A nitrogen-rich hydrothermal phase high-quality biomass-based carbon dot and its preparation method and application

Abstract

The present invention discloses a nitrogen-rich hydrothermal liquid-phase high-quality biomass-based carbon dot and its preparation method, belonging to the field of thermochemical processing. By combining an enzymatic reaction with hydrothermal carbonization technology, the present invention converts nitrogen-rich biomass into high-value-added hydrothermal carbon and aqueous solution. Enzymatic pretreatment prior to hydrothermal carbonization more efficiently transfers nitrogen from the raw material to the liquid product, while also improving the fluorescence quality of the liquid product. The liquid product can be used to produce high-quality carbon dot materials, realizing the resource utilization of nitrogen-rich biomass, with high ecological and economic benefits.

Description

Technical Field

[0001] The present invention belongs to the field of biomass thermochemical treatment, and in particular relates to enzymatic reaction and biomass hydrothermal treatment technology. Background Art

[0002] Carbon dots (Cdots) is a broad term used to define carbon-based nanomaterials with luminescent properties, typically smaller than 10 nanometers in size. Compared to traditional organic dyes and semiconductor quantum dots, Cdots offer advantages such as low preparation cost, good water solubility, environmental friendliness, stable luminescence, easy surface modification, and low biotoxicity. They have been widely used in a variety of fields, including biosensing, biomedical imaging, and analytical testing.

[0003] Based on the differences in precursors, carbon dot synthesis methods can be broadly categorized into two main groups: top-down approaches based on the synthesis of large-scale carbon materials and bottom-up approaches based on the synthesis of small-molecule hydrocarbons. Top-down approaches generally involve chemically and physically cutting relatively macroscopic carbon materials such as carbon nanotubes, graphene, carbon fibers, and carbon powder using methods such as electrochemistry, laser irradiation, and chemical oxidation. However, these methods require the destruction of large, highly crystalline carbon materials and require demanding preparation conditions, resulting in high costs and unsuitable for mass production. Bottom-up approaches, using small organic molecules, oligomers, or biomass as carbon sources, ultimately produce carbon dots through a series of chemical reactions. These methods primarily include microwave, hydrothermal, electrochemical, and ultrasonic methods. Hydrothermal methods are widely used due to their simplicity, safety, efficiency, minimal environmental impact, and high fluorescence quantum yield. To date, no methods have been reported for producing high-quality carbon dots through the hydrothermal carbonization of nitrogen-rich biomass catalyzed by an enzymatic reaction. Summary of the Invention

[0004] The present invention combines enzymatic reaction with hydrothermal carbonization technology to convert nitrogen-rich biomass into high-value-added hydrothermal carbon and aqueous solution. Through enzymatic pretreatment before hydrothermal carbonization, the nitrogen element in the raw material is more efficiently transferred to the liquid product, while the fluorescence quality of the liquid product is improved. The liquid product can be used to prepare high-quality carbon dot materials, realizing the resource utilization of nitrogen-rich biomass, and has high ecological and economic benefits.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A method for preparing high-quality biomass-based carbon dots in a nitrogen-rich hydrothermal phase, the method comprising the following steps:

[0007] S1. Raw material processing: drying and crushing the nitrogen-rich biomass to 60-100 mesh to obtain pre-treated raw materials;

[0008] S2. Enzymatic pretreatment: Mix the pretreated raw materials, protease and deionized water in a ratio of 1-10 g: 1-20 mg: 10-100 mL, add acid to adjust to acidity, and stir at a temperature of 45-85°C for 4-6 hours;

[0009] S3, hydrothermal carbonization: the product after the enzymatic reaction is transferred to a high-pressure hydrothermal reactor for hydrothermal carbonization to produce high-quality fluorescent carbon dots;

[0010] S4. Product collection: The solid and liquid phase products after hydrothermal carbonization were centrifuged and the supernatant was dialyzed using a dialysis membrane with a molecular weight cutoff of 1000-1400 Da for 48-56 h and freeze-dried to obtain a solid carbon dot sample.

[0011] In the technical solution of the present invention: the nitrogen-rich biomass described in step S1 is Spirulina platensis, Chlorella pyrenoidosa, soybean meal, water hyacinth, soybean straw or kitchen waste.

[0012] In the technical solution of the present invention: the raw materials described in step S1 are dried at 105° C. for 5-10 minutes and crushed to 80 mesh.

[0013] In the technical solution of the present invention: the ratio of the raw material, protease and deionized water after pretreatment in step S2 is (1-5) g: (5-15) mg: (50-100) mL;

[0014] In the technical solution of the present invention: the acidic condition in step S2 refers to a pH of 3 to 5.

[0015] In the technical solution of the present invention: the reaction temperature in step S2 is 50-80° C., and the stirring speed is 200-300 rpm.

[0016] In the technical solution of the present invention: the temperature of the hydrothermal carbonization reaction in step S3 is (140°C-300°C), the time is (5-100 minutes), and the reaction environment is a nitrogen atmosphere.

[0017] In the technical solution of the present invention: the hydrothermal reaction in step S3 is carried out under stirring conditions, and the stirring speed is 200-600 rpm.

[0018] In the technical solution of the present invention: in step S4, the supernatant is dialyzed with a dialysis membrane with a molecular weight cutoff of 1100-1300 Da for 48-55 hours, and the liquid product carbon liquid is used to prepare high-quality carbon dot material.

[0019] A nitrogen-rich hydrothermal liquid phase high-quality biomass-based carbon dot, the high-quality biomass-based carbon dot is prepared by the above method.

[0020] In the technical solution of the present invention, the nitrogen-rich hydrothermal liquid phase high-quality biomass-based carbon dots prepared above are used in breast cancer cell labeling.

[0021] In the technical solution of the present invention: the protease in step S2 is acidic protease (20,000 U / g), which is purchased from Guangxi Nanning Pangbo Bioengineering Co., Ltd.

[0022] Beneficial effects of the present invention:

[0023] 1. The prepared liquid product contains carbon nanoparticles with stable fluorescent properties. It has many advantages, such as high light resistance, high water solubility, good chemical inertness, easy modification, low toxicity, good biocompatibility, and good electronic properties as electron donors and acceptors. It has great application potential in the field of organic long-lasting luminescence.

[0024] 2. The present invention fully transfers the nitrogen element in nitrogen-rich biomass to the liquid phase product, which not only improves the quality of the fluorescent carbon material, but also reduces the emission of nitrogen-containing organic pollutants, effectively utilizes the nitrogen-rich biomass, and has high economic and ecological benefits.

[0025] 3. The prepared fluorescent carbon dot material uses nitrogen-rich biomass as raw material, which has a wide source. The present invention can convert nitrogen-rich biomass into high-value products. The technology and equipment are simple, and no complex transformation is required. It is cheap and efficient, and has an industrialization prospect that cannot be ignored. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 a and b are Example 2, the effects of different residence times on the total nitrogen in the hydrothermal liquid phase of soybean meal at a reaction temperature of 220°C, where a is hydrothermal treatment (the hydrothermal treatment conditions are the same as those of the hydrothermal carbonization reaction in Example 2), and b is enzymatic-hydrothermal treatment.

[0027] Figure 1 c and d are the effects of different reaction temperatures on the total nitrogen in the hydrothermal liquid phase of soybean meal when the residence time is 10 min in Example 1, where c is hydrothermal treatment (the hydrothermal treatment conditions are the same as those of the hydrothermal carbonization reaction in Example 1), and d is enzymatic-hydrothermal treatment.

[0028] Figure 2 In Example 1, the effect of different reaction temperatures on the 3D fluorescence spectra of soybean meal-based carbon dots at a residence time of 10 minutes is shown. (a) represents the hydrothermal treatment (the hydrothermal treatment conditions are identical to those in Example 1), and (b) represents the enzymatic-hydrothermal treatment. It can be seen that the liquid fluorescence intensity before and after enzymatic treatment is affected by both reaction temperature and total nitrogen in the liquid phase.

[0029] Figure 3TEM (a and b) and HRTEM (c and d) images of soybean meal-based carbon dots (CDOs) at 220°C and a residence time of 10 minutes, as shown in Example 2. Images a and c represent hydrothermal treatment (using the same hydrothermal carbonization conditions as in Example 2), while images b and d represent enzymatic-hydrothermal treatment. Enzymatic action enhances the concentration of small molecules in the liquid phase, leading to entanglement between CDOs and small molecule chains, increasing their size, and widening the interplanar spacing.

[0030] Figure 4 The XRD spectrum of soybean meal-based carbon dots is shown in Example 1, with a reaction temperature of 220°C and a residence time of 10 min. S-CDs are carbon dots obtained by hydrothermal treatment (the hydrothermal treatment conditions are the same as those of the hydrothermal carbonization reaction in Example 1), and SE-CDs are carbon dots obtained by enzymatic-hydrothermal treatment. The enzymatic action enhances the carbonization degree of the carbon dots, and the intensity of the characteristic peak of amorphous carbon is increased.

[0031] Figure 5 This is a diagram of the labeling of mouse breast cancer cells by soybean meal-based carbon dots obtained by enzymatic-hydrothermal treatment at a reaction temperature of 220°C and a residence time of 10 min, where a is before labeling and b is after labeling. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the embodiments, but the protection scope of the present invention is not limited thereto:

[0033] The raw materials in the examples were dried at 105° C. for 8 min.

[0034] The protease was acidic protease (20,000 U / g) purchased from Pangbo Bioengineering Co., Ltd., Nanning, Guangxi.

[0035] Example 1

[0036] (1) Enzymatic pretreatment

[0037] The soybean meal was dried and crushed to 80 mesh, 2 g of soybean meal powder, 8 mg of acidic protease and 70 mL of deionized water were accurately weighed, mixed evenly, and citric acid was added to adjust the pH to 4. The mixture was placed in a constant temperature water bath in a magnetic stirrer with a water bath temperature of 55 ° C and a stirring speed of 280 rpm for 5 hours.

[0038] (2) Hydrothermal carbonization reaction

[0039] The product after the enzymatic reaction was moved to a high-pressure hydrothermal reactor for hydrothermal carbonization reaction. The reaction environment was a nitrogen atmosphere. The reactor body was fixed in an electric heating furnace and sealed. The hydrothermal temperatures were set to 160°C, 180°C, 200°C, 220°C, 240°C, and 260°C, respectively. The residence time was 10 minutes and the stirring speed was 550 rpm. After the reaction was completed, the solid and liquid were centrifuged and separated. The supernatant was dialyzed for 52 hours using a dialysis membrane with a molecular weight cutoff of 1200Da. The obtained liquid product carbon liquid was used to prepare carbon quantum dot fluorescent material.

[0040] Example 2

[0041] (1) Enzymatic pretreatment

[0042] The soybean meal was dried and crushed to 80 mesh, 2 g of soybean meal powder, 8 mg of acidic protease and 70 mL of deionized water were accurately weighed, mixed evenly, and citric acid was added to adjust the pH to 4. The mixture was placed in a constant temperature water bath in a magnetic stirrer with a water bath temperature of 55 ° C and a stirring speed of 280 rpm for 5 hours.

[0043] (2) Hydrothermal carbonization reaction

[0044] The product after the enzymatic reaction was moved to a high-pressure hydrothermal reactor for hydrothermal carbonization reaction. The reaction environment was a nitrogen atmosphere. The reactor body was fixed in an electric heating furnace and sealed. The hydrothermal temperature was set to 220°C. The residence times were 5 min, 10 min, 20 min, 30 min, 60 min, and 90 min, respectively, and the stirring speed was 550 rpm. After the reaction was completed, the solid and liquid were centrifuged and separated. The supernatant was dialyzed for 52 h using a dialysis membrane with a molecular weight cutoff of 1200 Da. The obtained liquid product carbon liquid was used to prepare carbon quantum dot fluorescent material.

[0045] Example 3

[0046] (1) Enzymatic pretreatment

[0047] The plate was dried and crushed to 100 mesh size. 3 g of soybean meal powder, 12 mg of acidic protease and 100 mL of deionized water were accurately weighed and mixed evenly. After that, citric acid was added to adjust the pH to 4. The plate was placed in a constant temperature water bath in a magnetic stirrer at 80°C and a stirring speed of 340 rpm for 6 h.

[0048] (2) Hydrothermal carbonization reaction

[0049] The product after the enzymatic reaction was transferred to a high-pressure hydrothermal reactor for hydrothermal carbonization reaction. The reaction environment was a nitrogen atmosphere. The reactor body was fixed in an electric heating furnace and sealed. The hydrothermal temperature was set to 220°C, the residence time was 10 minutes, and the stirring speed was 550 rpm. After the reaction was completed, the solid and liquid were centrifuged and separated. The supernatant was dialyzed for 56 hours using a dialysis membrane with a molecular weight cutoff of 1400Da. The obtained liquid product carbon liquid was used to prepare carbon quantum dot fluorescent material.

[0050] Example 4

[0051] (1) Enzymatic pretreatment

[0052] The protein nucleus Chlorella was dried and crushed into 60 mesh, 1g soybean meal powder, 6mg acid protease and 50mL deionized water were accurately weighed, mixed evenly, and citric acid was added to adjust the pH to 4. The mixture was placed in a constant temperature water bath in a magnetic stirrer with a water bath temperature of 50°C and a stirring speed of 200 rpm for 4 hours.

[0053] (2) Hydrothermal carbonization reaction

[0054] The product after the enzymatic reaction was transferred to a high-pressure hydrothermal reactor for hydrothermal carbonization reaction. The reaction environment was a nitrogen atmosphere. The reactor body was fixed in an electric heating furnace and sealed. The hydrothermal temperature was set to 240°C, the residence time was 20 minutes, and the stirring speed was 400 rpm. After the reaction was completed, the solid and liquid were centrifuged and separated. The supernatant was dialyzed for 48 hours using a dialysis membrane with a molecular weight cutoff of 1000Da. The obtained liquid product carbon liquid was used to prepare carbon quantum dot fluorescent material.

[0055] Example 5

[0056] (1) Enzymatic pretreatment

[0057] The water hyacinth chlorella was dried and crushed into 80 mesh, and 1g soybean meal powder, 10mg acid protease and 70mL deionized water were accurately weighed. After mixing evenly, citric acid was added to adjust the pH to 4. The mixture was placed in a constant temperature water bath in a magnetic stirrer with a water bath temperature of 60°C and a stirring speed of 250rpm for 5 hours.

[0058] (2) Hydrothermal carbonization reaction

[0059] The product after the enzymatic reaction was transferred to a high-pressure hydrothermal reactor for hydrothermal carbonization reaction. The reaction environment was a nitrogen atmosphere. The reactor body was fixed in an electric heating furnace and sealed. The hydrothermal temperature was set to 260°C, the residence time was 30 minutes, and the stirring speed was 500 rpm. After the reaction was completed, the solid and liquid were centrifuged and separated. The supernatant was dialyzed for 50 hours using a dialysis membrane with a molecular weight cutoff of 1200Da. The obtained liquid product carbon liquid was used to prepare carbon quantum dot fluorescent material.

[0060] Performance testing:

[0061] Figure 1 Figures 1 and 2 show the effects of different residence times on total nitrogen in the hydrothermal liquid phase of soybean meal (a and b) and the effects of different reaction temperatures on total nitrogen in the hydrothermal liquid phase of soybean meal (c and d). Figures 1 and 2 represent hydrothermal treatments, while 2 and 3 represent enzymatic-hydrothermal treatments. Enzymatic-hydrothermal treatment involves enzymatic pretreatment of the soybean meal followed by hydrothermal carbonization of the enzymatic product; hydrothermal treatment involves direct hydrothermal carbonization of the soybean meal.

[0062] Figure 2 Figure 3 shows the effect of different reaction temperatures on the three-dimensional fluorescence spectra of soybean meal-based carbon dots (a) for hydrothermal treatment and b for enzymatic-hydrothermal treatment. It can be seen that the liquid fluorescence intensity before and after enzymatic treatment is affected by both reaction temperature and total nitrogen in the liquid phase.

[0063] Figure 3 Figures TEM (a and b) and HRTEM (c and d) of soybean meal-based carbon dots. (a and c) represent hydrothermal treatment, while (b and d) represent enzymatic-hydrothermal treatment. Enzymatic treatment enhances the concentration of small molecules in the liquid phase, leading to entanglement between the carbon dots and small molecule chains, increasing their size and widening the interplanar spacing.

[0064] Figure 4 This is the XRD spectrum of soybean meal-based carbon dots. The enzymatic action enhances the carbonization degree of the carbon dots, and the intensity of the characteristic peak of amorphous carbon increases.

[0065] Figure 5 This is a picture of soybean meal-based carbon dots labeling mouse breast cancer cells, where a is before labeling and b is after labeling.

Claims

1. A method for preparing high-quality biomass-based carbon dots in a nitrogen-rich hydrothermal phase, characterized by: The method comprises the following steps: S1. Raw material processing: drying and crushing nitrogen-rich biomass to 60-100 mesh to obtain pretreated raw materials; the nitrogen-rich biomass in step S1 is Spirulina platensis, Chlorella pyrenoidosa, soybean meal, water hyacinth, soybean straw or kitchen waste; S2. Enzymatic pretreatment: Mix the pretreated raw materials, protease and deionized water in a ratio of 1-10 g: 1-20 mg: 10-100 mL, add acid to adjust to acidity, and stir at 45-85°C for 4-6 hours; S3, hydrothermal carbonization: the product after the enzymatic reaction is transferred to a high-pressure hydrothermal reactor for hydrothermal carbonization reaction; S4. Product collection: The solid and liquid phase products after hydrothermal carbonization were centrifuged and the supernatant was dialyzed using a dialysis membrane with a molecular weight cutoff of 1000-1400 Da for 48-56 h and freeze-dried to obtain a solid carbon dot sample.

2. The method for preparing high-quality biomass-based carbon dots in a nitrogen-rich hydrothermal phase according to claim 1, characterized in that: The ratio of the pretreated raw material, protease, and deionized water in step S2 is (1-5) g: (5-15) mg: (50-100) mL.

3. The method for preparing high-quality biomass-based carbon dots in a nitrogen-rich hydrothermal phase according to claim 1, wherein: The acidic condition in step S2 refers to a pH of 3 to 5.

4. The method for preparing high-quality biomass-based carbon dots in a nitrogen-rich hydrothermal phase according to claim 1, wherein: The reaction temperature in step S2 is 50-80° C., and the stirring speed is 200-350 rpm.

5. The method for preparing high-quality biomass-based carbon dots in a nitrogen-rich hydrothermal phase according to claim 1, characterized in that: The temperature of the hydrothermal carbonization reaction in step S3 is 140° C.-300° C., the time is 5-100 min, and the reaction environment is a nitrogen atmosphere.

6. The method for preparing high-quality biomass-based carbon dots in a nitrogen-rich hydrothermal phase according to claim 1, wherein: The hydrothermal reaction in step S3 is carried out under stirring at a speed of 200-600 rpm.

7. The method for preparing high-quality biomass-based carbon dots in a nitrogen-rich hydrothermal phase according to claim 1, characterized in that: The liquid product carbon liquid in step S4 is used to prepare high-quality carbon dot materials.

8. A nitrogen-rich hydrothermal phase high-quality biomass-based carbon dot, characterized by: The high-quality biomass-based carbon dots are prepared using the preparation method described in any one of claims 1 to 7.

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