Preparation method and application of a carbonized silk fibroin-based nano-enzyme system

Abstract

The application relates to a preparation method of a carbonized fibroin-based double-enzyme system and application thereof, and belongs to the field of biological analysis detection (functional material technology). The application is used for solving the problems that a traditional glucose detection method is relatively complex in a detection process and is low in efficiency. The principle that the carbonized fibroin double-enzyme system is used for detecting glucose lies in that the catalytic activity of the glucose oxidase-like carbonized fibroin can oxidize glucose to generate gluconic acid and hydrogen peroxide at a natural pH, and the catalytic activity of the peroxidase-like carbonized fibroin can catalyze hydrogen peroxide to generate hydroxyl radicals at the natural pH, then the hydroxyl radicals oxidize a color developing agent TMB to generate blue oxTMB, a color developing reaction occurs, and an obvious absorption peak can be generated at a wavelength of 652 nm, so that the detection of glucose is realized. The carbon-based biological enzyme provided by the application is mainly used for detecting glucose.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical detection (functional materials technology), specifically relating to the preparation method and application of a carbonized silk fibroin glucose oxidation nanozyme and a hydrogen peroxide nanozyme system. Background Technology

[0002] Diabetes and its complications can seriously endanger a patient's health. Diabetic patients need to monitor their blood glucose levels in real time to intervene promptly and maintain stable blood sugar levels. Accurate and real-time blood glucose monitoring can help diabetic patients better adjust their lifestyle and dietary habits to maintain good health. Common blood glucose monitoring technologies are based on glucose oxidase sensors, which generally utilize the specific recognition of glucose by glucose oxidase and the transmission of a signal to achieve sensitive detection. For continuous monitoring technology, glucose sensors need to address the issues of demanding reaction conditions and short lifespan.

[0003] Due to the limitations of glucose oxidase, such as complex preparation process, poor stability, demanding storage conditions, and high cost, various enzyme mimics have emerged and developed. Among them, noble metal enzymes, as materials with biological enzyme catalytic activity, represent a new generation of enzyme mimics. They can utilize the glucose oxidase-like properties of noble metals to recognize glucose and transmit signals, achieving sensitive detection. Although noble metal enzymes can overcome the high environmental dependence of natural enzymes, their widespread application in glucose detection is hindered by the high cost of noble metals, complex manufacturing processes, and environmental pollution associated with them.

[0004] A good solution is to use carbon-based enzymes to replace natural glucose oxidase and precious metal enzymes. Carbon-based enzymes are micron- or nanometer-sized materials with similar catalytic efficiency and enzymatic reaction kinetics to natural enzymes. Compared to natural enzymes, they are more stable and maintain high catalytic activity under extreme conditions; compared to precious metal enzymes, they are simpler to manufacture, less expensive, and less environmentally burdensome. Therefore, using carbon-based enzymes with glucose oxidase activity to replace natural and precious metal enzymes in blood glucose detection can significantly reduce costs and improve economic efficiency.

[0005] Peroxidase is an enzyme that catalyzes the oxidation of substrates using hydrogen peroxide as an electron acceptor, and it currently has wide applications in the field of bioanalytical detection. As early as the beginning of the 21st century, studies discovered that Fe3O4 nanoparticles possess catalytic activity similar to natural peroxidases; in the presence of hydrogen peroxide, this material can induce a colorimetric reaction in chromogenic reagents. Besides Fe3O4 nanoparticles, many metal oxide nanomaterials (such as Co3O4 and ZnFe2O4) have been shown to possess peroxidase-like catalytic activity and have been applied to the detection of hydrogen peroxide and glucose. However, the complex synthesis of metal nanomaterials and the need to consider their biocompatibility limit their further applications.

[0006] A good solution is to use carbon-based enzymes to replace metal nano-enzymes. Compared to metal nano-enzymes, carbon-based enzymes not only possess the high catalytic activity of peroxidases but also exhibit good biocompatibility and a simpler fabrication process. Therefore, using carbon-based enzymes with peroxidase activity to replace metal nano-enzymes in hydrogen peroxide detection can broaden its application scenarios.

[0007] In summary, the preparation method and application of a carbon-based enzyme that simultaneously possesses the characteristics of glucose oxidase and peroxidase, capable of efficiently catalyzing both glucose oxidation and hydrogen peroxide oxidation, thereby enabling electrochemical and colorimetric detection of glucose, is highly in line with the current needs of the medical and health fields. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a method for preparing a dual-enzyme system based on carbonized silk fibroin and its application. This carbonized silk fibroin enzyme possesses characteristics mimicking glucose oxidase and catalase, and can efficiently catalyze the oxidation of glucose into gluconic acid and hydrogen peroxide, followed by further catalytic oxidation of the generated hydrogen peroxide, enabling electrochemical and colorimetric detection of glucose and improving the efficiency of one-step glucose detection.

[0009] A method for preparing a carbonized silk fibroin dual-enzyme system, the method comprising the following steps: Step 1: Extract silk fibroin to obtain a regenerated silk fibroin solution; Step 2: Freeze-dry the regenerated silk fibroin solution obtained in Step 1 to obtain freeze-dried silk fibroin; Step 3: Carbonize the freeze-dried silk fibroin obtained in Step 2 at high temperature to form carbonized silk fibroin; Step 4: Grind the carbonized silk fibroin formed in Step 3 into a powder sample to obtain the carbonized silk fibroin dual-enzyme system; Furthermore, the specific steps for extracting silk fibroin in step one to obtain the regenerated silk fibroin solution are achieved through the following steps: Step 11: First, cut the silkworm cocoons into pieces or spirals, or peel them into several layers into thin sheets; Step 1 and Step 2: Boil the fragmented silkworm cocoons obtained in Step 1 1 in a 0.5% sodium carbonate aqueous solution 2-3 times to remove sericin. After washing with deionized water several times, dry them in a 45℃ constant temperature forced-air drying oven to obtain silk fibroin fibers with sericin removed. Step 13: Place the silk fibroin fibers obtained in Step 12 in a water bath at 70-75℃. Place 20g of degummed silk fibroin in 200mL of a ternary mixed solution of calcium chloride / anhydrous ethanol / water (bath ratio 1:10) (molar ratio 1:2:8) and dissolve it under magnetic stirring for 1-2 hours until it is completely dissolved to obtain a silk fibroin protein mixed solution. Step 14: After cooling the silk fibroin mixture obtained in Step 13, centrifuge to remove various impurities. Dialyze this solution to ultrapure water (molecular weight 3.5-5 kDa) using a dialysis bag for 3 days, changing the water at intervals of 4 h, 8 h, 24 h, 48 h, and 96 h (a total of 3 days) to remove salt ions and impurities. Then collect and filter to obtain the silk fibroin solution and store it at 4℃. Furthermore, the silk fibroin solution obtained in step two is freeze-dried to obtain freeze-dried silk fibroin. The specific steps for this process are as follows: Step 21: Pre-freeze the silk fibroin solution obtained in Step 1 in an ultra-low temperature freezer (-80℃) for 24 hours; Step 22: Place the pre-frozen silk fibroin solution obtained in Step 21 into a vacuum freeze dryer and freeze dry for 48 hours to obtain fully freeze-dried silk fibroin. Furthermore, the freeze-dried silk fibroin obtained in step three is subjected to high-temperature carbonization to form carbonized silk fibroin. This is achieved through the following steps: Step 31: Place the freeze-dried silk fibroin obtained in Step 2 into a tube furnace and heat it at 900°C for 4 hours in nitrogen. After naturally cooling to room temperature, black carbonized silk fibroin is obtained. Furthermore, the specific steps for grinding the carbonized silk fibroin formed in step four into a powder sample to obtain the carbonized silk fibroin dual-enzyme system are achieved through the following steps: Step 41: Place the carbonized silk fiber obtained in Step 3 into agate slurry, grind it thoroughly into powder, and store it at 4°C for later use; An application of a carbonized silk fibroin-based dual-enzyme system prepared in a certain preparation method, specifically for the detection of glucose, is achieved through the following steps: Step A: Test the glucose oxidase-like activity of carbonized silk fibroin; Step B: Test the catalase-like activity of carbonized silk fibroin; Step C: Determine the uniqueness of the carbonized silk fibroin dual-enzyme system for glucose detection; Furthermore, the specific steps for testing the glucose oxidase-like activity of carbonized silk fibroin in step A are implemented through the following steps: Step A1: Weigh 12.5 mg of the powdered carbonized silk fibroin obtained in Step 4 and dissolve it in 5 mL of acetate buffer solution to obtain an acetate buffer solution containing carbonized silk fibroin (2.5 mg / mL). Step A2: Weigh 24 mg of 3,3',5,5'-tetramethylbenzidine (TMB) and dissolve it in 100 mL of acetate buffer solution to obtain an acetate buffer solution containing TMB (1 mM). Step A3: Add 1 mL of the acetate buffer solution containing TMB (1 mM) obtained in Step A2 to the 5 mL acetate buffer solution containing carbonized silk fibroin (2.5 mg / mL) obtained in Step A1, and vortex the mixture for 1 min. Step A4: Weigh out glucose (C6H) 12 Dissolve 0.99 g of O6·H2O in 100 mL of water to obtain a 50 mM glucose standard solution; Step A5: Add 2.4 μL, 14.4 μL, 29 μL, 46 μL, 60 μL, 74 μL, 89 μL, and 103 μL of the glucose standard solution (50 mM) obtained in Step A4 to 6 mL of the mixed solution obtained in Step A3, so that the glucose concentrations in the mixed solution are 0.04 mM, 0.24 mM, 0.48 mM, 0.76 mM, 1.00 mM, 1.24 mM, 1.48 mM, and 1.72 mM, respectively. Vortex the mixed solution containing glucose for 1 min, let it stand for 30 min, centrifuge at 5000 rpm for 5 min, and collect the supernatant. Step A6: Pour the supernatant obtained in step A5 into a cuvette and measure the absorbance at 652 nm in a UV spectrophotometer. Furthermore, the specific steps for testing the catalase-like activity of carbonized silk fibroin in step B are implemented through the following steps: Step B1: Weigh 12.5 mg of the powdered carbonized silk fibroin obtained in Step 4 and dissolve it in 5 mL of acetate buffer solution to obtain an acetate buffer solution containing carbonized silk fibroin (2.5 mg / mL). Step B2: Weigh 24 mg of 3,3',5,5'-tetramethylbenzidine (TMB) and dissolve it in 100 mL of acetate buffer solution to obtain an acetate buffer solution containing TMB (1 mM). Step B3: Add 1 mL of the acetate buffer solution containing TMB (1 mM) obtained in Step B2 to the 5 mL acetate buffer solution containing carbonized silk fibroin (2.5 mg / mL) obtained in Step B1, and vortex the mixture for 1 min. Step B4: Take 206 μL of hydrogen peroxide (30%), dilute to 100 mL, and obtain a 20 mM hydrogen peroxide standard solution; Step B5: Add the hydrogen peroxide standard solution (20 mM) obtained in step B4 to 6 mL of the mixed solution obtained in step B3 at concentrations of 0 μL, 18 μL, 42 μL, 66 μL, 90 μL, and 114 μL, respectively, so that the hydrogen peroxide concentrations in the mixed solution are 0 mM, 0.3 mM, 0.7 mM, 1.1 mM, 1.5 mM, and 1.8 mM, respectively. Vortex the mixed solution containing hydrogen peroxide for 1 min, let it stand for 30 min, centrifuge at 5000 rpm for 5 min, and take the supernatant. Step B6: Pour the supernatant obtained in step B5 into a cuvette and measure the absorbance at 652 nm in a UV spectrophotometer. Furthermore, the process of determining the uniqueness of the carbonized silk fibroin dual-enzyme system for glucose detection in step C is as follows: Step C1: Weigh 12.5 mg of the powdered carbonized silk fibroin obtained in step four and dissolve it in 5 mL of acetate buffer solution to obtain an acetate buffer solution containing carbonized silk fibroin (2.5 mg / mL). Step C2: Weigh 24 mg of 3,3',5,5'-tetramethylbenzidine (TMB) and dissolve it in 100 mL of acetate buffer solution to obtain an acetate buffer solution containing TMB (1 mM). Step C3: Add 1 mL of the acetate buffer solution containing TMB (1 mM) obtained in step B2 to the 5 mL acetate buffer solution containing carbonized silk fibroin (2.5 mg / mL) obtained in step C1, and vortex the mixture for 1 min. Step C4: Add 60 μL of 1 mM glucose solution, 10 mM uric acid solution, 10 mM ascorbic acid solution and 10 mM maltose solution to the mixed solution obtained in step C3 to form 4 equal volumes of control test solution. Step C5: Observe the color changes of the four equal volumes of control solutions in C4. If only the control solution with added 1 mM glucose solution shows a dramatic change in absorbance, while the absorbance of the other three equal volumes of control solutions changes very little, then the carbonized silk fibroin dual-enzyme system is determined to be unique in detecting glucose. Attached Figure Description

[0010] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 This is a flowchart illustrating the preparation process of the present invention; Figure 2 These are TEM images of silk fibroin before and after carbonization, where a is before carbonization and b is after carbonization. Figure 3 This is an elemental analysis diagram of the carbonized silk fibroin of the present invention; Figure 4 The results are from the colorimetric reaction of carbonized silk fibroin glucose oxidase. Figure 5 The absorbance of the solution at 652 nm is the result of the absorbance change after the addition of glucose solution; Figure 6 The results are from the colorimetric reaction of carbonized silk fibroin catalase. Figure 7 The absorbance of the solution at 652 nm is the result of the absorbance change after adding H2O2 solution; Figure 8 The results show the absorbance changes of the carbonized silk fibroin dual-enzyme system for glucose, uric acid, ascorbic acid, and maltose. Detailed Implementation

[0011] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0012] Figure 1 An exemplary preparation process of the carbonized silk fibroin dual-enzyme system of the present invention is shown. Specifically, it is achieved through the following steps: First, the silkworm cocoons are cut into pieces or spirals, or peeled into several layers into thin sheets. The obtained fragmented cocoons are boiled 2-3 times in a 0.5% sodium carbonate aqueous solution (liquor ratio 1:50) to remove sericin. After washing with deionized water several times, they are dried in a 45℃ constant temperature drying oven. This yields silk fibroin fibers with sericin removed. The obtained silk fibroin fibers were placed in a 70-75℃ water bath. 20 grams of degummed silk fibroin were placed in 200 mL of a ternary mixed solution of calcium chloride / anhydrous ethanol / water (liquid ratio 1:10) (molar ratio 1:2:8). The solution was dissolved under magnetic stirring for 1-2 hours until it was completely dissolved, resulting in a silk fibroin protein mixed solution. After natural cooling, the solution was centrifuged to remove various impurities. This solution was then dialyzed against ultrapure water (molecular weight 3.5-5 kDa) for 3 days using a dialysis bag. The water was changed at intervals of 4 h, 8 h, 24 h, 48 h, and 96 h (a total of 3 days) to remove salt ions and impurities. The solution was then collected, filtered, and stored at 4℃.

[0013] After pre-freezing the silk fibroin solution in an ultra-low temperature freezer (-80℃) for 24 hours, it was placed in a vacuum freeze dryer and freeze-dried for 48 hours to obtain fully freeze-dried silk fibroin.

[0014] The obtained freeze-dried silk fibroin was placed in a tube furnace and heated at 900°C for 4 hours in a nitrogen atmosphere. After naturally cooling to room temperature, black carbonized silk fibroin was obtained. It was then placed in agate slurry, ground thoroughly into powder, and stored at 4°C for subsequent experiments. Based on the above methods and steps, this application also involves characterizing the morphology of the prepared carbonized silk fibroin dual-enzyme system. Figure 2 The images show TEM images of the silk fibroin before and after carbonization according to the present invention. After vacuum freeze-drying, the silk fibroin solution exhibits a non-uniform three-dimensional porous structure. Figure 2 a) Uniform carbonized silk fibroin microparticles were obtained by high-temperature carbonization in a tube furnace. Figure 2 b). Compared to uncarbonized silk fibroin, carbonized silk fibroin has a larger specific surface area, potentially providing more reaction binding sites.

[0015] Based on the above methods and steps, this application also involves elemental scanning analysis of the prepared carbonized silk fibroin dual-enzyme system. Figure 3 The image shows the EDS diagram of carbonized silk fibroin according to the present invention. Figure 3 It can be observed that the elements are evenly distributed and have not evaporated due to carbonization, thus there may be more reaction binding sites.

[0016] This application also relates to the use of colorimetric reactions to test the glucose oxidase activity of the prepared carbonized silk fibroin dual-enzyme system. The specific characterization steps are as follows: To further explore the glucose oxidase activity of the carbonized silk fibroin dual-enzyme system, the glucose oxidase activity of the material was calibrated using colorimetric reactions. The test results are as follows: Figure 4 As shown in the figure, the color gradually deepens with increasing glucose concentration, which proves that carbonized silk fibroin does indeed have glucose oxidase-like catalytic activity.

[0017] After the above experiments, it was determined that the carbonized silk fibroin dual-enzyme system itself possesses glucose oxidase-like activity and can directly replace natural glucose oxidase for application in glucose detection. The absorption peak at 652 nm was measured using a visible-ultraviolet spectrophotometer. Figure 5 It can be seen that the absorption peak in the ultraviolet spectrum is significantly enhanced as the reaction proceeds and the concentration increases. This further proves that carbonized silk fibroin has glucose oxidase-like activity.

[0018] In addition to the determination of glucose oxidase-like activity, this application also involves the colorimetric reaction for testing the catalase-like activity of the prepared carbonized silk fibroin dual-enzyme system. The specific characterization steps are as follows: To further explore the catalase-like activity of the carbonized silk fibroin dual-enzyme system, the catalase-like activity of the material was calibrated using a colorimetric reaction. The test results are as follows: Figure 6 As shown in the figure, the color gradually deepens with increasing hydrogen peroxide concentration, which proves that carbonized silk fibroin does indeed have catalase-like catalytic activity.

[0019] After the above experiments, it was determined that the carbonized silk fibroin dual-enzyme system itself possesses catalase-like activity and can directly replace natural catalase for application in hydrogen peroxide detection. The absorption peak at 652 nm was measured using a visible-ultraviolet spectrophotometer. Figure 7 It can be seen that the absorption peak in the ultraviolet spectrum is significantly enhanced as the reaction proceeds and the concentration increases. This further proves that carbonized silk fibroin has catalase-like activity.

[0020] To improve the accuracy of glucose detection, this application also involves determining the uniqueness of the carbonized silk fibroin dual-enzyme system for glucose detection. The specific characterization steps are as follows: Equal volumes of the carbonized silk fibroin dual-enzyme system mixed buffer solution are mixed with equal volumes of glucose solution (1 mM), uric acid solution (10 mM), ascorbic acid solution (10 mM), and maltose solution (10 mM) to form four equal-volume control solutions. The absorbance changes of the four equal-volume control solutions are tested, such as... Figure 8 As shown, only the control solution with added 1mM glucose solution showed a dramatic change in absorbance, while the absorbance of the other three equal volumes of control solutions showed very little change. Therefore, it can be determined that the carbonized silk fibroin dual-enzyme system is unique in detecting glucose.

[0021] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An application of a two-enzyme system based on carbonized silk fibroin, characterized in that: The detection of glucose using a two-enzyme system of carbonized silk fibroin is achieved through the following steps: Step A: Test the glucose oxidase-like activity of carbonized silk fibroin; Step B: Test the catalase-like activity of carbonized silk fibroin; Step C: Determine the uniqueness of the carbonized silk fibroin dual-enzyme system for glucose detection; The preparation method of the carbonized silk fibroin dual-enzyme system is achieved through the following steps: Step 1: Extract silk fibroin to obtain a regenerated silk fibroin solution; Step 2: Freeze-dry the regenerated silk fibroin solution obtained in Step 1 to obtain freeze-dried silk fibroin; Step 3: Carbonize the freeze-dried silk fibroin obtained in Step 2 at high temperature to form carbonized silk fibroin; Step 4: Grind the carbonized silk fibroin formed in Step 3 into a powder sample to obtain the carbonized silk fibroin dual-enzyme system; The freeze-dried silk fibroin obtained in step three is subjected to high-temperature carbonization to form carbonized silk fibroin. This is achieved through the following steps: Step 31: Place the freeze-dried silk fibroin obtained in Step 2 into a tube furnace and heat it at 900°C for 4 hours in nitrogen. After naturally cooling to room temperature, black carbonized silk fibroin is obtained. The carbonized silk fibroin formed in step four is ground into a powder sample to obtain the carbonized silk fibroin dual-enzyme system. This is achieved through the following steps: Step 41: Place the carbonized silk fiber obtained in Step 3 into agate slurry, grind it thoroughly into powder, and store it at 4°C for later use.

2. The application according to claim 1, characterized in that, The extraction of silk fibroin in step one to obtain the regenerated silk fibroin solution is achieved through the following steps: Step 11: First, cut the silkworm cocoons into pieces or spirals, or peel them into several layers into thin sheets; Step 1 and Step 2: Boil the fragmented silkworm cocoons obtained in Step 1 1 in a 0.5% sodium carbonate aqueous solution 2-3 times to remove sericin. After washing with deionized water several times, dry them in a 45℃ constant temperature forced-air drying oven to obtain silk fibroin fibers with sericin removed. Step 13: Place the silk fibroin fibers obtained in Step 12 in a water bath at 70-75℃. Place 20g of degummed silk fibroin in 200mL of a ternary mixed solution of calcium chloride / anhydrous ethanol / water and dissolve it under magnetic stirring for 1-2 hours until it is completely dissolved to obtain a silk fibroin protein mixed solution. Step 14: After cooling the silk fibroin mixture obtained in Step 13, centrifuge it to remove various impurities. Dialyze this solution using a dialysis bag, changing the water at intervals of 4 h, 8 h, 24 h, 48 h, and 96 h to remove salt ions and impurities. Then collect and filter the solution to obtain the silk fibroin solution and store it at 4°C.

3. The application according to claim 1, characterized in that: The silk fibroin solution obtained in step two is freeze-dried to obtain freeze-dried silk fibroin. The specific steps are as follows: Step 21: Pre-freeze the silk fibroin solution obtained in Step 1 at -80℃ for 24 hours in an ultra-low temperature freezer; Step 22: Place the pre-frozen silk fibroin solution obtained in Step 21 into a vacuum freeze dryer and freeze dry for 48 hours to obtain fully freeze-dried silk fibroin.

4. The application according to claim 1, characterized in that: The specific process for testing the glucose oxidase-like activity of carbonized silk fibroin in step A is as follows: Step A1: Weigh 12.5 mg of the powdered carbonized silk fibroin obtained in Step 4 and dissolve it in 5 mL of acetate buffer solution to obtain an acetate buffer solution containing carbonized silk fibroin. Step A2: Weigh 24 mg of 3,3',5,5'-tetramethylbenzidine and dissolve it in 100 mL of acetate buffer solution to obtain an acetate buffer solution containing TMB. Step A3: Add 1 mL of the acetate buffer solution containing TMB obtained in Step A2 to the 5 mL acetate buffer solution containing carbonized silk fibroin obtained in Step A1, and vortex the mixture for 1 min. Step A4: Weigh 0.99 g of glucose and dissolve it in 100 mL of water to obtain a 50 mM glucose standard solution; Step A5: Add 50 mM glucose standard solution obtained in step A4 to 6 mL of the mixed solution obtained in step A3 at concentrations of 2.4 μL, 14.4 μL, 29 μL, 46 μL, 60 μL, 74 μL, 89 μL, and 103 μL, respectively, so that the glucose concentrations in the mixed solution are 0.04 mM, 0.24 mM, 0.48 mM, 0.76 mM, 1.00 mM, 1.24 mM, 1.48 mM, and 1.72 mM, respectively. Vortex the mixed solution containing glucose for 1 min, let it stand for 30 min, centrifuge at 5000 rpm for 5 min, and collect the supernatant. Step A6: Pour the supernatant obtained in step A5 into a cuvette and measure the absorbance at 652 nm in a UV spectrophotometer.

5. The application according to claim 1, characterized in that: The specific process for testing the catalase-like activity of carbonized silk fibroin in step B is as follows: Step B1: Weigh 12.5 mg of the powdered carbonized silk fibroin obtained in Step 4 and dissolve it in 5 mL of acetate buffer solution to obtain an acetate buffer solution containing carbonized silk fibroin. Step B2: Weigh 24 mg of 3,3',5,5'-tetramethylbenzidine and dissolve it in 100 mL of acetate buffer solution to obtain an acetate buffer solution containing TMB. Step B3: Add 1 mL of the acetate buffer solution containing TMB obtained in Step B2 to the 5 mL acetate buffer solution containing carbonized silk fibroin obtained in Step B1, and vortex the mixture for 1 min. Step B4: Take 206 μL of 30% hydrogen peroxide and dilute it to 100 mL to obtain a 20 mM hydrogen peroxide standard solution; Step B5: Add 20 mM of the hydrogen peroxide standard solution obtained in step B4 to 6 mL of the mixed solution obtained in step B3 at concentrations of 0 μL, 18 μL, 42 μL, 66 μL, 90 μL, and 114 μL, respectively, so that the hydrogen peroxide concentrations in the mixed solution are 0 mM, 0.3 mM, 0.7 mM, 1.1 mM, 1.5 mM, and 1.8 mM, respectively. Vortex the mixed solution containing hydrogen peroxide for 1 min, let it stand for 30 min, centrifuge at 5000 rpm for 5 min, and take the supernatant. Step B6: Pour the supernatant obtained in step B5 into a cuvette and measure the absorbance at 652 nm in a UV spectrophotometer.

6. The application according to claim 1, characterized in that: The process of determining the uniqueness of the carbonized silk fibroin dual-enzyme system for glucose detection in step C is as follows: Step C1: Weigh 12.5 mg of the powdered carbonized silk fibroin obtained in step four and dissolve it in 5 mL of acetate buffer solution to obtain an acetate buffer solution containing carbonized silk fibroin. Step C2: Weigh 24 mg of 3,3',5,5'-tetramethylbenzidine and dissolve it in 100 mL of acetate buffer solution to obtain an acetate buffer solution containing TMB. Step C3: Add 1 mL of the acetate buffer solution containing TMB obtained in step B2 to the 5 mL acetate buffer solution containing carbonized silk fibroin obtained in step C1, and vortex the mixture for 1 min. Step C4: Add 60 μL of 1 mM glucose solution, 10 mM uric acid solution, 10 mM ascorbic acid solution and 10 mM maltose solution to the mixed solution obtained in step C3 to form 4 equal volumes of control test solution. Step C5: Observe the color changes of the four equal volumes of control solutions in C4. If only the control solution with added 1 mM glucose solution shows a dramatic change in absorbance, while the absorbance of the other three equal volumes of control solutions changes very little, then the carbonized silk fibroin dual-enzyme system is determined to be unique in detecting glucose.

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