A carbon quantum dot using golden chrysanthemum as a carbon source, a preparation method thereof, and its application in the detection of ascorbic acid

Abstract

The present invention belongs to the technical field of pharmaceutical preparation and application technology, discloses a kind of carbon quantum dots with golden chrysanthemum as carbon source and its preparation method and detection application to ascorbic acid, in order to solve the technical problem of safety in ascorbic acid detection process in the prior art. The steps are as follows: golden chrysanthemum is removed from the calyx, cleaned, dried and crushed, dissolved in anhydrous ethanol, carbonized at high temperature, polyoxyethylene diamine alcohol solution is light-proof reaction, Na2CO3 solution is light-proof reaction, then dialyzed, rotary evaporated, freeze-dried and processed to obtain carbon quantum dots. The carbon quantum dots prepared by the present invention have the characteristics of low cytotoxicity, good stability and selectivity to ascorbic acid, and the aqueous solution of the carbon quantum dots is utilized, and fluorescence analysis is adopted, i.e., the carbon quantum dot fluorescence intensity numerical value change ΔF shows a good linear relationship with ascorbic acid 1.25-7.50 mmol / L concentration range, which can realize the detection of ascorbic acid.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparation and application, and particularly relates to carbon quantum dots using golden chrysanthemum as a carbon source, a preparation method thereof, and an application in the detection of ascorbic acid. Background Art

[0002] Carbon quantum dots (CQDs), a novel zero-dimensional carbon nanofluorescent material, consist of dispersed, spherical carbon particles. They not only possess the excellent optical properties and extremely small particle size (<10 nm) of conventional quantum dots, but also offer advantages such as low toxicity, excellent biocompatibility, widespread availability, ease of preparation, and good water solubility, making them a research hotspot. Due to their unique properties, CQDs have been successfully applied in pesticide residue detection, heavy metal detection, pollutant degradation, food safety testing, bioimaging, biosensing, anti-counterfeiting, biomedicine, and pharmaceutical analysis. In recent years, doped CQDs have also attracted considerable research attention. Common heteroatoms used to dope CQDs include nitrogen, sulfur, and phosphorus. Doping CQDs can adjust the transition energy levels of CQDs, making them more susceptible to fluorescence and prolonging their emission duration, avoiding fluorescence quenching, increasing fluorescence quantum yield, or enhancing their selectivity for metal ion detection. Furthermore, CQDs can be used to adjust the target fluorescence emission wavelength, avoiding ultraviolet light, thereby making their application in humans safer and reducing toxicity.

[0003] Chrysanthemum is a plant of the Asteraceae family. It can not only be planted for viewing, but also has a traditional edible culture. It tastes sweet, bitter and slightly cold. It has the effects of clearing away heat and relieving fire, clearing the liver and improving eyesight, detoxifying and reducing inflammation. It is a local medicinal material in Henan Province. It is rich in flavonoids, volatile oils, triterpenes, organic acids and other organic compounds. It has great medicinal value. Many studies have discovered its pharmacological effects in anti-aging, sedation and hypnosis, antibacterial, anti-tumor, liver damage protection, acute lung injury protection and increasing the body's immunity.

[0004] Ascorbic acid, also known as vitamin C, is found naturally in a variety of fruits and vegetables and is widely used in the food industry, agriculture, forestry, animal husbandry, and pharmaceuticals. It has antioxidant properties, participates in promoting collagen synthesis, delays cell aging and apoptosis, fights cancer and tumors, regulates cellular pathways, and bidirectionally regulates cholesterol metabolism and synthesis. It is an essential trace element for the human body and plays an indispensable role in maintaining life processes. Therefore, accurate and rapid determination of ascorbic acid content is of great significance for food safety testing and disease diagnosis.

[0005] Currently, a range of analytical methods for detecting AA have been developed, including colorimetry, electrochemical analysis, chromatography, titration, and fluorescence (Dhara, K.; Mahapatra, DR. Review on nanomaterials-enabled electrochemical sensors for ascorbic acid detection. Anal. Biochem. 2019, 586, 113415.). While appropriate methods can be selected under different conditions, some methods also have their limitations.For example, colorimetric methods have good sensitivity, low cost, simple operation, and strong practicality, but they still have disadvantages such as long catalyst preparation time (24 h), long measurement time (40-60 min), and poor selectivity (Jun, P.; Ling, J.; Zhang, XQ; Zhang, LY; Cao, QE; Ding, ZT A rapid, sensitive and selective colorimetric method for detection of ascorbic acid. Sens. Actuators B Chem. 2015, 221, 708–716.). Electrochemical methods have attracted much attention due to their high sensitivity, good selectivity, low cost, and good equipment portability. However, the presence of impurities with oxidation potentials close to that of AA can lead to poor resolution of their oxidation peaks on conventional electrodes, or even overlap (Jirimali, HD; Nagarale, RK; Saravanakumar, D.; Lee, JM; Shin, W. Hydroquinone modified chitosan / carbon film electrode for the selective detection of ascorbic acid. Carbohydr. Polym. 2013, 92, 641–644.); The fluorescence method is low-cost, highly sensitive, and simple to detect, but it is susceptible to interference, and the raw materials are mostly prepared using metal substances, which poses certain safety issues in the field of pharmaceutical preparation and application technology (Gidwani B., Sahu V., Shukla SS, Pandey R.,Joshi V., Jain VK, Vyas A. Quantum dots: Prospectives, toxicity, advancesand applications. J. Drug Deliv. Sci. Technol. 2021;61:102308.). The raw materials of the present invention are derived from plants, which improves the safety risk of metal raw materials in the fluorescence method. Summary of the Invention

[0006] To address the safety issues inherent in ascorbic acid detection in existing technologies, the present invention proposes carbon quantum dots (CQDs) using Goldenrod as a carbon source, a preparation method, and their application in ascorbic acid detection. This invention utilizes a solvothermal method, using Goldenrod, a plant rich in flavonoids, volatile oils, triterpenes, and organic acids, as a carbon source. The CQDs are synthesized in a single step using a green, low-toxic method, and then undergo surface passivation treatment.

[0007] To achieve the above object, the technical solution of the present invention is implemented as follows:

[0008] A method for preparing carbon quantum dots using golden chrysanthemum as a carbon source, comprising the following steps:

[0009] (1) Remove the calyx of golden chrysanthemum, clean it and put it into the oven for drying at a temperature of 75-85℃ for 18-30h;

[0010] (2) Crush the sample, weigh 2.00 g of crushed golden chrysanthemum powder, measure 60-80 mL of anhydrous ethanol, mix evenly with ultrasonic wave, and transfer to the reactor. Then put the reactor into the oven and carbonize at high temperature for 9-11 hours at 170-210 °C.

[0011] (3) After the reaction is completed, the mixture is cooled to room temperature, and the supernatant is collected after high-speed centrifugation. The filtrate is filtered through a microporous filter membrane and reacted with a polyoxyethylene diamine alcohol solution in the dark to obtain a reaction solution;

[0012] (4) The reaction solution obtained in step (3) was reacted with a Na2CO3 solution having a concentration of 0.08-0.12 mg / mL at a volume ratio of 1: (0.8-1.2) in the dark, and dialyzed. After the dialysis was completed, the obtained solution was subjected to rotary evaporation and freeze-drying to obtain a brown dry carbon quantum dot product.

[0013] Preferably, in step (2), 2.00 g of the golden chrysanthemum sample after removing the calyx is added to 70 mL of anhydrous ethanol.

[0014] Preferably, in step (2), the ultrasonic time is 10 min, and the mixture is poured into a reactor and then carbonized in an oven at 190°C for 10 h.

[0015] Preferably, in step (3), the reaction liquid after cooling to room temperature after the reaction is completed is a dark brown solution, which is then centrifuged at a speed of 6000-10000 r / min for 5-15 min; the microporous filter membrane used is 0.22 μm.

[0016] Preferably, in step (3), the polyoxyethylene diamine alcohol solution refers to an ethanol solution of polyoxyethylene diamine, and the concentration of the polyoxyethylene diamine alcohol solution is 0.40-0.80 mg / mL; the volume ratio of the filtrate to the polyoxyethylene diamine alcohol solution is 10:(1-1.2); the temperature of the light-proof reaction is 25-35°C, and the reaction time is 36-60 hours.

[0017] Preferably, in step (4), the concentration of the Na2CO3 solution reacting with the filtrate is 0.10 mg / mL, and the volume ratio of the reaction solution to the Na2CO3 solution is 1:1.

[0018] Preferably, in step (4), the temperature of the light-shielding reaction in step (4) is 25-35° C., and the time is 18-36 h; the molecular weight cutoff of the dialysis bag used for dialysis is 1000 Da, the dialysis time is 36-60 h, and the water is changed every 12 h during the dialysis period.

[0019] The carbon quantum dots are prepared by the above-mentioned method for preparing carbon quantum dots using golden chrysanthemum as a carbon source.

[0020] The above-mentioned carbon quantum dots are used in the detection of ascorbic acid.

[0021] Furthermore, the detection method is: dissolving carbon quantum dots in distilled water to prepare a solution, then adding ascorbic acid solution, measuring the linear relationship between the change in fluorescence intensity at an excitation wavelength of 450 nm and the ascorbic acid concentration, and using this linear relationship to detect ascorbic acid of unknown concentration.

[0022] The beneficial effects produced by the present invention are:

[0023] (1) The present invention adopts a solvent thermal method, uses golden chrysanthemum rich in flavonoids, volatile oils, triterpenes, organic acids and other compounds as a carbon source, adopts a green and low-toxic method to synthesize carbon quantum dots in one step, and passivates its surface to prepare a carbon quantum dot with green fluorescence. The carbon quantum dots are evenly dispersed and have a uniform particle size (average particle size is 2.67nm). The emission wavelength is dependent on the excitation wavelength, has an optimal excitation wavelength and an optimal concentration, can enter the cytoplasm to achieve fluorescence imaging of cells, and have low cytotoxicity.

[0024] (2) The carbon quantum dots have good selectivity for the fluorescence quenching of ascorbic acid, and the fluorescence is stable over a wide pH range and for a long period of time, with high optical stability. The carbon quantum dots can be used as a potential testing tool to achieve rapid detection of ascorbic acid within 1 minute. The fluorescence intensity displayed when mixed with ascorbic acid solutions of different concentrations is different. In the concentration range of 1.25-7.50mmol / L, the numerical change ΔF of the CQDs fluorescence intensity shows a good linear relationship with the ascorbic acid concentration. The fitted regression equation is y = 9.66857x–9.13333, and the linear correlation R 2 =0.9912. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 TEM spectrum of CQDs (a); particle size distribution (b); XRD spectrum (c); FTIR spectrum (d).

[0027] Figure 2 UV-visible absorption spectra of CQDs in PBS, H2O, and CH3CH2OH.

[0028] Figure 3 Fluorescence emission spectra of CQDs at different excitation wavelengths in solvents H2O (a), CH3CH2OH (b), and PBS (c).

[0029] Figure 4 Fluorescence emission spectra of CQDs in solvents H2O (a), CH3CH2OH (b), and PBS (c) at different concentrations (optimal excitation wavelength).

[0030] Figure 5 The fluorescence emission spectra of CQDs in PBS, H2O, and CH3CH2OH at the optimal concentration of 500 μg / mL and the optimal excitation wavelength.

[0031] Figure 6 The fluorescence properties of CQDs under different pH conditions (a); D1 and D2 when CQDs are dissolved in different solvents in a dark box 10 Photo (b).

[0032] Figure 7Fluorescence quenching spectra of CQDs by ascorbic acid solutions of different concentrations (a); linear fitting relationship between ΔF and ascorbic acid concentration (b).

[0033] Figure 8 Effects of different concentrations of CQDs on the viability of (a) Lo2 cells and (b) HepG2 cells.

[0034] Figure 9 Laser confocal fluorescence images of CQDs after acting on (a) Lo2 cells; (b) HepG2 cells for 1, 3, and 5 hours. DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all 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.

[0036] Example 1

[0037] The preparation method of carbon quantum dots using golden chrysanthemum as a carbon source in this embodiment comprises the following steps:

[0038] (1) Remove the calyx of the golden chrysanthemum, clean it and put it into the oven for drying at 80℃ for 24 hours;

[0039] (2) Crush and weigh 2.00 g of crushed golden chrysanthemum powder and add it to 70 mL of anhydrous ethanol. After ultrasonication for 10 min, pour it into the reactor and place it in an oven at 190 ° C for high temperature carbonization for 10 h;

[0040] (3) After the reaction is completed, the temperature of the reactor is lowered to room temperature, and the reaction liquid is removed to obtain a dark brown solution. The solution is centrifuged at 8000 r / min for 10 min. After the centrifugation, the supernatant is removed and filtered with a 0.22 μm microporous filter membrane to obtain 50 mL of filtrate. 5 mL of 0.60 mg / mL polyoxyethylene diamine alcohol solution is slowly added dropwise under ultrasound, and the solution is reacted at 25°C in the dark for 48 h to obtain a reaction solution.

[0041] (4) Add 0.10 mg / mL Na2CO3 solution to the reaction solution obtained in step (3) under ultrasound at a volume ratio of 1:1. After reacting at 25°C in the dark for 24 hours, transfer the solution to a dialysis bag (1000 Da) and dialyze for 48 hours. During the dialysis, change the water every 12 hours. After the dialysis is completed, the obtained solution is subjected to rotary evaporation and freeze-drying to obtain a brown dry carbon quantum dot product.

[0042] Figure 1(a) is the TEM spectrum of the carbon quantum dots prepared in Example 1. It can be seen from the figure that the carbon quantum dots are evenly distributed and uniform in size. They are spherical nanoparticles. The HR-TEM image shows that the lattice fringe spacing is 0.18nm, and the particle size distribution shows that the average particle size is 2.67nm ( Figure 1 (b)). Figure 1 (c) is the XRD spectrum of carbon quantum dots. It can be seen that the carbon quantum dots contain carbon structures. Figure 1 (d) is the FTIR spectrum of carbon quantum dots. The results show that the surface of CQDs contains functional groups such as carbonyl, amino, hydroxyl, and carboxyl. These functional groups play a key role in the excellent luminescence properties and good water solubility of CQDs.

[0043] Example 2

[0044] The preparation method of carbon quantum dots using golden chrysanthemum as a carbon source in this embodiment comprises the following steps:

[0045] (1) Remove the calyx of the golden chrysanthemum, clean it and put it into the oven for drying at 75℃ for 30h;

[0046] (2) Crush and weigh 2.00 g of crushed golden chrysanthemum powder and add it to 60 mL of anhydrous ethanol. After ultrasonication for 10 min, pour it into the reactor and place it in an oven at 210 ° C for high temperature carbonization for 9 h;

[0047] (3) After the reaction is completed, the temperature of the reactor is lowered to room temperature, and the reaction liquid is removed to obtain a dark brown solution. The solution is centrifuged at 6000 r / min for 5 min. After the centrifugation, the supernatant is removed and filtered with a 0.22 μm microporous filter membrane to obtain 40 mL of filtrate. 4.4 mL of 0.80 mg / mL polyoxyethylene diamine alcohol solution is slowly added dropwise under ultrasound, and the solution is reacted at 30°C in the dark for 36 h to obtain a reaction solution.

[0048] (4) Add 0.10 mg / mL Na2CO3 solution to the reaction solution obtained in step (3) under ultrasound at a volume ratio of 1:0.8. After reacting at 30°C in the dark for 18 h, transfer the solution to a dialysis bag (1000 Da) and dialyze for 36 h. During the dialysis, change the water every 12 h. After the dialysis is completed, the obtained solution is subjected to rotary evaporation and freeze-drying to obtain a brown dry carbon quantum dot product.

[0049] Example 3

[0050] The preparation method of carbon quantum dots using golden chrysanthemum as a carbon source in this embodiment comprises the following steps:

[0051] (1) Remove the calyx of the golden chrysanthemum, clean it and put it into the oven for drying at 85℃ for 18 hours;

[0052] (2) Crush and weigh 2.00 g of crushed golden chrysanthemum powder and add it to 80 mL of anhydrous ethanol. After ultrasonication for 10 min, pour it into the reactor and place it in an oven at 170 ° C for high temperature carbonization for 11 h;

[0053] (3) After the reaction is completed, the temperature of the reactor is lowered to room temperature, and the reaction liquid is removed to obtain a dark brown solution. The solution is centrifuged at a speed of 10,000 r / min for 15 min. After the centrifugation, the supernatant is removed and filtered with a 0.22 μm microporous filter membrane to obtain 60 mL of filtrate. 7.2 mL of 0.40 mg / mL polyoxyethylene diamine alcohol solution is slowly added dropwise under ultrasound, and the solution is reacted at 35°C in the dark for 60 h to obtain a reaction solution.

[0054] (4) Add 0.10 mg / mL Na2CO3 solution to the reaction solution obtained in step (3) under ultrasound at a volume ratio of 1:1.2. After reacting at 35°C in the dark for 36 hours, transfer the solution to a dialysis bag (1000 Da) and dialyze for 60 hours. During the dialysis, change the water every 12 hours. After the dialysis is completed, the obtained solution is subjected to rotary evaporation and freeze-drying to obtain a brown dry carbon quantum dot product.

[0055] Example 4

[0056] The preparation method of carbon quantum dots using golden chrysanthemum as a carbon source in this embodiment comprises the following steps:

[0057] (1) Remove the calyx of the golden chrysanthemum, clean it and put it into the oven for drying at 80℃ for 24 hours;

[0058] (2) Crush and weigh 2.00 g of crushed golden chrysanthemum powder and add it to 70 mL of anhydrous ethanol. After ultrasonication for 10 min, pour it into the reactor and place it in an oven at 180 ° C for high temperature carbonization for 10 h;

[0059] (3) After the reaction is completed, the temperature of the reactor is lowered to room temperature, and the reaction liquid is removed to obtain a dark brown solution. The solution is centrifuged at 8000 r / min for 10 min. After the centrifugation, the supernatant is removed and filtered with a 0.22 μm microporous filter membrane to obtain 50 mL of filtrate. 5 mL of 0.60 mg / mL polyoxyethylene diamine alcohol solution is slowly added dropwise under ultrasound, and the solution is reacted at 25°C in the dark for 60 h to obtain a reaction solution.

[0060] (4) Add 0.08 mg / mL Na2CO3 solution to the reaction solution obtained in step (3) under ultrasound at a volume ratio of 1:1. After reacting at 25°C in the dark for 24 hours, transfer the solution to a dialysis bag (1000 Da) and dialyze for 48 hours. During the dialysis, change the water every 12 hours. After the dialysis is completed, the obtained solution is subjected to rotary evaporation and freeze-drying to obtain a brown dry carbon quantum dot product.

[0061] Example 5

[0062] The preparation method of carbon quantum dots using golden chrysanthemum as a carbon source in this embodiment comprises the following steps:

[0063] (1) Remove the calyx of the golden chrysanthemum, clean it and put it into the oven for drying at 80℃ for 24 hours;

[0064] (2) Crush and weigh 2.00 g of crushed golden chrysanthemum powder and add it to 70 mL of anhydrous ethanol. After ultrasonication for 10 min, pour it into the reactor and place it in an oven at 200 ° C for high temperature carbonization for 11 h;

[0065] (3) After the reaction is completed, the temperature of the reactor is lowered to room temperature, and the reaction liquid is removed to obtain a dark brown solution. The solution is centrifuged at 8000 r / min for 10 min. After the centrifugation, the supernatant is removed and filtered with a 0.22 μm microporous filter membrane to obtain 50 mL of filtrate. 5.5 mL of 0.60 mg / mL polyoxyethylene diamine alcohol solution is slowly added dropwise under ultrasound, and the solution is reacted at 35°C in the dark for 36 h to obtain a reaction solution.

[0066] (4) At a volume ratio of 1:1, 0.12 mg / mL Na2CO3 solution was added dropwise to the reaction solution obtained in step (3) under ultrasound. The mixture was reacted at 25°C in the dark for 24 h and then transferred to a dialysis bag (1000 Da) for 48 h. The water was changed every 12 h during the dialysis. After the dialysis was completed, the obtained solution was subjected to rotary evaporation and freeze-drying to obtain a brown dry carbon quantum dot product.

[0067] Test Case

[0068] The carbon quantum dots prepared in Example 1 were tested and analyzed for optical properties, stability, selective detection of ascorbic acid, cytotoxicity, and cell imaging.

[0069] (1) Optical properties: The prepared carbon quantum dots were dissolved in different solvents, PBS, H2O, and CH3CH2OH, and their UV-visible absorption properties were tested. The results are as follows: Figure 2 As shown in the figure, the prepared CQDs have an absorption peak at 268nm in different solvents including PBS, H2O and CH3CH2OH.

[0070] Figure 3 The fluorescence emission spectra of CQDs in different solvents (all at 500 μg / mL) and different excitation wavelengths are shown. As can be seen from the figure, the CQDs in different solvents have different optimal excitation wavelengths when the fluorescence intensity is the strongest. In water, the optimal excitation wavelength is 410 nm, and the emission wavelength is 490 nm ( Figure 3(a)); In ethanol, the optimal excitation wavelength is 450nm, and the emission wavelength is 520nm ( Figure 3 (b)); In PBS, the optimal excitation wavelength is 370nm, and the emission wavelength is 460nm ( Figure 3 (c)).

[0071] Figure 4 The fluorescence emission spectra of CQDs under the optimal excitation wavelength corresponding to different solvents and different concentration conditions are shown in Figure 2. Figure 4 It can be seen from (a)-4(c) that the fluorescence intensity first increases and then decreases with the increase of carbon quantum dot concentration. When the concentration is 500μg / mL, the intensity is the strongest, that is, the optimal concentration of carbon quantum dots in the solvent is 500μg / mL.

[0072] Figure 5 The fluorescence emission spectra of CQDs in different solvents (H2O, PBS, CH3CH2OH) at the optimal concentration of 500μg / mL and the optimal excitation wavelength are obtained. The luminescence performance is best when CQDs are dissolved in CH3CH2OH.

[0073] (2) Stability: The fluorescence performance of the prepared CQDs under different pH conditions was tested. The specific test process is as follows: The CQDs mother solution was prepared at a concentration of 2500 μg / mL and added to PBS buffer with different pH values ​​(pH = 3.0, 5.0, 7.0, 9.0 and 11.0) to measure its fluorescence performance. Figure 6 As can be seen in (a), the fluorescence intensity of CQDs is less affected under different pH conditions, indicating that the luminescence properties of CQDs remain relatively stable over a wide pH range. Figure 6 (b) D1 and D2 when CQDs are dissolved in different solvents in a dark box 10 The photo shows that the CQDs solution still has strong fluorescence after 10 days of storage, which further proves the strong optical stability of the CQDs. These results show that the optical stability of CQDs is strong and is not affected by pH and time.

[0074] (3) Selective detection of ascorbic acid: The specific process of ascorbic acid detection is as follows: distilled water is used as the solvent to prepare 100 mmol / L ascorbic acid mother solution and 1000 μg / mL carbon quantum dot mother solution, 1 mL of carbon quantum dot mother solution is drawn into the fluorescence cell, different volumes (0-150 μL) of ascorbic acid mother solution are added, and diluted to 2 mL to obtain ascorbic acid solutions of different concentrations (0-7.50 mmol / L), which are then incubated for 1 min, and then the fluorescence intensity F is measured using a fluorescence spectrophotometer at an excitation wavelength of 450 nm.

[0075] Figure 7(a) is the fluorescence quenching spectrum of CQDs by ascorbic acid solutions of different concentrations. It can be seen that when the ascorbic acid concentration is 0, the carbon quantum dots emit strong green fluorescence at an excitation wavelength of 450nm. When ascorbic acid solutions of different concentrations (0-7.50mmol / L) are added to the carbon quantum dot solution, an obvious fluorescence quenching effect is exhibited, indicating that the fluorescence intensity of CQDs is affected by ascorbic acid of different concentrations (0-7.50mmol / L). Figure 7 (b) is the linear fitting relationship between the fluorescence intensity change ΔF and the ascorbic acid concentration. It can be seen that when the ascorbic acid concentration is in the range of 1.25-7.50mmol / L, the CQDs fluorescence intensity change ΔF shows a good linear relationship with the ascorbic acid concentration. The fitted regression equation is y = 9.66857x -9.13333, and the linear correlation R 2 = 0.9912; in addition, the detection limit (LOD) was calculated based on 3 times the blank standard deviation (number of measurements n = 11 times) divided by the slope of the measured sample to be 0.64mmol / L (3σ / kn=11).

[0076] (4) Cytotoxicity: The MTT assay was used to detect the cytotoxic effects of CQDs at different concentrations (50, 100, 150, 200, and 250 μg / mL) on Lo2 cells and HepG2 tumor cells. Figure 8 (a) and Figure 8 (b) The results show that even at a high concentration of 250 μg / mL, the cells still have strong viability, indicating that the prepared CQDs have low cytotoxicity.

[0077] (5) Cell imaging: The Lo2 cells and HepG2 tumor cells were imaged 1, 3, and 5 hours after staining using a laser confocal high-resolution live cell imaging system. The imaging results (e.g. Figure 9 (a) and Figure 9 (b) shows green fluorescence emitted by CQDs in the cytoplasm, indicating that the CQDs can enter the cytoplasm and display green fluorescence there. The cells also exhibit good morphology, demonstrating the low cytotoxicity and high biosafety of the CQDs. During the 1-5 hour incubation period, the green fluorescence intensity increases with increasing incubation time, indicating that cellular uptake of the CQDs is time-dependent.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An application of carbon quantum dots using golden chrysanthemum as a carbon source in the detection of ascorbic acid, characterized in that: The preparation method of the carbon quantum dots is as follows: (1) After removing the calyx of the golden chrysanthemum, washing, drying and crushing, golden chrysanthemum powder is obtained; (2) dispersing the golden chrysanthemum powder obtained in step (1) into anhydrous ethanol, and subjecting the mixture to a high-temperature carbonization treatment at 170-210° C. for 9-11 hours to obtain a reaction solution I; (3) Cooling the reaction solution I obtained in step (2) to room temperature, centrifuging, filtering the supernatant, and reacting the obtained filtrate with the polyoxyethylene diamine alcohol solution at 25-35°C in the dark for 36-60 hours to obtain reaction solution II; (4) Reacting the reaction solution II with Na2CO3 solution at 25-35°C in the dark for 18-36 hours, and obtaining carbon quantum dots by dialysis, rotary evaporation, and freeze drying.

2. The use according to claim 1, characterized in that The drying temperature in step (1) is 75-85° C. and the drying time is 18-30 hours.

3. The use according to claim 2, characterized in that In the step (2), the concentration of the golden chrysanthemum powder in anhydrous ethanol is 0.025-0.033 g / mL.

4. The use according to claim 3, characterized in that The centrifugal speed in step (3) is 6000-10000 r / min, and the time is 5-15 min; the pore size of the microporous filter membrane used for filtration is 0.22 μm.

5. The use according to claim 4, characterized in that The concentration of the polyoxyethylene diamine alcohol solution in step (3) is 0.40-0.80 mg / mL; the volume ratio of the filtrate to the polyoxyethylene diamine alcohol solution is 10:(1-1.2).

6. The use according to claim 5, characterized in that The concentration of the Na2CO3 solution in step (4) is 0.08-0.12 mg / mL; the volume ratio of reaction solution II to the Na2CO3 solution is 1:(0.8-1.2).

7. The use according to claim 6, characterized in that The molecular weight cut-off of the dialysis bag used for dialysis in step (4) is 1000 Da, and the dialysis time is 36-60 h.

8. The use according to claim 1, characterized in that The detection method comprises dissolving carbon quantum dots in distilled water to prepare a solution, then adding ascorbic acid solution, measuring the linear relationship between the fluorescence intensity value change at an excitation wavelength of 450 nm and the ascorbic acid concentration, and using this linear relationship to detect ascorbic acid of unknown concentration.

Images