Preparation method and application of yellow wine fluorescent carbon dots

Abstract

The present invention discloses a preparation method and application of yellow wine fluorescent carbon dots. The yellow wine is concentrated and dialyzed to obtain an external dialyzate. The collected external dialyzate is concentrated and dialyzed to obtain an internal dialyzate. The internal dialyzate is filtered and freeze-dried to obtain yellow wine fluorescent carbon dots. The yellow wine fluorescent carbon dots can be excited by ultraviolet light to emit bright blue fluorescence. The fluorescent carbon dots have an emission center at 432 nm, and the corresponding maximum excitation wavelength is 357 nm. The preparation method has the characteristics of easy availability of raw materials, low cost, simple and controllable operation. The prepared fluorescent carbon dots have good fluorescence properties and biocompatibility. The fluorescent carbon dots are used as fluorescent probes for cell bioimaging and Fe in water samples. 3+ and Cu 2+ The detection of content greatly reduces the detection cost and improves convenience and economy.

Description

Technical Field

[0001] The invention relates to a method for preparing carbon quantum dots, and in particular to a method for preparing yellow wine fluorescent carbon dots and applications thereof. Background Art

[0002] Carbon dots are a zero-dimensional nanomaterial with good fluorescent properties. Their particle size is generally less than 10 nm. Due to their good biocompatibility, water solubility, low toxicity and good photoluminescence properties, they are widely used in bioimaging, optoelectronic devices and photocatalysis.

[0003] Current methods for preparing carbon dots primarily rely on physical or chemical synthesis, such as electrochemical synthesis, solvothermal synthesis, and chemical oxidation. However, these methods suffer from drawbacks such as high energy consumption, demanding synthesis conditions, and significant pollution during the synthesis process. Recent research has discovered that some foods produce fluorescent carbon dots during their production and processing. For example, carbon dots composed of four elements, C, H, O, and N, can be extracted from beer. Carbon dots can be directly extracted from foods without requiring synthesis, eliminating some of the pain points of the carbon dot synthesis process and offering promising applications. Summary of the Invention

[0004] The present invention aims to solve the above problems and provides a preparation method and application of yellow wine fluorescent carbon dots.

[0005] A method for preparing yellow wine fluorescent carbon dots and its application, the method comprising the following steps:

[0006] (1) Concentrating the rice wine to a concentration ratio of 5 to 10 times;

[0007] (2) dialyzing the concentrated rice wine to obtain the dialyzate;

[0008] (3) The dialysate is concentrated and dialyzed to obtain the dialysate;

[0009] (4) The dialyzed liquid was filtered and freeze-dried.

[0010] In the technical solution of the present invention: in step (1), the rice wine is concentrated by vacuum rotary evaporation, the vacuum degree is between 0.09-0.1Mpa, and the rotary evaporation temperature is 40-70°C.

[0011] In the technical solution of the present invention: the molecular weight cut-off of the dialysis bag used for dialysis in step (2) is 1000-3000Da, and the dialysis time is 24h-48h.

[0012] In the technical solution of the present invention: in step (3), the dialyzed external fluid is concentrated by vacuum rotary evaporation, the vacuum degree is between 0.09-0.1Mpa, and the rotary evaporation temperature is 50-75°C.

[0013] In the technical solution of the present invention: the molecular weight cut-off of the dialysis bag used for dialysis in step (3) is 350-500Da, and the dialysis time is 24h-48h.

[0014] In the technical solution of the present invention: the filter membrane used for filtering the dialyzed liquid in step (4) is a 0.2-0.3 μm water filter membrane.

[0015] In the technical solution of the present invention: the freeze drying in step (4) adopts vacuum freeze drying, the vacuum degree is 0.08-0.133Mpa, and the freezing temperature is -80 to -85°C.

[0016] A fluorescent carbon dot is prepared by the above method.

[0017] In the technical solution of the present invention, the fluorescent carbon dots prepared by the above method are used in the field of biological imaging.

[0018] In the technical solution of the present invention, the fluorescent carbon dots prepared by the above method are used in the detection of metal ions.

[0019] Huangjiu (yellow rice wine) has long been one of China's most popular alcoholic beverages. Made from grains like glutinous rice and wheat, it undergoes a series of processes, including steaming, adding koji, saccharification and fermentation, pressing, and decoction. It contains numerous functional ingredients beneficial to the human body. During the brewing process, the breakdown of macromolecules like proteins and polysaccharides, as well as the aggregation of small molecules, can produce carbon dots (Cdots). We used a double dialysis method to quickly and easily extract fluorescent Cdots from yellow rice wine. The resulting Cdots exhibit excellent optical properties, exhibiting bright blue fluorescence under ultraviolet (UV) light.

[0020] Beneficial effects:

[0021] (1) The present invention establishes a method for preparing yellow wine fluorescent carbon dots;

[0022] (2) The present invention uses yellow rice wine as the main raw material for extracting fluorescent carbon dots. The raw material is easily available, the cost is low, and the preparation process is simple and efficient.

[0023] (3) The fluorescent carbon dots prepared by the present invention have good optical properties and high biosafety, and have broad application prospects in the fields of biological imaging and metal ion detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Attachment Figure 1 This is a fluorescence image of the fluorescent carbon dots extracted and prepared by the present invention under ultraviolet light;

[0025] Attachment Figure 2 The infrared spectrum of the fluorescent carbon dots extracted and prepared by the present invention;

[0026] Attachment Figure 3The fluorescence spectrum of the fluorescent carbon dots extracted and prepared by the present invention;

[0027] Attachment Figure 4 This is the ultraviolet spectrum of the fluorescent carbon dots extracted and prepared by the present invention;

[0028] Attachment Figure 5 The activity of Caco-2 cells under the action of fluorescent carbon dots extracted and prepared by the present invention;

[0029] Attachment Figure 6 The ABTS free radical scavenging rate of the fluorescent carbon dots extracted and prepared by the present invention;

[0030] Attachment Figure 7 The hydroxyl radical scavenging rate of the fluorescent carbon dots extracted and prepared by the present invention;

[0031] Attachment Figure 8 The total reducibility of the fluorescent carbon dots extracted and prepared by the present invention;

[0032] Attachment Figure 9 This is a Caco-2 cell imaging image of the fluorescent carbon dots extracted and prepared by the present invention;

[0033] Attachment Figure 10 This is a graph of the fluorescence intensity of the fluorescent carbon dots extracted and prepared by the present invention in the presence of different concentrations of iron ions;

[0034] Attachment Figure 11 This is a fluorescence intensity diagram of the fluorescent carbon dots extracted and prepared in the present invention in the presence of different concentrations of copper ions. DETAILED DESCRIPTION

[0035] The present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto:

[0036] Experimental Materials:

[0037] Yellow rice wine (Shazhou Youhuang) was purchased from Jiangsu Zhangjiagang Brewing Co., Ltd. (Jiangsu, China).

[0038] Example 1: Extraction and preparation of yellow wine fluorescent carbon dots.

[0039] (1) Using a vacuum rotary evaporator with a vacuum degree of 0.1 MPa and a rotary evaporation temperature of 60°C, 500 mL of rice wine was concentrated to 50 mL. The concentrated rice wine was dialyzed using a dialysis bag with a molecular weight cutoff of 3000 Da. The dialysate was deionized water. The dialysate was collected several times during the dialysis process until the dialysate had no obvious color. The dialysis was completed after about 48 hours. The dialysate was stored in a refrigerator at 4°C for later use.

[0040] (2) Using a vacuum rotary evaporator with a vacuum degree of 0.1 MPa and a rotary evaporation temperature of 60°C, the collected dialyzed external liquid was concentrated to 50 mL. The concentrated dialyzed external liquid was dialyzed using a dialysis bag with a molecular weight cutoff of 500 Da. The dialysate was deionized water and the dialysate was replaced every 5 hours. After dialysis for 48 hours, the dialysate was collected and filtered using a 0.22 μm filter membrane. The dialysate was then vacuum-freeze-dried with a vacuum degree of 0.133 MPa and a freezing temperature of -84°C. The powder collected by freeze-drying was the yellow wine fluorescent carbon dots, which were stored in a drying dish away from light to prevent moisture absorption.

[0041] Example 2: Characterization of the physicochemical properties of yellow wine fluorescent carbon dots.

[0042] (1) Fluorescence characteristics

[0043] Using ultraviolet light excitation, the fluorescence properties of rice wine fluorescent carbon dots were investigated and characterized.

[0044] The extracted yellow wine fluorescent carbon dots were dissolved in deionized water to prepare solutions with concentrations of 1000, 500, 250, 125, 62.5, 31.25, and 15.625 mg / mL. The solutions were transferred to a quartz cuvette and placed under ultraviolet light. Figure 1 As shown in the figure, it shows strong blue fluorescence under the excitation of ultraviolet light with a wavelength of 365nm, and the fluorescence intensity decreases with the decrease of carbon dot concentration, indicating that the carbon dots in the extracted rice wine are fluorescent carbon dots.

[0045] (2) Fourier transform infrared spectroscopy analysis

[0046] The surface functional group composition of yellow wine fluorescent carbon dots was studied using Fourier transform infrared spectrophotometer.

[0047] The yellow wine fluorescent carbon dots used in the experiment were prepared by freeze drying equipment. The freeze drying process was carried out in a Christ Alpha 1-4 freeze dryer (Christ, ALPHA 1-4 / 2-4LD plus, Shanghai). -1 The FT-IR spectrum of yellow wine fluorescent carbon dots was obtained in the wavenumber range of Figure 2 As shown, at 3275cm -1 The peak at 2919cm is the stretching vibration absorption peak of -NH2 and -OH. -1 The peaks near 1628cm indicate the existence of CH bonds of yellow wine fluorescent carbon dots; -1 The peaks near the NH2 stretching vibration peak at 1537 cm -1 Absorption at 1430 cm -1The peaks near the graphite may be related to C=C stretching, which indicates the presence of graphitized sp 2 Hybridization, these results indicate that there are amino, hydroxyl and carboxyl groups on the surface of yellow wine fluorescent carbon dots.

[0048] (3) Fluorescence and UV spectroscopy analysis

[0049] The prepared yellow wine fluorescent carbon dots were dissolved in deionized water to prepare a solution with a concentration of 500 mg / mL, and then fluorescence and UV spectroscopy were performed. Figure 3 As shown in Figure 2, the maximum fluorescence excitation wavelength of yellow wine fluorescent carbon dots is 357nm, and the maximum emission wavelength is 432nm. Figure 4 As shown, the ultraviolet spectrum of yellow wine fluorescent carbon dots has a peak at 273nm.

[0050] Example 3: Biocompatibility of Yellow Wine Fluorescent Carbon Dots

[0051] Caco-2 cells in the logarithmic growth phase were digested with trypsin and collected for counting. The initial cell concentration was adjusted to 5000 cells / well and seeded into 96-well plates. The cells were placed in a 5% CO2 incubator and incubated for 24 hours. Yellow wine fluorescent carbon dots were prepared into 6.25, 12.5, 25, 50, 100, 200, 400, 800, and 1600 mg / mL solutions using serum-free culture medium. The supernatant culture medium of the 96-well plate cells was aspirated with a pipette and the yellow wine fluorescent carbon dot solution was added. The control group was serum-free culture medium without yellow wine fluorescent carbon dots. The cells were cultured for another 24 hours and the cell viability was detected by MTT assay. The cell survival rate was obtained as follows: Figure 5 As shown in Figure 5 The results are the absorbance value at a detection wavelength of 570nm. The cell survival rate under the action of high concentration yellow wine fluorescent carbon dots is higher than 80%, indicating that yellow wine fluorescent carbon dots have good biocompatibility.

[0052] Example 4: Antioxidant properties of yellow wine fluorescent carbon dots

[0053] (1) ABTS free radical scavenging rate

[0054] The prepared yellow wine fluorescent carbon dots were dissolved in deionized water to prepare a solution with a concentration of 2.5 mg / mL for ABTS free radical scavenging rate detection. 10 mL of 7 mmol / L ABTS solution was mixed with 178 mL of 140 mmol / L potassium persulfate solution and incubated in the dark for 12 h to obtain the ABTS stock solution, which was then diluted with deionized water to an OD of 734 nm= 0.7 ABTS working solution, ABTS working solution and rice wine fluorescent carbon dot solution were mixed in equal proportions and incubated at room temperature in the dark for 1 hour. The control group was incubated with ABTS working solution and deionized water. Then the cells were transferred to a 96-well plate and the absorbance was measured at a detection wavelength of 734 nm using a microplate reader to calculate the ABTS clearance rate. The results are shown in Figure 2. Figure 6 As shown in the figure, the yellow wine fluorescent carbon dots exhibited an ABTS free radical scavenging rate of 80%, indicating that the yellow wine fluorescent carbon dots had significant antioxidant effects.

[0055] (2) Hydroxyl radical scavenging rate

[0056] The prepared yellow wine fluorescent carbon dots were dissolved in deionized water to prepare a solution with a concentration of 2.5 mg / mL for hydroxyl radical scavenging rate detection. 1 mL of yellow wine fluorescent carbon dot solution, 1 mL of 6 mmol / L FeSO4 solution, 1 mL of 6 mmol / L H2O2 and 1 mL of 6 mmol / L ethanol salicylic acid solution were mixed in sequence and incubated at 37°C in the dark for 1 hour. In the control group, the yellow wine fluorescent carbon dot solution was replaced with deionized water and then transferred to a 96-well plate. The absorbance was measured at a detection wavelength of 510 nm using an enzyme reader, and the hydroxyl radical scavenging rate was calculated. The results are shown in Figure 2. Figure 7 As shown in the figure, the yellow wine fluorescent carbon dots exhibited a hydroxyl radical scavenging rate of more than 50%, indicating that the yellow wine fluorescent carbon dots had a significant antioxidant effect.

[0057] (3) Total reducibility

[0058] The prepared yellow wine fluorescent carbon dots were dissolved in deionized water to prepare a solution with a concentration of 2.5 mg / mL for total reducing test. 2.5 mL of yellow wine fluorescent carbon dots solution, 2.5 mL of 0.02 mol / L PBS and 2.5 mL of 1% (w / v) potassium ferricyanide were mixed evenly and incubated at 55°C for 20 minutes. Then 2.5 mL of 10% (w / v) trichloroacetic acid solution was added to the mixture, mixed evenly and centrifuged at 1000 g for 15 minutes. 2.5 mL of supernatant was added to 2.5 mL of deionized water and 0.5 mL of 0.1% ferric chloride solution and mixed evenly, incubated at room temperature in the dark for 10 minutes, and then transferred to a 96-well plate and the absorbance was measured at a detection wavelength of 700 nm using an enzyme reader. The absorbance corresponds to the intensity of the total reducing property. Figure 8 As shown in the figure, the results show that the yellow wine fluorescent carbon dots have strong reducing ability.

[0059] Example 5: Application of Yellow Wine Fluorescent Carbon Dots in Bioimaging

[0060] Caco-2 cells in the logarithmic growth phase were digested with trypsin and collected for counting. The initial cell concentration was adjusted to 150,000 cells / well and seeded into 12-well plates. The plates were placed in a 5% CO2 incubator and incubated for 24 hours. Yellow wine fluorescent carbon dots were prepared into 100 and 400 mg / mL solutions using serum-free culture medium. The supernatant of the culture medium in the 12-well plates was aspirated with a pipette and then added to the yellow wine fluorescent carbon dots solution. The cells were cultured for 3 hours and then removed. The supernatant was discarded and the yellow wine fluorescent carbon dots that had not entered the cells were removed. The cells were rinsed with PBS buffer three times without blowing off the cells and then observed under a fluorescence microscope. The results are shown in Figure 2. Figure 9 As shown in the figure, as the concentration of yellow wine fluorescent carbon dots increases, the fluorescence intensity in the cells increases, and when the excitation wavelength is 460-550nm, the imaging effect of yellow wine fluorescent carbon dots is better.

[0061] Example 6: Application of Yellow Wine Fluorescent Carbon Dots in Metal Ion Detection

[0062] (1)Fe 3+ Ion detection

[0063] Prepare 4 mL of 1 mol / L Fe 3+ solution, and then take 2mL of 1mol / L Fe 3+ The solution was thoroughly mixed with 2 mL of deionized water to prepare 0.5 mol / L Fe 3+ solution, and then take 2mL of 0.5mol / L Fe 3+ The solution was thoroughly mixed with 2 mL of deionized water to prepare 0.25 mol / L Fe 3+ Solution, continue to Fe 3+ The solution was diluted to a final concentration of 9.54×10 - 7 mol / L, a 10 mg / mL yellow wine fluorescent carbon dot solution was prepared and mixed with different concentrations of Fe 3+ The solution was mixed to a final concentration of 0.1 mg / mL of yellow wine fluorescent carbon dots. After mixing, the solution was shaken vigorously at room temperature for 40 minutes and the fluorescence intensity of the solution was measured. When measuring the fluorescence intensity, the excitation wavelength was 357 nm, the emission wavelength was 450 nm, and the slit width was 5 nm. The results are shown in Figure 2. Figure 10 As shown, with Fe 3+ As the concentration increases, the fluorescence intensity of the yellow wine fluorescent carbon dots decreases, and the Fe 3+ concentration.

[0064] (2)Cu 2+ Ion detection

[0065] Prepare 4 mL of 1 mol / L Cu 2+solution, and then take 2mL of 1mol / L Cu 2+ The solution was thoroughly mixed with 2 mL of deionized water to prepare 0.5 mol / L Cu 2+ solution, and then take 2mL of 0.5mol / L Cu 2+ The solution was thoroughly mixed with 2 mL of deionized water to prepare 0.25 mol / L Cu 2+ Solution, continue to add Cu 2+ The solution was diluted serially to a final concentration of 2.38 × 10 - 7 mol / L, a 10 mg / mL yellow wine fluorescent carbon dot solution was prepared and mixed with different concentrations of Cu 2+ The solution was mixed to a final concentration of 0.1 mg / mL of yellow wine fluorescent carbon dots. After mixing, the solution was shaken vigorously at room temperature for 40 minutes and the fluorescence intensity of the solution was measured. When measuring the fluorescence intensity, the excitation wavelength was 357 nm, the emission wavelength was 450 nm, and the slit width was 5 nm. The results are shown in Figure 2. Figure 11 As shown, with Cu 2+ As the concentration increases, the fluorescence intensity of the yellow wine fluorescent carbon dots decreases, and the Cu 2+ concentration.

Claims

1. A method for preparing yellow wine fluorescent carbon dots, characterized by: The method comprises the following steps: (1) Concentrate the rice wine to a concentration ratio of 5-10 times; (2) dialyzing the concentrated rice wine to obtain the dialysis fluid; (3) The dialysis fluid is concentrated and dialyzed to obtain the dialysis fluid; (4) Filter the dialyzed fluid and freeze-dry it; Wherein, in step (1), the rice wine is concentrated by vacuum rotary evaporation, the vacuum degree is between 0.09-0.1 Mpa, and the rotary evaporation temperature is 40-70°C; The molecular weight cut-off of the dialysis bag used for dialysis in step (2) is 1000-3000 Da, and the dialysis time is 24 h-48 h; In step (3), the dialyzed liquid is concentrated by vacuum rotary evaporation, the vacuum degree is between 0.09-0.1 MPa, and the rotary evaporation temperature is 50-75°C; The molecular weight cut-off of the dialysis bag used for dialysis in step (3) is 350-500 Da, and the dialysis time is 24 h-48 h.

2. The method according to claim 1, wherein: The filter membrane used for filtration of the dialyzed liquid in step (4) is a 0.2-0.3 mm water filter membrane.

3. The method according to claim 1, wherein: In step (4), freeze drying is performed by vacuum freeze drying, the vacuum degree is 0.08-0.133 MPa, and the freezing temperature is -80 to -85°C.

4. A fluorescent carbon dot, characterized in that The fluorescent carbon dots are prepared by the method according to any one of claims 1 to 3.

5. Use of the fluorescent carbon dots prepared by the preparation method according to any one of claims 1 to 3 in the field of bioimaging for purposes other than disease diagnosis and treatment.

6. Use of the fluorescent carbon dots prepared by the preparation method according to any one of claims 1 to 3 in metal ion detection.

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