absorbance of the red fluorescent carbon dot solution at the excitation wavelength. std η represents the absorbance of Rhodamine B solution at the excitation wavelength. x η is the refractive index of the red fluorescent carbon dot solvent.std is the refractive index of ethanol.

[0050] 2. Experimental Results: (1) Morphology and dispersion analysis: Figure 1 The image shows the TEM characterization of the red fluorescent carbon dots. As can be seen from the image, the synthesized red fluorescent carbon dots exhibit a near-spherical morphology, uniform particle size, no obvious agglomeration, and good dispersibility. ImageJ software was used to analyze the particle size of 100 red fluorescent carbon dots, yielding an average particle size of 2.72 ± 0.64 nm and a particle size range of 2.08 nm to 3.36 nm, consistent with the product characteristics described in the invention. This demonstrates that the synthesis method of this invention can prepare red fluorescent carbon dot materials with uniform size and excellent dispersibility.

[0051] (2) Crystal structure analysis: Figure 2 The image shows the HR-TEM characterization of the red fluorescent carbon dots. As can be seen from the image, the red fluorescent carbon dot material exhibits clear lattice fringes with a lattice spacing of 0.21 nm, corresponding to sp... 2 The (100) crystal plane of the hybrid carbon core indicates that a carbon core with an ordered crystal structure was successfully constructed through a room-temperature Schiff base reaction and mild oxidation with air, providing a stable structural basis for fluorescence emission.

[0052] (3) Functional group analysis: Figure 3 The image shows the FT-IR spectrum of the red fluorescent carbon dots. As can be seen from the figure, at 3420 cm⁻¹... -1 A broad absorption peak appears nearby, corresponding to the stretching vibration of the hydroxyl group (-OH); 3280 cm⁻¹ -1 A characteristic absorption peak appears nearby, corresponding to the stretching vibration of the amino group (-NH2); 1635 cm⁻¹ -1 A strong absorption peak appears nearby, corresponding to the stretching vibration of the imine bond (C=N); 1580 cm⁻¹ -1 1490cm -1 The nearby absorption peaks correspond to the vibrations of the aromatic π-conjugated skeleton. The above characteristic peaks prove that 1,4-dihydroxyanthraquinone successfully undergoes a Schiff base reaction with ethylenediamine to form a structure containing C=N bonds; the surface of the red fluorescent carbon dots retains hydroxyl and amino functional groups and has a rigid aromatic π-conjugated structure, which is consistent with the innovative basis for the selection of raw materials and the reaction mechanism in the invention.

[0053] (4) Analysis of ultraviolet absorption characteristics: Figure 4The image shows the UV-Vis absorption spectrum of the red fluorescent carbon dots in ethanol solution. As can be seen, the red fluorescent carbon dots exhibit three distinct characteristic absorption peaks in the visible region, located at 517 nm, 545 nm, and 588 nm, respectively. These absorption peaks originate from the π→π* electronic transitions in the π-conjugated structure of the carbon dots, and correspond well with the subsequent fluorescence excitation spectrum, demonstrating that the red fluorescent carbon dots possess specific optical absorption characteristics, providing the electronic transition basis for red fluorescence emission.

[0054] (5) Fluorescence spectroscopy analysis: Figure 5 The excitation and emission spectra of the red fluorescent carbon dots in ethanol solution are shown. The excitation spectrum reveals that when the emission wavelength is monitored at 650 nm, the red fluorescent carbon dots exhibit three excitation peaks at 517 nm, 545 nm, and 588 nm, perfectly matching the positions of the ultraviolet absorption peaks, proving that this absorption peak can effectively excite the red fluorescent carbon dots to produce fluorescence. The emission spectrum shows that when the excitation wavelengths are 517 nm, 545 nm, and 588 nm, the red fluorescent carbon dots exhibit two main emission peaks, located at 624 nm and 650 nm, respectively. The 650 nm peak is the strongest, with the emission tail extending into the near-infrared I region (700 nm~800 nm), consistent with the optical characteristics of red fluorescent carbon dots. Furthermore, the symmetrical fluorescence peak shape indicates that the fluorescence emission of the red fluorescent carbon dots is stable and concentrated.

[0055] (6) Fluorescence quantum yield analysis: Figure 6 The fluorescence emission spectra of the red fluorescent carbon dots in DMSO, DMF, pyridine, ethyl acetate, ethanol, and dichloromethane, as well as the fluorescence emission spectrum of a Rhodamine B ethanol solution of the same concentration, are shown. Calculations using Rhodamine B as a reference show that the red fluorescent carbon dots exhibit stable fluorescence emission performance in all six solvents, with a fluorescence quantum yield (QY) of approximately 15%. Specifically, the QY in the ethanol solution is 15.2%, demonstrating that the synthesized red fluorescent carbon dots have high fluorescence quantum yield, strong solvent adaptability, and excellent optical stability, fully meeting the application requirements in the field of bioimaging.

[0056] II. Imaging capability of red fluorescent carbon dots: 1. Experimental Methods (1) HeLa cell fluorescence imaging experiment: ① Cell Culture: HeLa cells (human cervical cancer cells) were seeded in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics and cultured at 37°C in a 5% CO2 incubator until the logarithmic growth phase. ② Carbon Dot Incubation: HeLa cells in the logarithmic growth phase were incubated at 5 × 10⁻⁶ cells / year. 4Cells were seeded at a density of 1 cell / well in laser confocal microscopy-specific cell culture dishes and cultured for 24 hours to allow cell adhesion. The original culture medium was then removed, and fresh culture medium containing red fluorescent carbon dots (final concentration of red fluorescent carbon dots 50 μg / mL) was added. The cells were then incubated at 37°C and 5% CO2 for 4 hours to allow for full uptake of the red fluorescent carbon dots. ③ DAPI staining: After incubation, cells were washed three times with phosphate-buffered saline (PBS, pH 7.4) for 5 minutes each time to remove unuptaken free red fluorescent carbon dots. DAPI staining solution (final concentration 1 μg / mL) was added, and staining was performed at room temperature in the dark for 5 minutes to label the cell nuclei. After staining, cells were washed three times with PBS, and finally, 2 mL of fresh PBS was added to maintain cell viability. ④ Confocal Imaging: Imaging was performed using a laser confocal scanning microscope. The DAPI excitation wavelength was 405 nm, and the emission wavelength acquisition range was 420 nm to 480 nm (blue fluorescence). The excitation wavelength for red fluorescent carbon dots was 545 nm, and the emission wavelength acquisition range was 620 nm to 680 nm (red fluorescence). The same laser intensity, gain, and exposure time were set to acquire images of the DAPI channel, carbon dot channel, and merged image, respectively. Figure 7 ).

[0057] (2) Imaging experiment on organ distribution after oral administration of red fluorescent carbon dots to mice: ① Experimental animals: 6-8 week old SPF-grade C57 mice (weighing 20g-25g) were selected and acclimatized for 3 days before the experiment. ② Carbon dot administration: Red fluorescent carbon dots were dissolved in physiological saline to prepare a carbon dot suspension with a concentration of 1mg / mL. After fasting for 12 hours, the mice were administered the drug orally via gavage at a dose of 10mg / kg (calculated based on body weight). ③ Organ collection: 24 hours after administration, the mice were euthanized by cervical dislocation. The main organs, including the heart, liver, spleen, lungs, kidneys, and brain, were quickly dissected and separated. The organs were rinsed with physiological saline to remove surface blood, and the moisture was absorbed with filter paper to maintain their integrity. ④ In vivo imaging: The separated organs were arranged sequentially on a black glass slide. Fluorescence imaging was performed using a small animal in vivo imaging system. The Red imaging channel was selected, and the excitation wavelength was set to 590nm (narrow bandwidth excitation filter, bandwidth 30nm). An HB630 long-pass red filter was used (cutoff wavelength 630nm, transmission band 630nm~800nm). The exposure time was 10s, and fluorescence intensity images of each organ were acquired. Figure 8 This filter effectively filters out interference from 590nm excitation light, specifically acquiring the red fluorescence emission signal of red fluorescent carbon dots, avoiding the influence of the autofluorescence background of biological tissues. ⑤ Data Analysis: The fluorescence integral intensity of each organ is analyzed using the software built into the imaging system. The proportion of each organ's fluorescence intensity to the total fluorescence intensity is calculated, and a statistical bar chart of organ fluorescence integral intensity is plotted. Figure 9), to quantitatively evaluate the distribution characteristics of red fluorescent carbon dots in vivo.

[0058] 2. Experimental Results (1) Results of fluorescence imaging of HeLa cells: Figure 7 The images show laser confocal imaging of HeLa cells after DAPI staining and incubation with red fluorescent carbon dots. The left image shows the DAPI staining channel, where the cell nuclei exhibit clear blue fluorescence with intact outlines, indicating good staining. The middle image shows the red fluorescent carbon dot channel, where uniformly distributed red fluorescence appears within the cells, with strong and concentrated fluorescence signals and no significant background interference, demonstrating that the red fluorescent carbon dots can be effectively taken up by HeLa cells and maintain stable fluorescence emission within the cells. The right image shows the merged image, where the fluorescence boundary between the blue cell nuclei and the red fluorescent carbon dots is clear, with no obvious fluorescence overlap or quenching, indicating that the red fluorescent carbon dots are mainly distributed in the cytoplasm and that their red fluorescence has good specificity, enabling clear imaging at the cellular level and meeting the application requirements for cell tracking and localization.

[0059] (2) Imaging and statistical results of organ distribution in mice: Figure 8 Fluorescence imaging of various organs in mice 24 hours after oral administration of red fluorescent carbon dots. Figure 9 This is a bar chart showing the statistical analysis of the integrated fluorescence intensity of the corresponding organs. Figure 8 and Figure 9 The results show that, under the specific detection conditions of a 590nm excitation wavelength and an HB630 red filter, the red fluorescent carbon dots exhibit specific distribution characteristics in vivo: the fluorescence intensity in brain and kidney tissues is significantly higher than in other organs, accounting for 28.3% and 32.5% of the fluorescence integral intensity, respectively, demonstrating a significant enrichment effect; the fluorescence integral intensity in liver tissue accounts for 8.2%, showing a small amount of enrichment; only weak fluorescence was detected in the heart, spleen, and lungs, accounting for 4.1%, 3.7%, and 2.4%, respectively, all below 5%. These results clearly indicate that after oral administration, the red fluorescent carbon dots can efficiently reach and accumulate in kidney and brain tissues through in vivo metabolic pathways, while exhibiting a small distribution in liver tissue and a relatively small distribution in other organs. The effective enrichment in brain tissue, in particular, suggests its potential ability to cross the blood-brain barrier. This provides important experimental evidence for the subsequent design of fluorescent imaging probes for brain diseases (such as brain tumors and neurodegenerative diseases) and the development of targeted imaging materials that cross the blood-brain barrier, significantly expanding the application prospects of red fluorescent carbon dots in high-end bioimaging for the detection of brain lesions.

[0060] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0061] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

Claims

1. A method for rapid synthesis of red fluorescent carbon dots based on room temperature Schiff base reaction, characterized in that, Includes the following steps: Using 1,4-dihydroxyanthraquinone as a carbon source and ethylenediamine as a nitrogen source, the nitrogen source was added dropwise to the carbon source solution and stirred until homogeneous to obtain a precursor solution. An oxidant was added to the precursor solution, and a Schiff base reaction and in-situ carbon dot construction were carried out at room temperature for 50 min to 1 h to obtain the crude product system. After adding the antisolvent to the crude product system, the red fluorescent carbon dots were collected by centrifugation.

2. The method for rapid synthesis of red fluorescent carbon dots based on room temperature Schiff base reaction according to claim 1, characterized in that, The mass ratio of 1,4-dihydroxyanthraquinone to ethylenediamine is 0.8 g to 1.2 g : 1.5 mL to 2.5 mL.

3. The method for rapid synthesis of red fluorescent carbon dots based on room temperature Schiff base reaction according to claim 1, characterized in that, The antisolvent is a 25mM PBS aqueous solution with pH 7.2~7.4, and the volume ratio of the antisolvent to the solvent in the precursor solution is 1~3:

1.

4. The method for rapid synthesis of red fluorescent carbon dots based on room temperature Schiff base reaction according to claim 1, characterized in that, The oxidant is air under natural ventilation conditions.

5. A red fluorescent carbon dot, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 4.

6. The red fluorescent carbon dot according to claim 5, characterized in that, The red fluorescent carbon dots are composed of four elements: C, H, O, and N. The particle size range of the red fluorescent carbon dots is 2.08 nm to 3.36 nm, and the average particle size is 2.72 ± 0.64 nm.

7. The red fluorescent carbon dot according to claim 5, characterized in that, The fluorescence emission peak of the red fluorescent carbon dot is centered at 650 nm, and the emission tail peak extends into the near-infrared I region.

8. The red fluorescent carbon dot according to claim 5, characterized in that, The fluorescence quantum yield of the red fluorescent carbon dots is 15%.

9. The application of the red fluorescent carbon dot of claim 5 in the preparation of a biomedical imaging fluorescent probe.

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