Nitrogen-rich hydrothermal liquid-phase high-quality biomass-based carbon dots as well as preparation method and application thereof

Abstract

The invention discloses a nitrogen-rich hydrothermal liquid-phase high-quality biomass-based carbon dot and a preparation method thereof, and belongs to the field of thermochemical treatment. According to the method, an enzymatic reaction and a hydrothermal carbonization technology are combined, the nitrogen-rich biomass is converted into hydrothermal carbon and an aqueous solution with high added value, the nitrogen element in the raw material is more efficiently transferred into a liquid-phase product through enzymatic pretreatment before hydrothermal carbonization treatment, and meanwhile, the fluorescence quality of the liquid-phase product is improved; the liquid-phase product can be used for preparing a high-quality carbon dot material, so that resource utilization of nitrogen-rich biomass is realized, and the method has relatively high ecological benefits and economic benefits.

Description

Technical Field

[0001] The 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 are a broad term used to define carbon-based nanomaterials with luminescent properties, and their size is generally less than 10nm. Compared with traditional organic dyes and semiconductor quantum dots, carbon dots have the advantages of low preparation cost, good water solubility, environmental friendliness, stable luminescent properties, easy surface modification, and low biological toxicity. They have been widely used in many fields such as biosensing, biomedical imaging, and analytical detection.

[0003] According to the differences of precursors, the synthesis methods of carbon dots can be mainly divided into two categories: top-down methods based on the synthesis of large-size carbon materials and bottom-up methods based on the synthesis of small-molecule hydrocarbons. The top-down method generally uses electrochemical, laser irradiation and chemical oxidation methods to chemically and physically cut relatively macroscopic carbon materials such as carbon nanotubes, graphene, carbon fibers and carbon powder. However, this type of method requires the destruction of large pieces of carbon materials with high crystallinity and the preparation conditions are harsh, the cost is high, and it is not conducive to mass production. The bottom-up method uses small organic molecules, oligomers, and biomass as carbon sources, and finally obtains carbon dots through a series of chemical reactions, mainly including microwave method, hydrothermal method, electrochemical method, ultrasonic method, etc. Among them, the hydrothermal method is widely used because of its simple operation, safety and efficiency, less environmental damage, and high fluorescence quantum yield. So far, there has been no report on the method of preparing high-quality carbon dots based on hydrothermal carbonization of nitrogen-rich biomass under 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, realize 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 breaking the nitrogen-rich biomass to 60-100 mesh to obtain pre-treated raw materials;

[0008] S2. Enzymatic pretreatment: the pretreated raw material, protease and deionized water are mixed evenly in a ratio of 1-10 g: 1-20 mg: 10-100 mL, and then acid is added to adjust to acidity, and stirred 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 reaction to produce high-quality fluorescent carbon dots;

[0010] S4. Product collection: The solid and liquid products after hydrothermal carbonization are centrifuged and the supernatant is dialyzed with 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 material described in step S1 is dried at 105° C. for 5-10 minutes and crushed to 80 meshes.

[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 (5-100min), 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 for 48 to 55 hours using a dialysis membrane with a molecular weight cutoff of 1100 to 1300 Da, and the liquid product carbon liquid is used to prepare high-quality carbon dot materials.

[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 as above are used in breast cancer cell labeling.

[0021] In the technical solution of the present invention: the protease described in step S2 is an 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, which have 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 afterglow luminescence.

[0024] 2. The present invention fully transfers the nitrogen element in the 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 higher 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, and the technical equipment is simple, without the need for complex transformation. 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 is under the same conditions as 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 is under the same conditions as the hydrothermal carbonization reaction in Example 1), and d is enzymatic-hydrothermal treatment.

[0028] Figure 2 The effect of different reaction temperatures on the three-dimensional fluorescence spectra of soybean meal-based carbon dots when the residence time is 10 min in Example 1, where a is hydrothermal treatment (the hydrothermal treatment is the same as the hydrothermal carbonization reaction conditions in Example 1), and b is enzymatic-hydrothermal treatment. It can be seen that the liquid fluorescence intensity before and after enzymatic treatment is affected by the reaction temperature and the total nitrogen in the liquid phase.

[0029] Figure 3The TEM (a and b) and HRTEM (c and d) of soybean meal-based carbon dots at a reaction temperature of 220°C and a residence time of 10 min are shown in Example 2, where a and c are hydrothermal treatments (the hydrothermal treatments are the same as the hydrothermal carbonization reaction conditions in Example 2), and b and d are enzymatic-hydrothermal treatments. The enzymatic action enhances the concentration of small molecules in the liquid phase, causing the carbon dots to entangle with the small molecule chains, increasing the size of the carbon dots, and widening the interplanar spacing.

[0030] Figure 4 This is Example 1, with a reaction temperature of 220°C and a residence time of 10 min, and the XRD spectrum of soybean meal-based carbon dots. S-CDs are carbon dots obtained by hydrothermal treatment (the hydrothermal treatment is the same as the hydrothermal carbonization reaction conditions 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 characteristic peak intensity of amorphous carbon is improved.

[0031] Figure 5 This is Example 1, reaction temperature 220°C, residence time 10 min, and soybean meal-based carbon dots obtained by enzymatic-hydrothermal treatment for mouse breast cancer cell labeling, where a is before labeling and b is after labeling. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with 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 into 80 mesh, 2 g soybean meal powder, 8 mg acid protease and 70 mL deionized water were accurately weighed, mixed evenly, citric acid was added to adjust the pH to 4, and 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 min and the stirring speed was 550 rpm. After the reaction, the solid and liquid were centrifuged. The supernatant was dialyzed for 52 hours using a dialysis membrane with a molecular weight cutoff of 1200Da, and 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 into 80 mesh, 2 g soybean meal powder, 8 mg acid protease and 70 mL deionized water were accurately weighed, mixed evenly, citric acid was added to adjust the pH to 4, and 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. The stirring speed was 550 rpm. After the reaction, the solid and liquid were centrifuged. The supernatant was dialyzed for 52 hours 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] Dry and crush Spirulina platensis into 100 mesh size, accurately weigh 3 g soybean meal powder, 12 mg acid protease and 100 mL deionized water, mix well, add citric acid to adjust the pH to 4, place in a constant temperature water bath in a magnetic stirrer, the water bath temperature is 80°C, the stirring speed is 340 rpm, and the temperature is kept constant for 6 hours.

[0048] (2) Hydrothermal carbonization reaction

[0049] 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 time was 10 minutes, and the stirring speed was 550 rpm. After the reaction, the solid and liquid were centrifuged. The supernatant was dialyzed for 56 hours using a dialysis membrane with a molecular weight cutoff of 1400Da, and 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, citric acid was added to adjust the pH to 4, and placed in a constant temperature water bath in a magnetic stirrer with a water bath temperature of 50°C and a stirring speed of 200rpm for 4 hours.

[0053] (2) Hydrothermal carbonization reaction

[0054] 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 240°C, the residence time was 20 minutes, and the stirring speed was 400 rpm. After the reaction, the solid and liquid were centrifuged. The supernatant was dialyzed for 48 hours using a dialysis membrane with a molecular weight cutoff of 1000Da, and 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, 1g soybean meal powder, 10mg acid protease and 70mL deionized water were accurately weighed, mixed evenly, citric acid was added to adjust the pH to 4, and 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 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 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. The supernatant was dialyzed for 50 hours using a dialysis membrane with a molecular weight cutoff of 1200Da, and the obtained liquid product carbon liquid was used to prepare carbon quantum dot fluorescent material.

[0060] Performance testing:

[0061] Figure 1 The effects of different residence times on the total nitrogen in the hydrothermal liquid phase of soybean meal (a and b), and the effects of different reaction temperatures on the total nitrogen in the hydrothermal liquid phase of soybean meal (c and d), where a and c are hydrothermal treatments, and b and d are enzymatic-hydrothermal treatments. Enzymatic-hydrothermal treatment is to first subject the soybean meal to enzymatic pretreatment, and then subject the enzymatic product to hydrothermal carbonization; hydrothermal treatment is to directly subject the soybean meal to hydrothermal carbonization.

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

[0063] Figure 3 TEM (a and b) and HRTEM (c and d) of soybean meal-based carbon dots, where a and c are hydrothermal treatments, and b and d are enzymatic-hydrothermal treatments. Enzymatic action enhances the concentration of small molecules in the liquid phase, causing the carbon dots to entangle with the small molecule chains, increasing the size of the carbon dots 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 is improved.

[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 breaking the nitrogen-rich biomass to 60-100 mesh to obtain pre-treated raw materials; S2. Enzymatic pretreatment: the pretreated raw material, protease and deionized water are mixed evenly in a ratio of 1-10 g: 1-20 mg: 10-100 mL, and then acid is added to adjust to acidity, and stirred at a temperature of 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 products after hydrothermal carbonization are centrifuged and the supernatant is dialyzed with 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 nitrogen-rich hydrothermal phase according to claim 1, characterized in that: The nitrogen-rich biomass described in step S1 is Spirulina platensis, Chlorella pyrenoidosa, soybean meal, water hyacinth, soybean straw or kitchen waste.

3. The method for preparing high-quality biomass-based carbon dots in 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.

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

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

6. The method for preparing high-quality biomass-based carbon dots in 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.

7. The method for preparing high-quality biomass-based carbon dots in nitrogen-rich hydrothermal phase according to claim 1, characterized in that: The hydrothermal reaction in step S3 is carried out under stirring conditions, and the stirring speed is 200-600 rpm.

8. The method for preparing high-quality biomass-based carbon dots in 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.

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

10. Use of the nitrogen-rich hydrothermal liquid phase high-quality biomass-based carbon dots prepared in claim 1 in breast cancer cell labeling.

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