Preparation method of injectable functional fibroin-based hydrogel

Abstract

The invention belongs to the technical field of silk fibroin-based hydrogel, and particularly relates to a preparation method of injectable functional silk fibroin-based hydrogel. The preparation method of the hydrogel provided by the invention comprises the following steps: performing degumming treatment on raw silkworm cocoons in alkaline boiling water, washing and drying; performing infrared light irradiation on the dried raw silk; dissolving raw silk irradiated by infrared light in a lithium bromide solution to obtain a silk fibroin solution; carrying out dialysis treatment, filtration and centrifugal separation on the silk fibroin solution to obtain a clarified silk fibroin solution; mixing the clarified silk fibroin solution, sodium iodide and chloramine T, stirring for reaction, adding sodium metabisulfite, continuing stirring for reaction, and dialyzing to obtain iodine substituted silk fibroin liquid; and mixing the iodine-substituted silk fibroin liquid, an H2O2 solution and an HRP solution, and uniformly oscillating to obtain the hydrogel. The hydrogel prepared by the method is short in gelling time and high in mechanical strength.

Description

Technical Field

[0001] This invention belongs to the field of silk fibroin-based hydrogel technology, specifically relating to a method for preparing an injectable functional silk fibroin-based hydrogel, and further relating to a novel injectable natural protein-based hydrogel carrier for the treatment of hyperthyroidism. Background Technology

[0002] Injectable biomaterials play a vital role in clinical applications, being widely used in soft tissue enhancement, orthopedic repair, cardiac repair, nerve tissue injection, ophthalmic treatment, intravascular injection, intramuscular injection, women's health treatments, drug delivery systems, tissue engineering, cosmetic surgery, and dental treatments. These materials address a wide range of medical needs, from facial fillers to heart disease treatment and nerve repair, by providing minimally invasive treatment methods. With continuous technological advancements, the application scope of injectable biomaterials is expected to expand further, offering patients more treatment options and better outcomes.

[0003] Over the past decade, hydrogels have attracted worldwide attention due to their superior physical properties, chemical characteristics, biocompatibility, and broad application potential in injectable biomaterials. Hydrogels belong to the category of soft condensed matter, with water molecules and a three-dimensional cross-linked polymer network as their core components. However, natural-based hydrogels typically use bio-derived components as building blocks, such as gelatin, chitosan (CS), alginate, hyaluronic acid, and collagen. While these materials possess excellent intrinsic biocompatibility and biodegradability due to their natural origin, they suffer from drawbacks such as insufficient mechanical strength and limited elastic recovery.

[0004] Silk Fibroin (SF), a natural protein-based polymer, exhibits significant application potential in the biomedical field due to its excellent biocompatibility, unique mechanical properties, and programmable genetic sequence. Hyperthyroidism is a common endocrine disorder caused by overactive thyroid glands and excessive secretion of thyroid hormones. Its typical pathological features include diffuse or nodular hyperplasia of the thyroid gland, significantly elevated resting energy expenditure, and imbalance of the sympathetic and parasympathetic nervous systems. Current clinical intervention pathways for thyrotoxicosis mainly include three categories: medical treatment with antithyroid drugs (ATDs), subtotal thyroidectomy, and radioactive iodine ablation therapy. In modern treatment protocols for hyperthyroidism, oral ¹³¹I radioactive iodine therapy has been established as a classic treatment method. This therapy achieves precise destruction of thyroid tissue through targeted radiation with beta rays, thereby effectively inhibiting glandular secretion and achieving clinical therapeutic goals. However, this technology currently faces several application bottlenecks: the treatment course is relatively long, the treatment dose needs to be precisely controlled, and there is a risk of secondary pathological reactions caused by radiation doses exceeding the threshold. These factors collectively restrict the optimization process of this treatment regimen and urgently require breakthroughs through interdisciplinary innovation in materials science and radiation medicine.

[0005] There is no existing technology for directly iodizing silk fibroin and then preparing hydrogels. To solve this problem, this invention proposes this solution. Summary of the Invention

[0006] Therefore, the technical problem to be solved by this invention is to overcome the above-mentioned problems and thus provide a method for preparing an injectable functional silk fibroin-based hydrogel. This invention designs and develops a novel injectable natural protein-based hydrogel carrier for the treatment of hyperthyroidism. Based on the chemical modification of tyrosine residues in the silk fibroin (SF) molecular chain, functional modification is achieved through iodine atom-targeted grafting technology, and then a three-dimensional network structure is constructed through enzymatic oxidative cross-linking reactions, ultimately forming an injectable natural protein-based hydrogel carrier. This preparation strategy fully utilizes the structural tunability of natural biomolecules and provides a novel biomaterial solution for the clinical treatment of hyperthyroidism through molecular-level functional design.

[0007] A method for preparing an injectable functional silk fibroin-based hydrogel includes the following steps: 1) Degumming raw silkworm cocoons in alkaline boiling water. After degumming, remove the raw silkworm cocoons, wash and dry them to obtain dried raw silk. 2) Spread the dried raw silk into a single layer and irradiate it with infrared light in a vacuum environment; The vacuum level of the vacuum environment is 2-4 kPa, the infrared wavelength is 800-900 nm, the infrared irradiation time is 80-150 s, and the surface temperature of the raw silk layer is 45-55℃. 3) Dissolve raw silk irradiated with infrared light in lithium bromide solution to obtain silk fibroin solution; 4) Dialyze the silk fibroin solution, filter it after dialysis, and centrifuge to obtain a clear silk fibroin solution; 5) Mix the clarified silk fibroin solution, NaI and chloramine T, stir and react, then add sodium metabisulfite and continue stirring and reacting. After the reaction is complete, dialyze the mixture to obtain iodine-substituted silk fibroin liquid. 6) Mix iodine-substituted silk fibroin liquid, H2O2 solution and HRP solution, and vortex mix until homogeneous to obtain hydrogel.

[0008] Preferably, the alkaline boiling water is obtained by boiling a mixture of water and sodium carbonate, wherein the ratio of water to sodium carbonate is 1:(2.0-2.2), and the unit of ratio is L:g; The ratio of raw silkworm cocoons to alkaline boiling water is (7-10):(2.8-3.2), with the unit being g:L.

[0009] Preferably, the degumming step involves adding the raw silkworm cocoons to boiling water and boiling for 20-40 minutes to degumme them. After degumming, the raw silkworm cocoons are removed, washed with water 3-6 times, wrung out, loosened, and then placed in a vacuum oven at 45-55℃ to dry for 6-12 hours.

[0010] Preferably, the concentration of the lithium bromide solution in step 3) is 9.1-9.4 mol / L, the ratio of raw silk to lithium bromide solution is (4-8):(20-25), the ratio unit is g:ml, the dissolution temperature is 55-60℃, and the dissolution time is 4-6h.

[0011] Preferably, in step 4), the silk fibroin solution is placed into a dialysis bag for dialysis treatment, with a molecular weight cutoff of 3500 Da, and the dialysis time is 24-72 hours. The water is changed every 8-10 hours during the dialysis process.

[0012] Preferably, the centrifugation speed is 10000-12000 rpm, the centrifugation temperature is 4-5℃, the centrifugation time is 20-40 min, and the number of centrifugations is 2-5.

[0013] Preferably, in step 5), firstly, prepare a 50.0 mmol / L phosphate buffer solution with a pH of 7.4 and a 0.1 mol / L phosphate buffer solution with a pH of 7.4; then, using the aforementioned 50.0 mmol / L phosphate buffer solution, prepare a 1.0-1.2 mg / mL chloramine T solution, a 1.8-2.2 mg / mL sodium metabisulfite solution, a 1.5-2.5 mg / mL KI solution, and a 0.0002-0.0004 mg / mL NaI solution; and use the aforementioned 50.0 mmol / L phosphate buffer solution and clarified silk fibroin solution to prepare a 1.8-2.2 wt% silk fibroin solution; then, add 5-8 mL of the 1.8-2.2 wt% silk fibroin solution and 2-4 mL of NaI solution to the aforementioned 50-60 mL of 0.1 mol / L phosphate buffer solution with a pH of 7.4, and 50-60 mL of... Add chloramine T solution and stir at room temperature for 20-40 min. Add 30-35 mL of sodium metabisulfite solution to the system and stir at room temperature for 10-20 min. Add 10-15 mL of KI solution to dilute the residual iodide. Finally, transfer the resulting mixture into a dialysis bag and dialyze it in water.

[0014] In this invention, NaI or radioactive Na can be selected as needed. 131 I. In this invention, radioactive Na is preferred. 131 I.

[0015] Preferably, in step 5), the molecular weight cutoff of the dialysis bag is 3500 Daltons, the dialysis time is 24-48 hours, and the water is changed every 20-24 hours during the dialysis process.

[0016] Preferably, the concentration of the H2O2 solution is 0.3-0.5 mol / L, the concentration of the HRP solution is 0.7-0.9 mg / mL, and the concentration of the iodine-substituted silk fibroin liquid is 1.5-2.5 wt%.

[0017] Preferably, the volume ratio of iodine-substituted silk fibroin liquid, H2O2 solution and HRP solution is (500-550):(40-60):(40-60).

[0018] The technical solution of this invention has the following advantages: The method for preparing functional silk fibroin-based hydrogels provided by this invention involves first degumming raw silkworm cocoons, then drying them in a vacuum oven, followed by irradiation with infrared light under vacuum, then dissolving the infrared-irradiated raw silkworm silk in a lithium bromide solution to obtain a silk fibroin solution, which is then dialyzed and separated to obtain a clear silk fibroin solution. Next, iodine is directly modified onto tyrosine residues in the silk fibroin using chloramine-T to form iodoform silk fibroin, and finally, an enzymatic reaction of HRP and H2O2 is used to form a hydrogel.

[0019] The present invention prepares iodine-modified silk fibroin hydrogel, which can be used for targeted treatment of hyperthyroidism, and is expected to better meet the treatment needs of hyperthyroidism patients and provide new options for clinical treatment.

[0020] In addition, during the preparation of iodine-modified silk fibroin hydrogels, the inventors discovered that because iodine replaced some of the reaction sites of the phenolic hydroxyl groups in the tyrosine of silk fibroin, the gelation time was longer, and the mechanical strength of the obtained silk fibroin hydrogel was also lower. In order to overcome this problem, the inventors cleverly controlled the amount of iodine substitution and used a mixture of iodine-modified and unmodified silk fibroin, combined with vacuum infrared processing technology, which not only effectively shortened the gelation time, but also improved its mechanical strength to a certain extent. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The infrared spectrum of the iodine-substituted silk fibroin prepared in step 5 of Example 1 is shown.

[0023] Figure 2 The image shows an electron microscope image of the white powder obtained by freeze-drying the iodine-substituted silk fibroin prepared in step 5 of Example 1.

[0024] Figure 3 This is a scan of the oscillation frequency of the hydrogel prepared in Example 1.

[0025] Figure 4 This is a scan of the oscillation frequency of the hydrogel prepared in Example 2.

[0026] Figure 5 The image shows the oscillation frequency scan of the hydrogel prepared in Comparative Example 2. Detailed Implementation

[0027] The following embodiments are provided to further understand the present invention and are not limited to the preferred embodiments described herein, nor do they constitute a limitation on the scope of protection of the present invention.

[0028] Experimental steps or conditions not specified in the following embodiments of this invention can be implemented according to conventional experimental steps and conditions used in existing literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagents or instruments. Example

[0029] This embodiment provides a method for preparing an injectable functional silk fibroin-based hydrogel, comprising the following steps: 1) Pour 3L of deionized water into a stainless steel bucket, heat the water to a boil, add 6.36g of sodium carbonate to the boiling water, and after it is completely dissolved, add 7.8g of raw silkworm cocoons to the boiling water and boil for 20 minutes to degumme. After degumming, take out the raw silkworm cocoons, wash them with water 4 times, wring them dry, loosen them, and put them in a 50℃ vacuum oven to dry for 8 hours. 2) Spread the dried raw silk into a single layer and irradiate it with infrared light in a vacuum environment of 3 kPa. The infrared light wavelength is 850 nm, the irradiation time is 120 s, and the surface temperature of the raw silk layer is 50 ℃. 3) Weigh 5g of raw silk that has been irradiated with infrared light, tear it until fluffy, and dissolve it in 22ml of 9.3mol / L lithium bromide solution at 60℃ for 4.5h. After dissolution is complete, take it out and cool it to room temperature to obtain silk fibroin solution. 4) Place the silk fibroin solution into a dialysis bag (molecular weight cutoff 3500 Da), clamp both ends with dialysis clamps, and place it in 5.5 L of deionized water. Dialyze at room temperature for three days, changing the water every 8 hours. After dialysis, first filter the solution to remove larger impurities with gauze, then aliquot the filtrate into centrifuge tubes (mass error should not exceed 0.05 g) and centrifuge using a high-speed centrifuge to obtain a clear silk fibroin solution. The centrifugation speed of the high-speed centrifuge is 11000 rpm, the centrifugation temperature is 4℃, and the centrifugation time is 20 min. Centrifuge twice, and store the obtained clear silk fibroin solution in a 4℃ refrigerator for later use. 5) Prepare 50.0 mmol / L and 0.1 mol / L pH 7.4 phosphate buffers (PB), respectively; Weigh 50.0 mg of chloramine T and dissolve it in 50.0 mmol / L pH 7.4 phosphate buffer to prepare a 1.0 mg / mL chloramine T solution; Weigh 60.0 mg of sodium metabisulfite and dissolve it in 50.0 mmol / L pH 7.4 phosphate buffer to prepare a 2.0 mg / mL sodium metabisulfite solution; Weigh 1.835 g of clarified silk fibroin solution and dissolve it in 50.0 mmol / L pH 7.4 phosphate buffer to prepare a 2.0 wt% silk fibroin polymer solution; Potassium iodide (KI) was prepared into a KI solution of 2 mg / mL using 50.0 mmol / L pH 7.4 phosphate buffer. Weigh 10.0 mg NaI and dissolve it in 500.0 mL of 50.0 mmol / L pH 7.4 phosphate buffer to prepare NaI stock solution. Then, use a pipette to transfer 5.0 mL of NaI stock solution into a 500.0 mL volumetric flask and dilute to volume to prepare NaI solution. In 50.0 mL of 0.1 mol / L, pH 7.4 phosphate buffer, 5.0 mL of 2.0 wt% silk fibroin polymer solution and 2 mL of NaI solution were added. After adding 50.0 mL of chloramine T solution, the mixture was stirred at room temperature for 20 min. Then, 30.0 mL of sodium metabisulfite solution was added to the system, and the mixture was stirred at room temperature for 10 min. Finally, 10.0 mL of KI solution was added dropwise to dilute the residual iodide. The resulting mixture was transferred into a dialysis bag with a molecular weight cutoff of 3500 Daltons and immersed in deionized water for dialysis for 2 days, with the water changed daily. The product, iodine-substituted silk fibroin, was obtained as a transparent liquid, yielding 2.0 wt% iodine-substituted silk fibroin liquid. 6) Take 550.0 µL of 2.0 wt% iodine-substituted silk fibroin liquid and place it in a 10 mL sample bottle. Add 50 µL of 0.4 mol / L H2O2 solution and 50 µL of 0.8 mg / mL HRP (horseradish peroxidase) solution. Vortex the mixture until homogeneous to obtain a hydrogel. Example

[0030] This embodiment provides a method for preparing a functional silk fibroin-based hydrogel, comprising the following steps: 1) Pour 3L of deionized water into a stainless steel bucket, heat the water to a boil, add 6.36g of sodium carbonate to the boiling water, and after it is completely dissolved, add 7.8g of raw silkworm cocoons to the boiling water and boil for 20 minutes to degumme. After degumming, take out the raw silkworm cocoons, wash them with water 4 times, wring them dry, loosen them, and put them in a 50℃ vacuum oven to dry for 8 hours. 2) Spread the dried raw silk into a single layer and irradiate it with infrared light in a vacuum environment of 3 kPa. The infrared light wavelength is 850 nm, the irradiation time is 120 s, and the surface temperature of the raw silk layer is 50 ℃. 3) Weigh 5g of raw silk that has been irradiated with infrared light, tear it until fluffy, and dissolve it in 22ml of 9.3mol / L lithium bromide solution at 60℃ for 4.5h. After dissolution is complete, take it out and cool it to room temperature to obtain silk fibroin solution. 4) Place the silk fibroin solution into a dialysis bag (molecular weight cutoff 3500 Da), clamp both ends with dialysis clamps, and place it in 5.5 L of deionized water. Dialyze at room temperature for three days, changing the water every 8 hours. After dialysis, first filter the solution to remove larger impurities with gauze, then aliquot the filtrate into centrifuge tubes (mass error should not exceed 0.05 g) and centrifuge using a high-speed centrifuge to obtain a clear silk fibroin solution. The centrifugation speed of the high-speed centrifuge is 11000 rpm, the centrifugation temperature is 4℃, and the centrifugation time is 20 min. Centrifuge twice, and store the obtained clear silk fibroin solution in a 4℃ refrigerator for later use. 5) Prepare 50.0 mmol / L and 0.1 mol / L pH 7.4 phosphate buffers (PB) respectively; weigh 50.0 mg of chloramine T and dissolve it in 50.0 mmol / L pH 7.4 phosphate buffer to prepare a 1.0 mg / mL chloramine T solution; weigh 60.0 mg of sodium metabisulfite and dissolve it in 50.0 mmol / L pH 7.4 phosphate buffer to prepare a 2.0 mg / mL sodium metabisulfite solution; weigh 1.835 g of clarified silk fibroin solution and dissolve it in 50.0 mmol / L pH 7.4 phosphate buffer to prepare a 2.0 wt% silk fibroin polymer solution; prepare a 2 mg / mL potassium iodide (KI) solution using 50.0 mmol / L pH 7.4 phosphate buffer; weigh 10.0 mg of NaI and dissolve it in 500.0 mL of 50.0 mmol / L pH 7.4 phosphate buffer to prepare a NaI stock solution, then pipette 5.0 mL of the solution. The NaI stock solution was diluted to 500.0 mL in a volumetric flask to prepare a NaI solution. In 50.0 mL of 0.1 mol / L, pH 7.4 phosphate buffer, 5.0 mL of 2.0 wt% silk fibroin polymer solution and 4 mL of NaI solution were added. After adding 50.0 mL of chloramine T solution, the mixture was stirred at room temperature for 20 min. Then, 30.0 mL of sodium metabisulfite solution was added to the system, and the mixture was stirred at room temperature for 10 min. Finally, 10.0 mL of KI solution was added dropwise to dilute the residual iodide. The resulting mixture was transferred into a dialysis bag with a molecular weight cutoff of 3500 Daltons and immersed in deionized water for dialysis for 2 days, with the water changed daily. The product, iodine-substituted silk fibroin, was obtained as a transparent liquid, yielding 2.0 wt% iodine-substituted silk fibroin liquid. 6) Take 550.0 µL of 2.0 wt% iodine-substituted silk fibroin liquid and place it in a 10 mL sample bottle. Add 50 µL of 0.4 mol / L H2O2 solution and 50 µL of 0.8 mg / mL HRP (horseradish peroxidase) solution. Vortex the mixture until homogeneous to obtain a hydrogel.

[0031] Comparative Example 1 This comparative example provides a method for preparing a silk fibroin-based hydrogel, which differs from Example 1 in that in step 5), 8 ml of NaI solution is added to 50.0 mL of 0.1 mol / L, pH 7.4 phosphate buffer.

[0032] Comparative Example 2 This comparative example provides a method for preparing silk fibroin-based hydrogels. The difference between this method and Example 1 is that the drying time in step 1) is increased to 8.04 h, and step 2) is omitted. The dried raw silk is directly processed in step 3).

[0033] Test Example 1 The iodine-substituted silk fibroin prepared in step 5) of Example 1 was characterized by Fourier transform infrared spectroscopy (FT-IR), see below. Figure 1 In this spectrum, SF (silk fibroin) represents the infrared spectrum of silk fibroin, and I-SF represents the infrared spectrum of iodine-substituted silk fibroin. Based on the infrared spectra, the following changes can be observed: in the wavenumber range of 500-600 cm⁻¹ -1 This region is typically associated with the stretching vibrations of halogenated hydrocarbons (CI). The shift in this region observed in I-SF polymers compared to SF is due to the addition of CI bonds, demonstrating that a portion of the hydrogen atom at the phenolic ortho position of tyrosine in SF is replaced by iodine.

[0034] The white powder obtained after freeze-drying iodine-substituted silk fibroin in step 5 of Example 1 was observed under a scanning electron microscope as follows: Figure 2 As shown, by Figure 2 It can be seen that silk fibroin fibers are composed of interlocking nanofiber bundles with a diameter of 30-35 nm, and have a micro-nano-scale hierarchical structure.

[0035] Test Example 2 Example 1 Example 2 Comparative Example 1 Comparative Example 2 Gel formation time (s) 90 490 Unable to form glue 130 The gelation mechanism of silk fibroin hydrogel is to prepare hydrogel by using HRP and H2O2 to mediate the coupling of tyrosine in SF polymer. New carbon-carbon bonds can be generated through free radical coupling at the ortho position of phenolic hydroxyl groups in SF tyrosine. As the molar ratio with NaI increases, the phenolic hydroxyl groups in tyrosine are replaced by iodine, and the gelation rate slows down. When 8 ml of NaI solution is added, it can hardly gel.

[0036] Test Example 3 The storage modulus (G') and loss modulus (G'') of the hydrogels obtained in Examples 1-2 and Comparative Example 2 were measured using a rheometer in frequency scanning mode. These values ​​were plotted as the ordinate and the scanning frequency as the abscissa to create an oscillation frequency scan diagram of the hydrogels. Figure 3-5 As shown, where Figure 3 The oscillation frequency scan diagram of the hydrogel prepared in Example 1 is shown. Figure 4 The oscillation frequency scan diagram of the hydrogel prepared in Example 2 is shown. Figure 5 The image shows a scan of the oscillation frequency of the hydrogel prepared in Comparative Example 2. Figure 3-4 It can be seen that the hydrogel has a high storage modulus, indicating its excellent mechanical properties. Figure 5 It can be seen that its energy storage modulus is slightly lower than that of Example 1, indicating that infrared light irradiation is beneficial to improving the mechanical strength of the hydrogel.

[0037] Obviously, those skilled in the art can make other modifications based on the above embodiments, and the obvious modifications therefrom are still within the protection scope of this invention.

Claims

1. A method for preparing an injectable functional silk fibroin-based hydrogel, characterized in that, Includes the following steps: 1) Degumming raw silkworm cocoons in alkaline boiling water. After degumming, remove the raw silkworm cocoons, wash and dry them to obtain dried raw silk. 2) Spread the dried raw silk into a single layer and irradiate it with infrared light in a vacuum environment; The vacuum level of the vacuum environment is 2-4 kPa, the infrared wavelength is 800-900 nm, the infrared irradiation time is 80-150 s, and the surface temperature of the raw silk layer is 45-55℃. 3) Dissolve raw silk irradiated with infrared light in lithium bromide solution to obtain silk fibroin solution; 4) Dialyze the silk fibroin solution, filter it after dialysis, and centrifuge to obtain a clear silk fibroin solution; 5) Mix the clarified silk fibroin solution, NaI and chloramine T, stir and react, then add sodium metabisulfite and continue stirring and reacting. After the reaction is complete, dialyze the mixture to obtain iodine-substituted silk fibroin liquid. 6) Mix iodine-substituted silk fibroin liquid, H2O2 solution and HRP solution, and vortex mix until homogeneous to obtain hydrogel.

2. The preparation method according to claim 1, characterized in that, The alkaline boiling water is obtained by boiling a mixture of water and sodium carbonate, wherein the ratio of water to sodium carbonate is 1:(2.0-2.2), and the unit of ratio is L:g; The ratio of raw silkworm cocoons to alkaline boiling water is (7-10):(2.8-3.2), with the unit being g:L.

3. The preparation method according to claim 1 or 2, characterized in that, The degumming process involves adding raw silkworm cocoons to boiling water and boiling for 20-40 minutes to remove the glue. After degumming, remove the raw silkworm cocoons, wash them with water 3-6 times, wring them dry, loosen them, and then dry them in a vacuum oven at 45-55℃ for 6-12 hours.

4. The preparation method according to claim 1, characterized in that, In step 3), the concentration of the lithium bromide solution is 9.1-9.4 mol / L, the ratio of raw silk to lithium bromide solution is (4-8):(20-25), the ratio unit is g:ml, the dissolution temperature is 55-60℃, and the dissolution time is 4-6h.

5. The preparation method according to claim 1, characterized in that, In step 4), the silk fibroin solution is placed into a dialysis bag for dialysis treatment. The molecular weight cutoff is 3500 Da, and the dialysis time is 24-72 hours. The water is changed every 8-10 hours during the dialysis process.

6. The preparation method according to claim 1, characterized in that, During centrifugation, the centrifugation speed is 10000-12000 rpm, the centrifugation temperature is 4-5℃, the centrifugation time is 20-40 min, and the number of centrifugations is 2-5.

7. The preparation method according to claim 1, characterized in that, In step 5), firstly, prepare 50.0 mmol / L phosphate buffer (pH 7.4) and 0.1 mol / L phosphate buffer (pH 7.4); then, using the 50.0 mmol / L phosphate buffer, prepare 1.0-1.2 mg / mL chloramine T solution, 1.8-2.2 mg / mL sodium metabisulfite solution, 1.5-2.5 mg / mL KI solution, and 0.0002-0.0004 mg / mL NaI solution; and use the 50.0 mmol / L phosphate buffer and clarified silk fibroin solution to prepare a 1.8-2.2 wt% silk fibroin solution; then, add 5-8 mL of the 1.8-2.2 wt% silk fibroin solution and 2-4 mL of NaI solution to 50-60 mL of the 0.1 mol / L phosphate buffer (pH 7.4), and 50-60 mL of... Chloramine T solution was stirred at room temperature for 20-40 min. 30-35 mL of sodium metabisulfite solution was added to the system and stirred at room temperature for 10-20 min. 10-15 mL of KI solution was added to dilute the residual iodide. Finally, the resulting mixture was transferred into a dialysis bag and dialyzed in water.

8. The preparation method according to claim 7, characterized in that, In step 5), the molecular weight cutoff of the dialysis bag is 3500 Daltons, the dialysis time is 24-48 hours, and the water is changed every 20-24 hours during the dialysis process.

9. The preparation method according to claim 7, characterized in that, The concentration of H2O2 solution is 0.3-0.5 mol / L, the concentration of HRP solution is 0.7-0.9 mg / mL, and the concentration of iodine-substituted silk fibroin liquid is 1.5-2.5 wt%.

10. The preparation method according to claim 9, characterized in that, The volume ratio of iodine-substituted silk fibroin liquid, H2O2 solution and HRP solution added is (500-550): (40-60): (40-60).

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