A nitrogen-doped carbon dot and its preparation method and application

Abstract

The present invention belongs to the technical field of carbon luminescent nanomaterials, and provides a kind of nitrogen-doped carbon point and its preparation method and application, after tremella powder and triethylamine are mixed according to a mass ratio of 1: 3-10, nitrogen-doped carbon point is synthesized by a one-step hydrothermal reaction at 150-250 DEG C. The method is simple to prepare carbon point process, and the raw material source is extensive and the price is convenient to announce, the preparation condition requirement is low, and the obtained carbon point yield is higher. The prepared carbon point is used as a probe for the detection of tetracycline antibiotics, and can also be applied in living cell fluorescence imaging. The present invention can obtain carbon point solution by a one-step hydrothermal method, and the synthesis method is simple and effective, the raw materials are cheap and easy to obtain, the reaction conditions are mild and environmentally friendly, and can be completed in a general laboratory, and is easy to promote. The prepared carbon point can be used for the detection of tetracycline antibiotics as a probe, and can also be used as the application of fluorescent probe in cell fluorescence imaging.

Description

Technical Field

[0001] The present invention belongs to the technical field of carbon luminescent nanomaterials, and in particular relates to nitrogen-doped carbon dots, a preparation method and applications thereof. Background Art

[0002] Tetracycline antibiotics are a class of broad-spectrum antibiotics produced by actinomycetes, including chlortetracycline, oxytetracycline, tetracycline, and doxycycline. They are widely used to treat infections caused by various bacteria, as well as rickettsiae, chlamydiae, and mycoplasmas. However, excessive levels of tetracycline antibiotics in the body can cause adverse reactions such as alternating bacterial flora, liver damage, nephrotoxicity, and vitamin deficiency. Therefore, the detection of tetracycline antibiotics in vivo is of great significance.

[0003] Fluorescence detection, as a traditional analytical method, has been widely used in numerous fields, including analytical chemistry, environmental biology, biochemistry, and medicine, due to its high sensitivity, low-cost equipment, instantaneous response, and real-time detection. In recent years, carbon dots, as a nanomaterial, have attracted widespread attention due to their excellent photostability, water solubility, and biocompatibility. Therefore, the development of carbon dot-based detection methods for tetracycline antibiotics is urgent. Summary of the Invention

[0004] The present invention provides nitrogen-doped carbon dots, a preparation method thereof, and applications thereof. The preparation process of the carbon dots is simple, the raw materials are natural and inexpensive, the preparation conditions are low, and the process is environmentally friendly. The carbon dots can be synthesized in general laboratories and are easy to promote.

[0005] The present invention is achieved by the following technical solution: a nitrogen-doped carbon dot is prepared by mixing tremella powder and triethylamine in a mass ratio of 1:3-10, and then reacting the mixture at 150-250°C through a one-step hydrothermal method to synthesize the nitrogen-doped carbon dot.

[0006] Furthermore, the mass ratio of the tremella powder to triethylamine is 1:5-8.

[0007] Furthermore, the mass ratio of the tremella powder to triethylamine is 1:7.28.

[0008] The method for preparing the nitrogen-doped carbon dots comprises the following steps: crushing tremella to 300 mesh for standby use; dissolving tremella powder and triethylamine in water in proportion at room temperature, transferring the solution to a 50 ml hydrothermal reactor, controlling the heating rate at 5°C / min, heating to 150-250°C, reacting for 2-7 hours, filtering insoluble matter to obtain a yellow solution; dialyzing the solution through a 500-1000 Da dialysis bag for 3-5 days to obtain an aqueous solution of pure carbon dots; and freeze-drying the solution to obtain the target carbon dots.

[0009] Furthermore, the reaction temperature is 170-200° C., and the reaction time is 3-6.5 hours.

[0010] Furthermore, the reaction temperature is 200° C. and the reaction time is 6 hours.

[0011] The present invention also provides the use of the nitrogen-doped carbon dots as fluorescent probes in detecting tetracycline antibiotics in aqueous solution.

[0012] The specific application method is as follows: dry carbon dot powder is prepared into a 0.25 mg / ml carbon dot solution with PBS buffer; tetracycline antibiotics are gradually added to the carbon dot solution, mixed thoroughly, and incubated at room temperature for 3 minutes, and the changes in fluorescence intensity are recorded.

[0013] The present invention also provides the use of the nitrogen-doped carbon dots as fluorescent probes in cell fluorescence imaging.

[0014] The carbon dots prepared by the method of the present invention can be used as fluorescent probes to detect tetracyclines in aqueous solutions. According to the formula cmin=3sb / S, the detection limit of the carbon dots in tetracycline is 1.33 nM, and the linear range is 2.38-66.64 nM; the detection limit in chlortetracycline is 1.38 nM, and the linear ranges are 2.38-19.04 nM and 19.04-76.16 nM; the detection limit in oxytetracycline is 1.19 nM, and the linear ranges are 2.38-14.28 nM and 14.28-38.08 nM; and the detection limit in doxycycline is 1.26 nM, and the linear ranges are 2.38-9.52 nM and 9.52-85.68 nM.

[0015] The present invention utilizes a one-step hydrothermal method to produce a carbon dot solution. The synthesis method is simple and effective, using readily available and inexpensive raw materials under mild and environmentally friendly reaction conditions. It can be performed in a standard laboratory and is readily available. The prepared carbon dots can be used as probes for the detection of tetracycline antibiotics and as fluorescent probes for cell fluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The UV-visible absorption spectrum and fluorescence spectrum of the carbon dots prepared in Example 1;

[0017] Figure 2 The fluorescence spectrum of the carbon dots prepared in Example 1 shows the fluorescence emission curve as the excitation wavelength changes;

[0018] Figure 3 This is the infrared spectrum of the carbon dots prepared in Example 1;

[0019] Figure 4 This is the X-ray photoelectron spectrum of the carbon dots prepared in Example 1;

[0020] Figure 5 Figure 1 shows the selectivity results of the carbon dots prepared in Example 1 for the detection of tetracycline antibiotics. In the figure, A shows the results of the cation selectivity test; B shows the results of the anion selectivity test.

[0021] Figure 6 Figure 1 is a graph showing the anti-interference performance of the carbon dots prepared in Example 1 for the detection of tetracycline antibiotics. In the figure, A is the result of the cationic anti-interference test; B is the result of the anionic anti-interference test.

[0022] Figure 7 is the fluorescence emission spectrum of carbon dots with increasing tetracycline concentration;

[0023] Figure 8 This is the linear fitting graph of the carbon dots detecting tetracycline concentration;

[0024] Figure 9 is the fluorescence emission spectrum of carbon dots with the increase of chlortetracycline concentration;

[0025] Figure 10 This is a linear fitting diagram of carbon dots detecting chlortetracycline concentration. In the figure: A shows that the carbon dots have a good linear relationship when the chlortetracycline concentration is 2.38-19.04 nM; B shows that the carbon dots have a good linear relationship when the chlortetracycline concentration is 19.04-76.16 nM.

[0026] Figure 11 is the fluorescence emission spectrum of carbon dots with the increase of oxytetracycline concentration;

[0027] Figure 12 The linear fitting diagram of carbon dots detecting oxytetracycline concentration. In the figure: A shows that the carbon dots have a good linear relationship when the concentration of oxytetracycline is 2.38-14.28 nM; B shows that the carbon dots have a good linear relationship when the concentration of oxytetracycline is 14.28-38.08 nM.

[0028] Figure 13 is the fluorescence emission spectrum of carbon dots with increasing doxycycline concentration;

[0029] Figure 14 The linear fitting graph of carbon dots detecting doxycycline concentration. In the figure: A shows that the carbon dots have a good linear relationship when the doxycycline concentration is 2.38-9.52 nM; B shows that the carbon dots have a good linear relationship when the doxycycline concentration is 9.52-85.68 nM.

[0030] Figure 15 This is a laser confocal microscopy image of the carbon dots prepared in Example 1 quenched by tetracycline antibiotics, and the cells are PC-12 cells. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure herein and the materials they cite are hereby incorporated by reference.

[0033] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0034] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0035] Example 1: Preparation of nitrogen-doped carbon dots, comprising the following steps:

[0036] Step 1: Dissolve 0.1 g of Tremella fuciformis powder and 1 mL (0.728 g) of triethylamine in 20 ml of water at room temperature, stir thoroughly, and ultrasonicate to obtain a clear solution.

[0037] Step 2: Transfer the solution to a 50 ml hydrothermal reactor.

[0038] Step 3: Place the hydrothermal kettle in an oven, control the heating rate to 5°C / min, heat to 200°C and react for 6 hours to obtain a red solution.

[0039] Step 4: After filtering the insoluble matter, a red solution is obtained. The red solution is dialyzed in a glass container through a 1000 Da dialysis bag for at least 3 days to obtain a pure aqueous solution of carbon dots.

[0040] Step 5: freeze-drying the fluorescent carbon dot aqueous solution to obtain fluorescent carbon dot powder, the relative quantum yield of which (based on rhodamine B) is 38.3%.

[0041] Example 2: Preparation of nitrogen-doped carbon dots, except that the mass of triethylamine was 0.314 g, all other aspects were the same as in Example 1. The relative quantum yield (based on rhodamine B) was 22.6%.

[0042] Example 3: Preparation of nitrogen-doped carbon dots, except that the mass of triethylamine was 0.471 g, all other conditions were the same as those in Example 1. The relative quantum yield (based on rhodamine B) was 26.4%.

[0043] Example 4: Preparation of nitrogen-doped carbon dots: Tremella powder and triethylamine were mixed in a mass ratio of 1:3; the temperature was raised to 150°C at a heating rate of 5°C / min for 7 hours, and the mixture was dialyzed through a 500Da dialysis bag for 5 days. The remaining procedures were the same as those described in Example 1.

[0044] Example 5: Preparation of nitrogen-doped carbon dots: Tremella powder and triethylamine were mixed in a mass ratio of 1:5; the temperature was raised to 180°C at a heating rate of 5°C / min for 6 hours, and the mixture was dialyzed through a 700Da dialysis bag for 4 days. The remaining procedures were the same as those described in Example 1.

[0045] Example 6: Preparation of nitrogen-doped carbon dots: Tremella powder and triethylamine were mixed in a mass ratio of 1:8; the temperature was raised to 220°C at a heating rate of 5°C / min for 4 hours, and the mixture was dialyzed through a 600Da dialysis bag for 4 days. The remaining procedures were the same as those described in Example 1.

[0046] Example 7: Preparation of nitrogen-doped carbon dots: Tremella powder and triethylamine were mixed in a mass ratio of 1:10; the temperature was raised to 250°C at a heating rate of 5°C / min for 2 hours, and the mixture was dialyzed through a 500Da dialysis bag for 3 days. The remaining procedures were the same as those described in Example 1.

[0047] Example 8: Preparation of nitrogen-doped carbon dots: Tremella powder and triethylamine were mixed in a mass ratio of 1:9; the rest of the method was the same as that described in Example 7.

[0048] Experimental Example 1: Optical characterization of the fluorescent carbon dots prepared in Example 1 of the present invention Figure 1 and 2 The UV-visible absorption spectrum of the carbon dots exhibits an absorption peak around 331 nm, corresponding to the π→π* transition of the carbon core state. Excitation at 360 nm reveals a blue fluorescence emission wavelength of 452 nm. Figure 2 Figure 3 is the emission spectrum of the carbon dots at different excitation wavelengths, indicating that the carbon dots are excitation wavelength dependent.

[0049] Experimental Example 2: The surface property spectrum of the fluorescent carbon dots prepared in Example 1 of the present invention is as follows Figure 3 and 4 As shown. Infrared spectrum shows that O-CDs has a wavelength of 3349 cm −1 and 3245 cm −1 The peak at 2985 cm corresponds to the -NH / -OH stretching vibration.−1 and 2905cm −1 The peak at 1639 cm corresponds to the stretching vibration of CH. −1 ,1061 cm −1 and 1408 cm −1 The peaks at 284.2, 399.2, and 534.4 eV correspond to the stretching vibrations of C=O, CO, and CN=, respectively. The full XPS spectrum shows three peaks at 284.2, 399.2, and 534.4 eV, attributed to C1s, N1s, and O1s, respectively. These results confirm that the carbon dots are composed of three elements: C, N, and O.

[0050] Experimental Example 3: The carbon dots obtained in Example 1 were added with water to prepare a 0.25 mg / ml carbon dot aqueous solution. 2 mL of the carbon dot aqueous solution and 10 μL of tetracycline antibiotics of different concentrations were added to a 1 cm quartz cuvette in sequence, and their emission spectra were measured.

[0051] Common cations and anions were used to replace tetracycline antibiotics to study the selectivity of carbon dots for tetracycline antibiotics. The specific cations are: Pb 2+ Cr 3+ Cr 6+ 、Cu 2+ 、Co 2+ 、Ni 2+ 、Zn 2+ 、Hg 2+ 、Fe 2+ 、Fe 3+ 、Sn 2+ 、Al 3+ ;Specific anions: F - 、Cl - Br - , I - , ClO - 、SCN - 、S 2- 、NO3 - 、NO2 - 、CO3 2- 、SO3 2- .

[0052] The results are as follows Figure 5 As shown, Figure 5 The results show that common cations and anions cannot change the emission spectrum of carbon dots, so carbon dots have good selectivity for the detection of tetracycline antibiotics.

[0053] After adding common cations and anions to the carbon dot solution, tetracycline antibiotics were added to study the anti-interference ability of carbon dots to tetracycline antibiotics. Figure 6The results showed that common cations and anions could not interfere with the emission spectrum of carbon dots, so carbon dots had good anti-interference ability for the detection of tetracycline antibiotics.

[0054] The sensing performance of the fluorescent carbon dots prepared in Example 1 of the present invention to tetracycline is as follows: Figure 7 As shown, Figure 8 The carbon dots showed a good linear relationship in the tetracycline concentration range of 2.38-66.64 nM, with a detection limit of 1.33 nM.

[0055] The sensing effect of the fluorescent carbon dots prepared in Example 1 on chlortetracycline is as follows: Figure 9 As shown, Figure 10 A shows that the carbon dots have a good linear relationship when the concentration of chlortetracycline is 2.38-19.04 nM. Figure 10 B shows that the carbon dots have a good linear relationship when the concentration of chlortetracycline is 19.04-76.16 nM, and the detection limit is 1.38 nM.

[0056] The sensing performance of the fluorescent carbon dots prepared in Example 1 of the present invention to oxytetracycline is as follows: Figure 11 As shown, Figure 12 A shows that the carbon dots have a good linear relationship when the concentration of oxytetracycline is 2.38-14.28 nM. Figure 12 B shows that the carbon dots have a good linear relationship when the concentration of oxytetracycline is 14.28-38.08 nM, and the detection limit is 1.19 nM.

[0057] The sensing performance of the fluorescent carbon dots prepared in Example 1 of the present invention for doxycycline is as follows: Figure 13 As shown, Figure 14 A shows that the carbon dots have a good linear relationship when the concentration of doxycycline is 2.38-9.52 nM. Figure 14 B shows that the carbon dots have a good linear relationship when the concentration of doxycycline is 9.52-85.68 nM, and the detection limit is 1.26 nM.

[0058] Experimental Example 4: PC12 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C and 5.0% CO2 for 24 h. After incubation with carbon dots, carbon dots@tetracycline, carbon dots@chlortetracycline, carbon dots@oxytetracycline, and carbon dots@doxycycline for 60 min at 37°C, the cells were washed three times in PBS (pH 7.4) to remove excess carbon dots. Fluorescence images were collected under a confocal laser scanning microscope. Figure 15 As shown, the cells have good morphology and the carbon dots have no cytotoxicity, which can be used for live cell labeling. Figure 15This is a cell imaging image of the fluorescent carbon dots prepared in Example 1 being quenched by tetracycline antibiotics. After the addition of tetracycline antibiotics, the fluorescence of the carbon dots is quenched, indicating that the fluorescent carbon dots can be used to construct a fluorescence sensing platform for tetracycline antibiotics in cells.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of nitrogen-doped carbon dots as fluorescent probes for detecting tetracycline antibiotics in aqueous solution, characterized in that: The preparation method of the nitrogen-doped carbon dots comprises: dissolving tremella powder and triethylamine in water at a mass ratio of 1:3-10, and performing a one-step hydrothermal reaction at 150-250° C. for 2-7 hours to synthesize the nitrogen-doped carbon dots.

2. Application of nitrogen-doped carbon dots as fluorescent probes for the detection of tetracycline antibiotics in cells, characterized by: The preparation method of the nitrogen-doped carbon dots comprises: mixing tremella powder and triethylamine in a mass ratio of 1:3-10, dissolving the mixture in water, and performing a one-step hydrothermal reaction at 150-250° C. for 2-7 hours to synthesize the nitrogen-doped carbon dots.

3. The use according to claim 1 or 2, characterized in that: The mass ratio of the tremella powder to triethylamine is 1:5-8.

4. The use according to claim 3, characterized in that: The mass ratio of the tremella powder to triethylamine is 1:7.

28.

5. The use according to claim 1 or 2, characterized in that: The preparation method of nitrogen-doped carbon dots comprises the following steps: dissolving Tremella fuciformis powder and triethylamine in water according to a mass ratio at room temperature, transferring the solution to a 50 ml hydrothermal reactor, heating the temperature to 150-250° C. at a controlled heating rate of 5° C. / min, reacting for 2-7 hours, filtering insoluble matter to obtain a yellow solution; dialyzing the solution through a 500-1000 Da dialysis bag for 3-5 days to obtain an aqueous solution of pure carbon dots; and freeze-drying the solution to obtain the target carbon dots.

6. The use according to claim 5, characterized in that: The reaction temperature is 170-200° C., and the reaction time is 3-6.5 hours.

7. The use according to claim 6, characterized in that: The reaction temperature is 200° C. and the reaction time is 6 h.

8. The use according to claim 1, characterized in that: The specific application method is as follows: dry carbon dot powder is prepared into a 0.25 mg / ml carbon dot solution with PBS buffer; tetracycline antibiotics are gradually added to the carbon dot solution, mixed thoroughly, and incubated at room temperature for 3 minutes, and the changes in fluorescence intensity are recorded.

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