A single-atom manganese-doped carbon quantum dot and its preparation method and application

Abstract

The present invention discloses a single-atom manganese-doped carbon quantum dot and a preparation method and application thereof. The preparation method uses water as a solvent, EDTA, manganese chloride tetrahydrate and ethylenediamine as raw materials to perform a hydrothermal reaction to obtain the single-atom manganese-doped carbon quantum dot; the mass ratio of the manganese chloride tetrahydrate to EDTA is 10:73. In the present invention, EDTA is used as a carbon source and a chelating agent, water is used as a solvent, and manganese chloride tetrahydrate and ethylenediamine (EDA) are used as raw materials to perform a hydrothermal reaction to obtain Mn-N2 coordinated single-atom manganese-doped carbon quantum dots, which are small in size, good in water solubility, and have good fluorescence and near-infrared imaging performance and efficient peroxidase-like activity.

Description

Technical Field

[0001] The present invention relates to the technical field of single-atom nanozyme materials, and in particular to single-atom manganese-doped carbon quantum dots and a preparation method and application thereof. Background Art

[0002] In recent years, single-atom nanozymes (SAzymes) have atomically dispersed active sites that can maximize the atomic utilization efficiency and active site density, thereby having ultra-high catalytic activity, which has attracted widespread attention from researchers and become promising candidate materials for the treatment of tumor-related diseases, antibacterial therapy, and biosensor detection. However, due to their high surface energy, isolated atoms inherently tend to migrate and aggregate into clusters, so it is still challenging to obtain atomically dispersed and stably formed single atoms from synthetic precursors. In order to solve this core problem, a series of synthetic methods have been successfully developed to prepare SAzymes. Depending on the starting metal precursor, the synthetic route of SAzymes can be divided into two types: "bottom-up" with mononuclear metal complexes as precursors and "top-down" with bulk metals or nanoparticles (NPs) as precursors. Therefore, the synthesis of SAzymes is crucial by selecting appropriate precursors and methods.

[0003] The widespread application of SAzymes faces a big obstacle, which is poor biocompatibility. Most SAzymes have poor water solubility and biocompatibility after synthesis, and require further modification with PEG, which undoubtedly hinders the application of SAzymes. Therefore, it is necessary to select suitable materials as carriers of SAzymes to improve the biocompatibility of SAzymes in vitro or in vivo and diversify their functions. Carbon-based quantum dots (CDs) are one of the effective candidate carrier materials with unique luminescence properties, good stability and tunable physicochemical properties. In addition, near-infrared carbon dots (NIR-CDs) have high penetrability, which is conducive to high-resolution imaging, providing a good foundation for their application in the biomedical field. At the same time, the excellent water solubility of CDs ensures its biosafety. Therefore, it is of great research significance to select suitable ligands to chelate with the central metal atom to realize the combination of SAzymes and CDs to make them multifunctional.

[0004] Ethylenediaminetetraacetic acid (EDTA) is an organic compound, also known as edetic acid, with a chemical formula of C 10 H 16 N2O8 is a white powder at room temperature and pressure. 2+ , Ca 2+ , Mn 2+ , Fe 2+Chelating agents combined with divalent metal ions such as chelating agents have been used in the preparation of carbon quantum dots. For example, a Chinese patent application document with publication number CN105112052A discloses a method for preparing fluorescence-magnetic resonance dual-modal carbon quantum dots, including the following method: dissolving a manganese-containing compound and ethylenediaminetetraacetic acid in a molar ratio of 1:0.05 to 1:2 in an amino compound and ethylene glycol solvent in a volume ratio of 1:10 to 10:1, and stirring magnetically at a high temperature of more than 100°C for 4h to 48h; placing the obtained solution into a permeable Dialysis is performed in a dialysis bag for 48 h to 72 h, with water replaced every 6 h. The dialysate obtained is subjected to vacuum rotary evaporation to a solid state to obtain manganese-doped carbon quantum dots, denoted as Mn-CQDs. The manganese-doped carbon quantum dots prepared by the method have diverse targeted imaging, realizing the combined use of fluorescence imaging and MRI magnetic resonance imaging technology, and the changes in fluorescence and magnetic resonance signals are intuitive and easy to operate. However, near-infrared imaging function has not been reported, and there is a lack of efficient peroxidase-like activity to achieve catalytic treatment of tumors. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to obtain a carbon quantum dot capable of near-infrared imaging and having efficient peroxidase-like activity.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] A method for preparing single-atom manganese-doped carbon quantum dots, which uses water as a solvent and EDTA, manganese chloride tetrahydrate and ethylenediamine as raw materials to carry out a hydrothermal reaction to obtain the single-atom manganese-doped carbon quantum dots; wherein the mass ratio of the manganese chloride tetrahydrate to EDTA is 10:73.

[0008] Beneficial effects: In the present invention, EDTA is used as a carbon source and a chelating agent, water is used as a solvent, and manganese chloride tetrahydrate and ethylenediamine (EDA) are used as raw materials for a hydrothermal reaction to obtain Mn-N2 coordinated single-atom manganese-doped carbon quantum dots, which have small size, good water solubility, good fluorescence and near-infrared imaging performance, and efficient peroxidase-like activity.

[0009] Preferably, the method for preparing the single-atom manganese-doped carbon quantum dots comprises the following steps: mixing manganese chloride tetrahydrate, EDTA, ethylenediamine and water to obtain a reaction solution, and subjecting the reaction solution to a hydrothermal reaction to obtain the single-atom manganese-doped carbon quantum dots.

[0010] Preferably, the mass volume ratio of the manganese chloride tetrahydrate to water is ≤2 mg:1 mL.

[0011] Preferably, the mass volume ratio of manganese chloride tetrahydrate to ethylenediamine is ≤2 mg:5 μL.

[0012] Preferably, the dosage ratio of manganese chloride tetrahydrate, EDTA, ethylenediamine and water is 40 mg: 292 mg: 100 μL: 20 ml.

[0013] Preferably, the temperature of the hydrothermal reaction is 180-200° C., and the time is ≥15 h.

[0014] Preferably, the hydrothermal reaction time is 15-20 hours.

[0015] Preferably, the water is ultrapure water.

[0016] Preferably, the method further comprises dialyzing and drying the product after the hydrothermal reaction.

[0017] Preferably, the preparation method of the single-atom manganese-doped carbon quantum dots comprises the following steps: mixing MnCl2•4H2O, EDTA, EDA and water in a dosage ratio of 40 mg: 292 mg: 100 μL: 20 mL to obtain a reaction solution; placing the obtained reaction solution in a hydrothermal reactor, reacting at 180°C for 20 hours, cooling to room temperature after the reaction, aspirating the product into a dialysis bag with a molecular weight cutoff of 3000D, placing it in water for dialysis, collecting the dialyzed aqueous solution, and freeze-drying it until the water completely disappears to obtain the single-atom manganese-doped carbon quantum dots.

[0018] The present invention also provides a single-atom manganese-doped carbon quantum dot, which is prepared by using the single-atom manganese-doped carbon quantum dot preparation method.

[0019] The present invention also proposes an application of the single-atom manganese-doped carbon quantum dots in the preparation of anti-tumor drugs.

[0020] The present invention also proposes an application of the single-atom manganese-doped carbon quantum dots in the preparation of tumor imaging agents.

[0021] The advantages of the present invention are:

[0022] (1) The single-atom manganese-doped carbon quantum dots generated by the present invention have good optical properties and optical stability and can be used for near-infrared imaging;

[0023] (2) The raw material cost used in the present invention is low, the synthesis time is short, the steps are simpler, and a large number of carbon quantum dots can be quickly synthesized in a short time, which has great prospects for application in industrial large-scale manufacturing in the future;

[0024] (3) The single-atom manganese-doped carbon quantum dots generated by the present invention are confirmed to exist in a Mn-N2 coordination structure, which ensures efficient peroxidase-like activity and has good biosafety. They show excellent ability to kill tumor cells and shrink tumor tissue in vitro and in vivo, and have great prospects in tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the synthesis steps for preparing carbon quantum dots in Example 1 of the present invention;

[0026] Figure 2 Transmission electron microscopy characterization of carbon quantum dots prepared in Example 1 of the present invention; the inset is a high-resolution image (upper right) and size distribution (lower right) of carbon quantum dots;

[0027] Figure 3 Atomic force microscopy characterization of the carbon quantum dots prepared in Example 1 of the present invention;

[0028] Figure 4 This is a high-angle annular dark-field spherical aberration electron microscope image of the carbon quantum dots prepared in Example 1 of the present invention;

[0029] Figure 5 This is the Raman spectrum of the carbon quantum dots prepared in Example 1 of the present invention;

[0030] Figure 6 This is the infrared spectrum of the carbon quantum dots prepared in Example 1 of the present invention;

[0031] Figure 7 is the X-ray photoelectron spectrum of the carbon quantum dots prepared in Example 1 of the present invention;

[0032] Figure 8 is the X-ray photoelectron spectrum Mn 2p spectrum of the carbon quantum dots prepared in Example 1 of the present invention;

[0033] Fig. 9 is the X-ray photoelectron spectrum N1 s spectrum of the carbon quantum dots prepared in Example 1 of the present invention;

[0034] Fig.10 XANEs, EXAFs characterization and Fourier transform fitting spectrum of carbon quantum dots prepared in Example 1 of the present invention (from top to bottom);

[0035] Fig.11 The UV-visible absorption spectrum and emission spectrum of the carbon quantum dots prepared in Example 1 of the present invention are characterized, and the inset is an optical image of the dispersion of the carbon quantum dots without (left picture) / irradiated with a wavelength of 365 nm (right picture);

[0036] Fig.12 The photoluminescence of the carbon quantum dots prepared in Example 1 of the present invention excited in the range of 300-440 nm;

[0037] Fig.13 After the carbon quantum dots prepared in Example 1 of the present invention are engulfed by cells, the cells generate fluorescence signals at different excitation wavelengths (405, 488, 546, and 640 nm from left to right);

[0038] Fig.14 The electron paramagnetic resonance spectrum of the carbon quantum dots prepared in Example 1 of the present invention for generating OH;

[0039] Fig.15 The cell viability of 4T1 cells treated with carbon quantum dots prepared in Example 1 of the present invention (pH = 7.4 and 6.2);

[0040] Fig.16 are digital photos of ex vivo tumors with various treatment procedures during the in vivo anti-tumor test of the present invention;

[0041] Fig.17 is the relative tumor volume of mice treated with different carbon quantum dots prepared in Example 1 of the present invention;

[0042] Fig.18 The mass and volume of the in vitro tumors of mice treated with carbon quantum dots prepared in Example 1 of the present invention;

[0043] Fig.19 This is a picture of IVIS imaging of mice in vivo under 680 nm wavelength excitation of the carbon quantum dots prepared in Example 1 of the present invention;

[0044] Fig. 20 This is a high-angle annular dark-field spherical aberration electron microscope image of the carbon quantum dots prepared in Example 2 of the present invention;

[0045] Fig.21 The electron paramagnetic resonance spectrum of the carbon quantum dots prepared in Example 2 of the present invention for generating OH;

[0046] Fig. 22 This is a high-angle annular dark-field spherical aberration electron microscope image of the carbon quantum dots prepared in Example 3 of the present invention;

[0047] Fig.23 The electron paramagnetic resonance spectrum of the carbon quantum dots prepared in Example 3 of the present invention for generating OH;

[0048] Fig.24 This is a high-angle annular dark-field spherical aberration electron microscope image of the carbon quantum dots prepared in Comparative Example 1 of the present invention;

[0049] Fig.25 This is the electron paramagnetic resonance spectrum of the carbon quantum dots prepared in Comparative Example 2 of the present invention;

[0050] Fig.26 This is the electron paramagnetic resonance spectrum of the carbon quantum dots prepared in Comparative Example 3 of the present invention;

[0051] Fig. 27 Optical images of the product prepared in Comparative Example 4 of the present invention without (left picture) / with irradiation at a wavelength of 365 nm (right picture);

[0052] Fig.28 This is a high-angle annular dark-field spherical aberration electron microscope image of the carbon quantum dots prepared in Comparative Example 5 of the present invention;

[0053] Fig.29 Optical images of carbon quantum dots prepared in Comparative Example 5 of the present invention without (left picture) / with irradiation at a wavelength of 365 nm (right picture);

[0054] Fig.30 This is the electron paramagnetic resonance spectrum of the carbon quantum dots prepared in Comparative Example 6 of the present invention;

[0055] Fig.31 This is a high-angle annular dark-field spherical aberration electron microscope image of the carbon quantum dots prepared in Comparative Example 7 of the present invention. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0058] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0059] Example 1

[0060] A method for preparing single-atom manganese-doped carbon quantum dots comprises the following steps: 40 mg of MnCl2•4H2O, 292 mg of EDTA and 100 μL of EDA are weighed respectively and dissolved in 20 mL of ultrapure water, a reaction solution is obtained by ultrasonication, the reaction solution is transferred to a 25 mL hydrothermal reactor, placed in a vacuum drying oven, heated to 180° C. and reacted for 20 hours, cooled to room temperature after the reaction is completed, the solution inside is sucked out into a dialysis bag with a molecular weight cutoff of 3000 D, and dialyzed in ultrapure water for 48 hours; during the dialysis process, the solution outside the dialysis bag is collected and new ultrapure water is added every 24 hours; the collected aqueous solution is freeze-dried until the water completely disappears, so as to obtain the prepared single-atom manganese-doped carbon quantum dots.

[0061] Figure 1The figure is a schematic diagram of the synthesis of Example 1 of the present invention. EDTA is used as the carbon source and chelating agent, water is used as the solvent, MnCl2•4H2O and EDA are used as raw materials, and the optimal reaction conditions are 180°C for 20 h in a 25 ml reactor. The synthesis mechanism is shown in the figure.

[0062] Figure 2 TEM characterization of the carbon quantum dots prepared in Example 1 of the present invention. It can be seen that the obtained carbon quantum dots are evenly dispersed, with a size distribution of about 5 nm. The high-resolution transmission electron microscope image shows a 0.22 nm interplanar spacing, which corresponds to the (100) crystal plane of graphite carbon. Figure 3 The atomic force microscopy characterization of the carbon quantum dots prepared in Example 1 of the present invention shows that the height of the carbon quantum dots is about 5 nm; Figure 4 This is a high-angle annular dark-field spherical aberration electron microscope image of the carbon quantum dots prepared in Example 1 of the present invention. It can be observed that the single atomic bright spots representing manganese atoms are evenly dispersed on the carbon quantum dots; Figure 5 This is the Raman spectrum of the carbon quantum dots prepared in Example 1 of the present invention, and the D peak and G peak belonging to the carbon material can be clearly seen; Figure 6 The infrared spectrum of the carbon quantum dots prepared in Example 1 of the present invention is shown at 3400 cm -1 The broad absorption bands were attributed to OH / NH stretching vibrations, and the remaining functional groups were identified as CN (1402 cm -1 )、C=C / C=N (1637 cm -1 ) and C-H (2919 cm -1 ).

[0063] Figure 7 The X-ray photoelectron spectrum of the carbon quantum dots prepared in Example 1 of the present invention shows the presence of Mn, C, N and O elements, and the atomic percentages (at%) of the corresponding elements are 0.94%, 73.71%, 9.79% and 15.56%, respectively; Figure 8 The X-ray photoelectron spectrum of the carbon quantum dots prepared in Example 1 of the present invention shows two main peaks at 653.1 and 641.1 eV and one characteristic peak at 645.9 eV, which belong to Mn 2p 1 / 2 、Mn 2p 3 / 2 and satellite peaks. 3 / 2 The two peaks at 641.3 and 643 eV correspond to Mn 2+ and Mn 3+ valence state; Fig. 9The X-ray photoelectron spectrum N 1s spectrum of the carbon quantum dots prepared in Example 1 of the present invention can be deconvoluted into three peaks at 399.4, 400.6 and 401.7 eV, corresponding to pyridine-N, pyrrole-N and graphite-N, respectively; Fig.10 They are respectively XANEs, EXAFs characterization and Fourier transform fitting spectrum of the carbon quantum dots prepared in Example 1 of the present invention. The XANES spectrum shows that the adsorption peak position of the carbon quantum dots is between the Mn foil and Mn2O3, which further confirms that the valence state of the Mn element is between 2 and 3 valences, which is consistent with the results of the X-ray photoelectron spectroscopy. EXAFS characterization further verifies the bonding type in the carbon quantum dots. As shown in the figure, the main peak of the Mn foil at 2.33 Å belongs to the Mn-Mn coordination, while the FT-EXAFS spectrum of Mn2O3 has obvious characteristic peaks at 1.41 Å and 2.81 Å, corresponding to Mn-O and Mn-Mn coordination, respectively, and the main peak of the EXAFS spectrum of the carbon quantum dots prepared in Example 1 is 1.78 Å, which belongs to the Mn-N coordination. After that, the Fourier change spectrum of the carbon quantum dots was fitted by Artemis, and the curve fitting results were in good agreement with the experimental data, and the carbon quantum dots in Example 1 were obtained to exist in the Mn-N2 coordination structure.

[0064] Fig.11 The UV-visible absorption spectrum and emission spectrum of the carbon quantum dots prepared in Example 1 of the present invention are characterized. The illustration is an optical image of the dispersion of carbon quantum dots without (left picture) / irradiated at a wavelength of 365 nm (right picture). It can be seen that the UV-visible absorption spectrum has obvious absorption peaks at 277 nm and 345 nm. When the excitation wavelength is 380 nm, the fluorescence intensity is the largest, close to 456 nm. The synthesized carbon quantum dot aqueous solution is light yellow and emits blue fluorescence under ultraviolet light. Fig.12 The carbon quantum dots prepared in Example 1 of the present invention are photoluminescently excited in the range of 300-440 nm. As the excitation wavelength increases from 300 nm to 440 nm, the fluorescence peak moves from 440 nm to 530 nm, and the fluorescence emission position red-shifts. Fig.13 After the carbon quantum dots prepared in Example 1 of the present invention are engulfed by cells, the cells generate fluorescence signals under different excitation wavelengths (405, 488, 546, 640 nm); it can be seen that blue, green, red and pink fluorescence are generated under different wavelengths of irradiation, indicating the near-infrared imaging capability of carbon quantum dots.

[0065] Fig.14The electron paramagnetic resonance spectrum of ·OH produced by the carbon quantum dots prepared in Example 1 of the present invention is shown. The prepared carbon quantum dots are loaded into an EPR capillary, and DMPO is used as a capture agent to detect the characteristic peaks of carbon quantum dots catalyzing H2O2 to produce ·OH at different pH values. The clear characteristic signal of 1:2:2:1 indicates the production of ·OH under acidic conditions. At the same time, the control group is taken without adding carbon quantum dots under pH 6.2. Fig.15 To measure the cell viability of 4T1 cells treated with carbon quantum dots prepared in Example 1 of the present invention, 4T1 cells were seeded in a 96-well plate and incubated in cell culture medium at pH 6.2 and 7.4, respectively. 4T1 cells were treated with different concentrations of carbon quantum dots (0 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml), the fluorescence intensity was measured using a microplate reader, and the cell activity was evaluated by the CCK-8 method. Under neutral conditions, even if the concentration was increased to 100 μg mL -1 , the cells still maintained high viability. In contrast, under acidic conditions, when the concentration of carbon quantum dots was the same, the cell viability decreased significantly (16.5%).

[0066] In vivo antitumor test: Four-week-old female nude mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., China) were used as test subjects. 4T1 cells suspended in matrix gel were injected subcutaneously into the right flank of nude mice for tumor model. 3 Nude mice bearing 4T1 tumors were treated with different procedures. Animal experiments were performed according to protocols approved by the Institutional Animal Care and Use. 4T1 nude mice were randomly divided into two groups: 1: treated with 200 μL PBS buffer (direct tail vein injection), 2: treated with 200 μL PBS solution of carbon quantum dots prepared in Example 1 (1 mg mL -1 , i.v., tail vein injection) treatment, repeated every two days for the first six days of the treatment cycle. Tumor size and mouse weight were measured every other day for 14 days. Ex vivo tumors can be obtained on day 14 for further analysis.

[0067] Fig.16 The digital photos of in vitro tumors with various treatment procedures in the in vivo anti-tumor test of the present invention are shown, wherein Ⅰ represents the PBS treatment group, Ⅱ represents the carbon quantum dot treatment group prepared in Example 1, and the tumors of mice in the PBS treatment group grew rapidly. In contrast, the tumor growth of the group treated with carbon quantum dots prepared in Example 1 was significantly reduced, indicating that carbon quantum dots can effectively inhibit tumor growth.

[0068] Fig.17 is the relative tumor volume of mice treated with the carbon quantum dots prepared in Example 1 of the present invention; Fig.18The in vitro tumor mass and tumor volume of mice treated with carbon quantum dots prepared in Example 1 of the present invention are calculated as follows: The length (L), width (w) and body weight of the tumor were measured every two days. V = (L × w 2 ) / 2 to calculate the tumor volume. The relative tumor volume was measured as V / V0 (V0: initial volume before treatment). Compared with the PBS treatment group, the tumor volume and mass of mice treated with the carbon quantum dots prepared in Example 1 were significantly reduced.

[0069] Fig.19 This is a picture of IVIS imaging of mice in vivo under 680 nm wavelength excitation of the carbon quantum dots prepared in Example 1 of the present invention. Mice injected with the carbon quantum dots prepared in Example 1 (1 day after injection) produced bright near-infrared fluorescence in tumor tissue under 680 nm excitation, demonstrating its potential for treating cancer under the guidance of near-infrared imaging.

[0070] Example 2

[0071] A method for preparing single-atom manganese-doped carbon quantum dots comprises the following steps: 40 mg MnCl2•4H2O, 292 mg EDTA and 100 μL EDA are weighed respectively and dissolved in 20 mL ultrapure water, a reaction solution is obtained by ultrasonication, the reaction solution is transferred to a 25 mL hydrothermal reactor, placed in a vacuum drying oven, heated to 200°C for reaction for 20 h, cooled to room temperature after the reaction is completed, the solution inside is sucked out into a dialysis bag with a molecular weight cutoff of 3000 D, and dialyzed in ultrapure water for 48 h; during the dialysis process, the solution outside the dialysis bag is collected and new ultrapure water is added every 24 h; the collected aqueous solution is freeze-dried until the water completely disappears, so as to obtain the prepared single-atom manganese-doped carbon quantum dots.

[0072] Fig. 20 This is a high-angle annular dark-field spherical aberration electron microscope image of the carbon quantum dots prepared in Example 2 of the present invention. It can also be observed that the single atomic bright spots representing manganese atoms are evenly dispersed on the carbon quantum dots; Fig.21 The electron paramagnetic resonance spectrum of the carbon quantum dots prepared in Example 2 of the present invention for producing ·OH, and the clear characteristic signal of 1:2:2:1 indicates that the carbon quantum dots prepared in Example 2 under acidic conditions can also catalyze the production of ·OH. Fig. 20 and Fig.21 It can be demonstrated that the carbon quantum dots prepared in Example 2 are similar to the carbon quantum dots prepared in Example 1.

[0073] Example 3

[0074] A preparation method of single-atom manganese-doped carbon quantum dots comprises the following steps: 40 mg MnCl2•4H2O, 292 mg EDTA and 100 μL EDA are weighed respectively and dissolved in 20 mL ultrapure water, a reaction solution is obtained by ultrasonication, the reaction solution is transferred to a 25 mL hydrothermal reactor, placed in a vacuum drying oven, heated to 180°C for reaction for 15 h, cooled to room temperature after the reaction, the solution inside is sucked out into a dialysis bag with a molecular weight cutoff of 3000 D, and dialyzed in ultrapure water for 48 h; during the dialysis process, the solution outside the dialysis bag is collected and new ultrapure water is added every 24 h; the collected aqueous solution is freeze-dried until the water completely disappears, so as to obtain the prepared single-atom manganese-doped carbon quantum dots.

[0075] Fig. 22 This is a high-angle annular dark-field spherical aberration electron microscopy image of the carbon quantum dots prepared in Example 3 of the present invention, and the distribution of single atomic bright spots representing manganese atoms can also be observed; Fig.23 This is the electron paramagnetic resonance spectrum of the carbon quantum dots prepared in Example 3 of the present invention for generating ·OH. The characteristic ·OH signal of 1:2:2:1 indicates that the carbon quantum dots prepared in Example 3 under acidic conditions have the same peroxidase-like activity. Fig. 22 and Fig.23 It can be demonstrated that the carbon quantum dots prepared in Example 3 are similar to the carbon quantum dots prepared in Example 1.

[0076] Comparative Example 1

[0077] 150 mg MnCl2•4H2O, 292 mg EDTA and 100 μL EDA were weighed respectively and dissolved in 20 mL ultrapure water. The reaction solution was obtained by ultrasonication. The above reaction solution was transferred to a 25 mL hydrothermal reactor, placed in a vacuum drying oven, heated to 180 °C for reaction for 15 h, and cooled to room temperature after the reaction. The solution inside was aspirated into a dialysis bag with a molecular weight cutoff of 3000 D, and dialyzed in ultrapure water for 48 h. During the dialysis process, the solution outside the dialysis bag was collected and new ultrapure water was added every 24 h. The collected aqueous solution was freeze-dried until the water completely disappeared to obtain the prepared carbon quantum dots.

[0078] Fig.24 The high-angle annular dark-field spherical aberration electron microscope image of the carbon quantum dots prepared in Comparative Example 1 of the present invention shows that no bright spots can be observed, proving that the manganese element in the carbon quantum dots prepared at this time is no longer distributed in the form of atoms.

[0079] Comparative Example 2

[0080] 40 mg CoCl2•6H2O, 292 mg EDTA and 100 μL EDA were weighed respectively and dissolved in 20 mL ultrapure water. The reaction solution was obtained by ultrasonication. The above reaction solution was transferred to a 25 mL hydrothermal reactor, placed in a vacuum drying oven, heated to 180°C for reaction for 20 h, cooled to room temperature after the reaction, and the solution inside was sucked out into a dialysis bag with a molecular weight cutoff of 3000 D, and dialyzed in ultrapure water for 48 h; during the dialysis process, the solution outside the dialysis bag was collected and new ultrapure water was added every 24 h; the collected aqueous solution was freeze-dried until the water completely disappeared to obtain the prepared carbon quantum dots.

[0081] Fig.25 This is the electron paramagnetic resonance spectrum of the carbon quantum dots prepared in Comparative Example 2 of the present invention. It can be seen that there is no characteristic signal of ·OH at this time, indicating that the carbon quantum dots prepared in Comparative Example 2 do not have peroxidase-like activity.

[0082] Comparative Example 3

[0083] 292 mg EDTA and 100 μL EDA were weighed and dissolved in 20 mL ultrapure water, and the reaction solution was obtained by ultrasonication. The reaction solution was transferred to a 25 mL hydrothermal reactor, placed in a vacuum drying oven, heated to 180 °C for reaction for 20 h, cooled to room temperature after the reaction, and the solution inside was sucked out into a dialysis bag with a molecular weight cutoff of 3000 D, and dialyzed in ultrapure water for 48 h; during the dialysis process, the solution outside the dialysis bag was collected and new ultrapure water was added every 24 h; the collected aqueous solution was freeze-dried until the water completely disappeared to obtain the prepared carbon quantum dots.

[0084] Fig.26 This is the electron paramagnetic resonance spectrum of the carbon quantum dots prepared in Comparative Example 3 of the present invention. It can be seen that there is no characteristic signal of ·OH at this time, indicating that the carbon quantum dots prepared in Comparative Example 3 do not have peroxidase-like activity.

[0085] Comparative Example 4

[0086] 40 mg MnCl2•4H2O, 292 mg EDTA and 100 μL EDA were weighed respectively and dissolved in 20 mL ethanol. The reaction solution was obtained by ultrasonication. The reaction solution was transferred to a 25 mL hydrothermal reactor, placed in a vacuum drying oven, heated to 180 °C and reacted for 20 h. After the reaction, it was cooled to room temperature, and the solution inside was sucked out into a dialysis bag with a molecular weight cutoff of 3000 D, and dialyzed in ultrapure water for 48 h. During the dialysis process, the solution outside the dialysis bag was collected and new ultrapure water was added every 24 h. The collected aqueous solution was freeze-dried until the water completely disappeared to obtain the prepared product.

[0087] Fig. 27 These are optical images of the aqueous solution of the product prepared in Comparative Example 4 of the present invention without (left picture) / with irradiation at a wavelength of 365 nm (right picture). No fluorescence is generated at this time, indicating that the prepared product is not carbon quantum dots.

[0088] Comparative Example 5

[0089] 40 mg MnCl2•4H2O and 292 mg EDTA were weighed and dissolved in 20 mL ultrapure water, and the reaction solution was obtained by ultrasonication. The reaction solution was transferred to a 25 mL hydrothermal reactor, placed in a vacuum drying oven, heated to 180 °C and reacted for 20 h. After the reaction, it was cooled to room temperature, and the solution inside was sucked out into a dialysis bag with a molecular weight cutoff of 3000 D, and dialyzed in ultrapure water for 48 h. During the dialysis process, the solution outside the dialysis bag was collected and new ultrapure water was added every 24 h. The collected aqueous solution was freeze-dried until the water completely disappeared to obtain the prepared manganese-doped carbon quantum dots. Fig.28 This is a high-angle annular dark-field spherical aberration electron microscope image of the carbon quantum dots prepared under this condition. It can be seen that no bright spots can be observed, proving that the manganese element in the carbon quantum dots prepared at this time is no longer distributed in the form of atoms; Fig.29 The optical images of the aqueous solution of the product prepared in this example without (left picture) and with (right picture) irradiation at a wavelength of 365 nm. It can be seen from the figure that the fluorescence generated at this time is weak. When applied to in vivo tumor imaging, the penetration depth is insufficient, which will result in failure to achieve the purpose of near-infrared in vivo tumor imaging.

[0090] Comparative Example 6

[0091] 30 mg MnCl2•4H2O, 292 mg EDTA and 100 μL EDA were weighed respectively and dissolved in 20 mL ultrapure water. The reaction solution was obtained by ultrasonication. The above reaction solution was transferred to a 25 mL hydrothermal reactor, placed in a vacuum drying oven, heated to 180°C for reaction for 20 hours, cooled to room temperature after the reaction, and the solution inside was sucked out into a dialysis bag with a molecular weight cutoff of 3000 D, and dialyzed in ultrapure water for 48 hours; during the dialysis process, the solution outside the dialysis bag was collected and new ultrapure water was added every 24 hours; the collected aqueous solution was freeze-dried until the water completely disappeared to obtain the prepared single-atom manganese-doped carbon quantum dots. Fig.30 This is the electron paramagnetic resonance spectrum of the carbon quantum dots prepared under this condition. It can be seen that there is no characteristic signal of ·OH at this time. Because the dosage of manganese chloride tetrahydrate is low, there are few catalytic active sites at this time, and there is no peroxidase-like property, which makes it impossible to achieve tumor catalytic therapy.

[0092] Comparative Example 7

[0093] 50 mg MnCl2•4H2O, 292 mg EDTA and 100 μL EDA were weighed respectively and dissolved in 20 mL ultrapure water, and the reaction solution was obtained by ultrasonication. The above reaction solution was transferred to a 25 mL hydrothermal reactor, placed in a vacuum drying oven, heated to 180 ° C and reacted for 20 hours. After the reaction, it was cooled to room temperature, and the solution inside was sucked out into a dialysis bag with a molecular weight cutoff of 3000 D, and dialyzed in ultrapure water for 48 hours; during the dialysis process, the solution outside the dialysis bag was collected and new ultrapure water was added every 24 hours; the collected aqueous solution was freeze-dried until the water completely disappeared to obtain the prepared manganese-doped carbon quantum dots. Fig.31 This is a high-angle annular dark-field spherical aberration electron microscope image of the carbon quantum dots prepared under this condition. It can be seen that no bright spots can be observed, proving that the manganese element in the carbon quantum dots prepared at this time is no longer distributed in atomic form.

[0094] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing single-atom manganese-doped carbon quantum dots, characterized in that: The method uses water as solvent and EDTA, manganese chloride tetrahydrate and ethylenediamine as raw materials to carry out hydrothermal reaction to obtain the single-atom manganese-doped carbon quantum dots; wherein the mass ratio of manganese chloride tetrahydrate to EDTA is 10:73; the mass volume ratio of manganese chloride tetrahydrate to ethylenediamine is 2 mg:5 μL; the temperature of the hydrothermal reaction is 180-200° C., and the time is 15-20 hours.

2. The method for preparing single-atom manganese-doped carbon quantum dots according to claim 1, characterized in that: The following steps are involved: Manganese chloride tetrahydrate, EDTA, ethylenediamine and water are mixed to obtain a reaction solution, and the reaction solution is subjected to a hydrothermal reaction to obtain the single-atom manganese-doped carbon quantum dots.

3. The method for preparing single-atom manganese-doped carbon quantum dots according to claim 1, characterized in that: The mass volume ratio of the manganese chloride tetrahydrate and water is ≤2 mg:1 mL.

4. The method for preparing single-atom manganese-doped carbon quantum dots according to any one of claims 1 to 3, characterized in that: The dosage ratio of the manganese chloride tetrahydrate, EDTA, ethylenediamine and water is 40 mg: 292 mg: 100 μL: 20 ml.

5. A single-atom manganese-doped carbon quantum dot, characterized in that: The carbon quantum dots are prepared by the method for preparing single-atom manganese-doped carbon quantum dots as described in any one of claims 1 to 4.

6. Use of the single-atom manganese-doped carbon quantum dots as claimed in claim 5 in the preparation of an anti-4T1 tumor drug, wherein the use utilizes the peroxidase-like activity of the carbon quantum dots to treat tumors.

7. Use of the single-atom manganese-doped carbon quantum dots as claimed in claim 5 in the preparation of tumor imaging agents.

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