阅读说明：本技术 一种干纺制备丝蛋白基纤维的方法及由其制备的丝蛋白基纤维及其应用 (Method for preparing silk protein-based fiber by dry spinning, silk protein-based fiber prepared by same and application thereof ) 是由 郭成辰 江瑞 孙子扬 于 2021-09-09 设计创作，主要内容包括：本发明提供一种干纺制备丝蛋白基纤维的方法及由其制备的丝蛋白基纤维及其应用。本发明的干纺制备丝蛋白基纤维的方法包括如下步骤：(1)在不需要加入外来物质的前提下浓缩丝蛋白基水溶液；(2)采用挤出设备将上述制得的丝蛋白基水溶液作为纺丝原液制备成纤维。本发明的方法具有操作简单,绿色环保,高效快捷等优点,并且本发明制备的丝蛋白基纤维尺寸均一且结构可控,同时还表现出改善的断裂强度和断裂伸长率。(The invention provides a method for preparing silk protein-based fibers by dry spinning, silk protein-based fibers prepared by the method and application of the silk protein-based fibers. The method for preparing the silk protein-based fiber by dry spinning comprises the following steps: (1) concentrating the silk protein-based aqueous solution without adding foreign substances; (2) and (3) preparing the prepared fibroin-based aqueous solution into fibers by using extrusion equipment as spinning solution. The method has the advantages of simple operation, environmental protection, high efficiency, rapidness and the like, and the silk protein-based fiber prepared by the method has uniform size and controllable structure and simultaneously shows improved breaking strength and breaking elongation.)

1. A method of dry spinning to produce a silk protein based fiber, the method comprising the steps of:

(1) concentrating a silk protein based aqueous solution comprising the steps of:

1': placing the initial silk protein aqueous solution into a dialysis bag for dialysis, centrifuging the dialyzed solution at a first centrifugal rotation speed under ambient pressure, and discarding the precipitate to obtain a silk protein-based aqueous solution with a concentration of 4.0-8.0 w/v%, preferably 4.5-6.5 w/v%;

2': placing the silk protein-based aqueous solution obtained in the step 1' in a vacuum environment, and performing centrifugal concentration at a second centrifugal rotating speed to obtain a silk protein-based aqueous solution with the concentration of more than 10 w/v%;

(2) preparing fibers: and (2) preparing the silk fibroin-based aqueous solution prepared in the step (1) into fibers by using extrusion equipment as spinning solution.

2. The method of claim 1, wherein the starting silk protein aqueous solution in step 1' is a natural silk protein aqueous solution; an aqueous solution of a regenerated silk protein derivative obtained by chemical modification; physically mixing to obtain a regenerated silk protein/additive composite silk protein-based solution; or an aqueous solution of recombinant silk protein.

3. The method according to claim 1, wherein in step 1', the first centrifugation rotation speed is 5000-;

in step 2', the vacuum centrifugation is performed under the following conditions: the second centrifugation rotation speed is 100-.

4. The method according to claim 1, wherein prior to step 2' or (2), an additive is added to the aqueous fibroin solution, the additive being selected from the group consisting of glycerol, inorganic salts, bioactive molecules, and the additive being present at a concentration of 0.1-50 wt%.

5. The method according to claim 1, wherein, in the step (2), the spinning dope is transferred into a syringe, a port of the syringe is connected with a stainless steel needle having a diameter of 0.2 to 2.0mm, and a solution flow rate is controlled by a syringe pump and set to 1 to 2000 μ L/h; the diameter of the collecting device is 5-1000mm, the rotating speed is 1-5000rpm, the horizontal distance between the needle tip and the collecting device is 1-1000mm, the temperature is 5-200 ℃ in the injection process, and the humidity is 5-90%.

6. The method of claim 1, wherein after step (2), the method further comprises step (3): post-treating the fiber prepared in the step (2) in methanol water solution, ethanol vapor or acetic acid vapor, or post-drafting the fiber in air, alcohol bath, ethanol vapor or acetic acid vapor,

alternatively, in the step (3), in the case of the post-treatment with an aqueous methanol solution, the concentration of methanol is 50 to 95 v/v% and the treatment time is 1 to 600 min;

alternatively, in the step (3), in the case of the back drawing in acetic acid vapor, the concentration of acetic acid vapor is 0.1 to 17.5M and the treatment time is 1 to 180 s;

alternatively, in step (3), the prepared fiber was first placed in a 3.5M acetic acid vapor atmosphere for 30s, and then the fiber was immersed in a 95 v/v% methanol solution for 120 min.

7. A silk protein based fibre prepared by the method of any one of claims 1 to 6.

8. A medical device made from the silk protein-based fiber of claim 7.

9. The medical device of claim 8, made from the silk fibroin-based fibers by a post-processing process of twisting, sizing, or weaving.

10. The medical device of claim 8, selected from any one of a surgical suture, an artificial ligament, a tissue scaffold, and a wound dressing.

Technical Field

The invention belongs to the field of protein-based high polymer materials, and particularly relates to a method for preparing high-performance silk protein-based fibers by a dry method, silk protein-based fibers prepared by the method, and medical instruments prepared from the silk protein-based fibers.

Background

The protein-based fiber is a fiber material formed by taking protein-based materials such as silk protein, collagen, keratin and the like as raw materials through different processing modes, and the basic structural unit of the fiber material is amino acid. The common protein-based fibers in nature mainly comprise silk, spider silk, animal hair, collagen microfibers and the like. These natural protein-based fibers generally have excellent mechanical properties, biocompatibility and bioabsorbability, and thus have been receiving wide attention in recent years and are being applied to biomedical fields such as surgical sutures and tissue engineering scaffolds, etc. In addition to directly utilizing the native natural protein-based fiber, the reverse engineering technology is used for dissolving the natural protein to prepare the regenerated protein-based fiber with specific functions, which is an important development direction, and the limitations of the native natural protein-based fiber in the aspects of biological function, degradation performance regulation and the like can be improved to a great extent.

At present, four methods for preparing regenerated protein-based fibers are mainly used, namely an electrostatic spinning method, a wet spinning method, a dry spinning method and a dry-jet wet spinning method. Among them, electrostatic spinning and wet spinning are more relevant researches, and dry spinning is relatively less research. The principle of the dry spinning method is that under the action of a drawing force, spinning solution is directly drawn out from a spinneret orifice and is solidified to form fibers after being rapidly volatilized by a solvent. The spinning mode is that of silkworms or spiders in nature, and the obtained fiber has excellent performance. Therefore, the development of an artificial dry spinning method for preparing the high-performance regenerated protein-based fiber by simulating the natural spinning mode of silkworms and spiders has important significance.

At present, the methods for preparing regenerated protein-based dry spinning solution mainly comprise two methods: (1) dissolving solid protein with organic solvent (such as hexafluoroisopropanol) to obtain high-concentration spinning solution; (2) concentrating the protein water solution to obtain spinning solution with high protein concentration, wherein inorganic salt ions and TiO are required to be added in the method₂And (3) waiting for metal oxide or buffer solution for adjusting the pH value to enable the metal oxide or the buffer solution to meet the spinning requirement.

CN101724920B, CN102134757B, and CN102220661B disclose that regenerated silk protein fiber is prepared by adding metal ions into silk protein aqueous solution and adjusting pH. CN108396425A discloses dissolving silk protein by using organic acid/salt ion system to prepare silk protein/carbon nanotube filament yarn. CN103572395B discloses mixing and concentrating silk protein aqueous solution and graphene oxide, and adding calcium ions for adjustment to prepare composite fibers.

In the method, inorganic salt is added or an organic solvent is utilized in the process of preparing the spinning solution, and the prepared fibroin aqueous solution is not a pure fibroin aqueous solution, so that the performance of the prepared regenerated fibroin fiber is influenced by a certain difference from a natural spinning mode.

Disclosure of Invention

Based on the problems, the invention successfully realizes the continuous preparation of the high-performance silk fibroin-based fiber filament by preparing the high-concentration pure silk fibroin aqueous solution and designing and optimizing the dry spinning method. The prepared fiber filament has excellent mechanical property, biocompatibility and bioabsorbability. In addition, by adding active biomolecules such as enzymes, drugs, antibiotics, etc. to the spinning solution, a fibroin-based fiber having biological activity, biocompatibility, and bioabsorbability can be prepared and applied to the related biomedical field.

One technical object of the present invention is to provide a method for dry-preparing silk protein-based fibers.

It is another technical object of the present invention to provide a silk protein-based fiber prepared by the above method.

It is another technical object of the present invention to provide a medical device made of the above silk protein-based fiber.

In one aspect, the present invention provides a method of dry spinning silk protein-based fibers, the method comprising the steps of:

(1) concentrating a silk protein based aqueous solution comprising the steps of:

1': placing the initial silk protein aqueous solution into a dialysis bag for dialysis, centrifuging the dialyzed solution at a first centrifugal rotation speed under ambient pressure, and discarding the precipitate to obtain a silk protein-based aqueous solution with a concentration of 4.0-8.0 w/v%, preferably 4.5-6.5 w/v%;

2': placing the silk protein-based aqueous solution obtained in the step 1' in a vacuum environment, and performing centrifugal concentration at a second centrifugal rotating speed to obtain a silk protein-based aqueous solution with the concentration of more than 10 w/v%;

(2) preparing fibers: and (3) preparing the prepared fibroin-based aqueous solution into fibers by using extrusion equipment as spinning solution.

In particular embodiments, the starting silk protein aqueous solution in step 1' can be a natural silk protein aqueous solution; an aqueous solution of a regenerated silk protein derivative obtained by chemical modification; a regenerated silk protein/additive (such as medicine, growth factor, antibiotic and the like) composite silk protein base solution obtained by physical mixing; recombinant silk protein aqueous solution, etc.

In a specific embodiment, the starting aqueous native silk protein solution is prepared by: placing the cut silkworm cocoon in 0.02M Na₂CO₃Boiling in water solution at 60-100 deg.C for 30-180min to degum silkworm cocoon, and drying the obtained glue solution to obtain degummed silk; then dissolving the degummed silk in 9-10M, preferably 9.3M LiBr water solution, and putting the solution into an oven at 60-150 ℃ for 2-8h to completely dissolve the degummed silk.

In a specific embodiment, in step 1', the starting fibroin aqueous solution is dialyzed under the following dialysis conditions: the dialysis time is 2-5 days, the dialysate is deionized water, and the cut-off molecular weight of the dialysis bag is 3500 Dalton (Da).

In a specific embodiment, in step 1', the first centrifugation rotation speed is 5000-. The foregoing centrifugation operation is mainly used to remove precipitates generated during dialysis.

In a specific embodiment, before step 2' or step (2), an additive for improving the properties of the silk fibroin-based material product may be added to the silk fibroin-based water solution, the additive being selected from the group consisting of glycerol, inorganic salts, bioactive molecules (e.g., enzymes, antibiotics, drugs, etc.), inorganic materials, organic molecules. The concentration of the additive may be 0.1 to 50 wt%. The properties of the silk fibroin-based material product, such as physicochemical and biological properties, can be improved by adding additives without affecting the concentration effect.

In a specific embodiment, in step 2', the vacuum centrifugation is performed under the following conditions: the second centrifugation rotation speed is 100-. The main purpose of this step is to concentrate the silk protein aqueous solution.

In a specific embodiment, the concentration of the silk protein based aqueous solution obtained in step 2' is 10-50 w/v%, preferably 25-40 w/v%, preferably the concentration of the silk protein based aqueous solution obtained in step (2) is 6 times the concentration of the silk protein based aqueous solution obtained in step (1).

In a specific embodiment, the method further comprises: after step 2', a third centrifugation operation at a third centrifugation rotational speed is performed to separate precipitated silk proteins.

In a specific embodiment, the third centrifugation operation is performed under the following conditions: the centrifugation time is 5-30min, the centrifugation temperature is 4-30 ℃, the third centrifugation rotating speed is 5000-. The third centrifugation operation is mainly used to remove the precipitate generated during step 2'.

In a specific embodiment, in step (2), the fibroin-based aqueous solution obtained in step (1) is prepared into fibers as a spinning dope by: transferring the spinning solution into an injector, wherein the port of the injector is connected with a stainless steel needle, the diameter of the needle is 0.2-2.0mm, and the solution flow rate is controlled by an injection pump and is set to be 1-2000 mu L/h; the diameter of the collecting device is 5-1000mm, the rotating speed is 1-5000rpm, the horizontal distance between the needle tip and the collecting device is 1-1000mm, the temperature is 5-200 ℃ in the injection process, and the humidity is 5-90%.

The method of the present invention may further comprise: (3) and (3) post-treatment: the prepared fiber is placed in methanol water solution, ethanol vapor or acetic acid vapor for post-treatment, or is post-drafted in the air, alcohol bath, ethanol vapor or acetic acid vapor environment, so that the fiber structure is regulated.

In the invention, the content of beta-sheet in the fiber can be increased to a certain extent through the post-treatment or the post-drawing treatment in the step (3), so that the mechanical property of the fiber is further improved, and the application range of the fiber is enlarged.

In a specific embodiment, in the step (3), in the case of the post-treatment with an aqueous methanol solution, the concentration of methanol is 50 to 95 v/v% and the treatment time is 1 to 600 min.

In a specific embodiment, in step (3), in the case of the post-treatment with acetic acid vapor, the acetic acid vapor concentration is 0.1 to 17.5M and the treatment time is 1 to 180s, for example, 10s, 30 s.

In a specific embodiment, in step (3), the prepared fiber is first placed in a 3.5M acetic acid vapor atmosphere for 30s, and then the fiber is immersed in a 95 v/v% methanol solution for 120 min.

In another aspect, the present invention provides a silk protein-based fiber prepared by the above method.

In yet another aspect, the present invention provides a medical device made from the above-described silk protein-based fiber.

In a specific mode, the medical appliance is made of the silk protein-based fibers through a post-processing technology of twisting, sizing or weaving.

In particular embodiments, the medical device is selected from one of a surgical suture, an artificial ligament, a tissue scaffold, and a wound dressing.

Advantageous effects

Compared with the prior art, the invention has the advantages that:

(1) the time efficiency of the preparation of the spinning solution is high, and the properties are stable;

(2) continuous spinning can be carried out;

(3) the prepared fiber filament has uniform size, controllable structure and more excellent mechanical property.

(5) The method also has the advantages of simple operation, environmental protection, high efficiency, rapidness and the like.

(6) The fiber obtained by dry spinning in the application has the breaking strength of 0.05-1.0GPa and the breaking elongation of 1-800%, and chemical substances such as inorganic salt or organic solvent which can have adverse effects on the fiber properties or additives which can be added are not added in the preparation process of the fiber, so that the fiber is convenient to be processed into a biocompatible material.

(7) The fiber preparation method of the invention can also be applied to the preparation of fibers by using collagen and keratin as raw materials, so as to prepare fibers similar to the fibers in the application, and the prepared fibers are also expected to be applied to medical instruments or other fields.

Drawings

FIG. 1 is a scanning electron microscope image of the surface of the dry-process regenerated yarn obtained in step (2) of example 1.

FIG. 2 is a sectional scanning electron microscope photograph of the dry-process regenerated yarn obtained in step (2) of example 2.

FIG. 3 is an infrared spectrum of the degummed silk, the dry-process regenerated silk and the dry-process regenerated silk after being soaked in methanol solution for 120min in example 2.

FIG. 4 is a tensile stress-strain curve of the degummed and dry-laid recycled filaments of example 3.

FIG. 5 is an infrared spectrum of dry-laid recycled filaments from example 4 and their fibers after post-draw treatment under ethanol vapor (example 4) and acetic acid vapor 5 (example).

FIG. 6 is a tensile stress-strain curve of the degummed yarn, the dry process regenerated yarn, and the dry process regenerated yarn after 30min treatment with a methanol solution in example 7.

Detailed Description

The term "silk protein" herein may be used interchangeably with "silk fibroin".

Hereinafter, the technical contents of the present invention are described in detail by specific examples to enable those skilled in the art to better understand the present invention, however, these examples are not intended to limit the contents of the present invention.

See table 1 below for the instruments and sources of reagents used in the examples and test examples below.

TABLE 1 Main test reagents and test instruments

Example 1

(1) Placing silkworm cocoon in 0.02M Na₂CO₃Boiling in water solution at 60-100 deg.C for 30-180min to degum silkworm cocoon, and drying the obtained glue solution to obtain degummed silk.

The dried degummed silk was then dissolved in 9.3M LiBr aqueous solution, and the completely dissolved solution was then placed in a dialysis bag with a molecular weight cut-off of 3500 daltons (Da), wherein the dialysis solution was deionized water, the dialysis time was 3 days, the dialyzed solution was centrifuged at 9000rpm at normal pressure, the supernatant was aspirated and the precipitate was separated to give an initial concentration of 4.5 wt.% silk protein aqueous solution.

The solution is then placed under vacuum for centrifugal concentration to prepare a high concentration silk protein aqueous solution. Wherein the concentration temperature is 30 deg.C, the centrifugal rotation speed is 2000rpm, the vacuum degree is 20mbar, and the concentration time is 13 h.

Centrifuging the concentrated solution at 4 deg.C and 6000rpm for 10 min. Finally, a silk protein concentrated solution with a mass fraction of 25 wt.% is obtained.

(2) The silk fibroin concentrated solution with the mass fraction of 25 wt.% is added into a 5mL syringe, the port of the syringe is connected with a stainless steel needle, and the diameter of the needle is 0.4 mm. The solution flow rate was controlled by a syringe pump at a rate of 40. mu.L/h. The diameter of the collecting device was 12mm and the rotational speed was 140 rpm. The distance between the needle tip and the collection means was 150 mm. The temperature during the experiment was 25 ℃ and the humidity was 45%. Finally, the dry-process regenerated silk is prepared, and the fiber diameter is 3.6 +/-0.3 mu m.

(3) Immersing the dry-method regenerated silk prepared in the method into a 95% (v/v) methanol solution, treating at room temperature for 30min, and then drying.

Example 2

(1) Placing silkworm cocoon in 0.02M Na₂CO₃Boiling in water solution at 60-100 deg.C for 30-180min to degum silkworm cocoon, and drying the obtained glue solution to obtain degummed silk.

The dried degummed silk was then dissolved in 9.3M LiBr aqueous solution, and the completely dissolved solution was then placed in a dialysis bag with a molecular weight cut-off of 3500 daltons (Da), wherein the dialysis solution was deionized water, the dialysis time was 3 days, the dialyzed solution was centrifuged at 9000rpm at normal pressure, the supernatant was aspirated and the precipitate was separated to obtain silk protein aqueous solution with an initial concentration of 5.3 wt.%.

The solution is then placed under vacuum for centrifugal concentration to prepare a high concentration silk protein aqueous solution. Wherein the concentration temperature is 45 deg.C, the centrifugal rotation speed is 2000rpm, the vacuum degree is 20mbar, and the concentration time is 14 h.

Centrifuging the concentrated solution at 4 deg.C and 6000rpm for 10 min. Finally, a silk protein concentrated solution with a mass fraction of 27 wt.% was obtained.

(2) The silk fibroin concentrated solution with the mass fraction of 27 wt.% is added into a 5mL syringe, the port of the syringe is connected with a stainless steel needle, and the diameter of the needle is 0.4 mm. The solution flow rate was controlled by a syringe pump at a rate of 40. mu.L/h. The diameter of the collecting device was 12mm and the rotational speed was 140 rpm. The distance between the needle tip and the collection means was 150 mm. The temperature during the experiment was 25 ℃ and the humidity was 35%. Finally, the dry-process regenerated silk is prepared, and the fiber diameter is 6.5 +/-0.7 mu m.

(3) Immersing the dry-method regenerated silk prepared in the method into a 95% (v/v) methanol solution, treating at room temperature for 120min, and then drying.

Example 3

(1) Placing silkworm cocoon in 0.02M Na₂CO₃Boiling in water solution at 60-100 deg.C for 30-180min to degum silkworm cocoon, and drying the obtained glue solution to obtain degummed silk.

The dried degummed silk was then dissolved in 9.3M LiBr aqueous solution, and the completely dissolved solution was then placed in a dialysis bag with a molecular weight cut-off of 3500 daltons (Da), wherein the dialysis solution was deionized water, the dialysis time was 3 days, the dialyzed solution was centrifuged at 9000rpm at normal pressure, the supernatant was aspirated and the precipitate was separated to obtain silk protein aqueous solution with an initial concentration of 5.8 wt.%.

The solution is then placed under vacuum for centrifugal concentration to prepare a high concentration silk protein aqueous solution. Wherein the concentration temperature is 45 deg.C, the centrifugal rotation speed is 1500rpm, the vacuum degree is 20mbar, and the concentration time is 17 h.

Centrifuging the concentrated solution at 4 deg.C and 9000rpm for 15 min. Finally, a silk protein concentrated solution with a mass fraction of 40 wt.% is obtained.

(2) The silk fibroin concentrated solution with the mass fraction of 40 wt.% is added into a 5mL syringe, the port of the syringe is connected with a stainless steel needle, and the diameter of the needle is 0.6 mm. The solution flow rate was controlled by a syringe pump at a rate of 60. mu.L/h. The diameter of the collecting device was 12mm and the rotational speed was 80 rpm. The distance between the needle tip and the collection means was 150 mm. The temperature during the experiment was 27 ℃ and the humidity was 40%. Finally, the dry-process regenerated silk is prepared, and the fiber diameter is 20.7 +/-1.5 mu m.

(3) Immersing the dry-method regenerated silk prepared in the method into a 95% (v/v) methanol solution, treating at room temperature for 60min, and then drying.

Example 4

(1) The silk fibroin concentrated solution prepared in the step (1) of example 1, having a mass fraction of 25 wt.%, was added to a 5mL syringe, the port of which was connected to a stainless steel needle having a diameter of 0.4 mm. The solution flow rate was controlled by a syringe pump at a rate of 40. mu.L/h. The diameter of the collecting device was 12mm and the rotational speed was 165 rpm. The distance between the needle tip and the collection means was 150 mm. The temperature during the experiment was 25 ℃ and the humidity was 45%. Finally, the dry-process regenerated silk is prepared, and the fiber diameter is 3.0 +/-0.2 mu m.

(2) The produced regenerated silk produced by the dry method is subjected to post-drawing treatment under the condition of 95% (v/v) ethanol steam for 30s, and then is dried.

Example 5

(1) The dry-process regenerated silk obtained in step (1) of example 4 was post-drawn under 17.5M acetic acid vapor for 30 seconds, and then dried.

Example 6

(1) The silk fibroin concentrated solution prepared in the step (1) of example 1, having a mass fraction of 25 wt.%, was added to a 5mL syringe, the port of which was connected to a stainless steel needle having a diameter of 0.4 mm. The solution flow rate was controlled by a syringe pump at a rate of 40. mu.L/h. The diameter of the collecting device was 12mm and the rotational speed was 180 rpm. The distance between the needle tip and the collection means was 150 mm. The temperature during the experiment was 25 ℃ and the humidity was 45%. Finally, the dry-process regenerated silk is prepared, and the fiber diameter is 2.7 +/-0.3 mu m.

(2) The dry-process regenerated silk prepared in the above way is placed in an acetic acid vapor environment of 3.5M for 30s, and then is immersed in a methanol solution of 95% (v/v) for 120min, and then is dried.

Example 7

(1) Placing silkworm cocoon in 0.02M Na₂CO₃Boiling in water solution at 60-100 deg.C for 30-180min to degum silkworm cocoon, and drying the obtained glue solution to obtain degummed silk.

The dried degummed silk was then dissolved in 9.3M LiBr aqueous solution, and the completely dissolved solution was then placed in a dialysis bag with a molecular weight cut-off of 3500 daltons (Da), wherein the dialysis solution was deionized water, the dialysis time was 3 days, the dialyzed solution was centrifuged at 9000rpm at normal pressure, the supernatant was aspirated and the precipitate was separated to obtain silk protein aqueous solution with an initial concentration of 5.5 wt.%.

Then, the solution of glycerin and calcium chloride is added into 5.5 wt.% silk protein water solution, so that the mass ratio of glycerin, calcium chloride and silk protein in the solution is 4: 1: 15. And then placed under a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. Wherein the concentration temperature is 30 deg.C, the centrifugal rotation speed is 2000rpm, the vacuum degree is 20mbar, and the concentration time is 15 h.

Centrifuging the concentrated solution at 4 deg.C and 6000rpm for 10 min. Finally, the composite silk protein concentrated solution with the mass fraction of 45 wt.% is obtained.

(2) The silk fibroin concentrated solution with the mass fraction of 45 wt.% is added into a 5mL syringe, the port of the syringe is connected with a stainless steel needle, and the diameter of the needle is 0.4 mm. The solution flow rate was controlled by a syringe pump at a rate of 40. mu.L/h. The diameter of the collecting device was 12mm and the rotational speed was 100 rpm. The distance between the needle tip and the collection means was 150 mm. The temperature during the experiment was 23 ℃ and the humidity was 50%. Finally, the dry-process regenerated silk is prepared, and the fiber diameter is 13 +/-1.5 mu m.

(3) Immersing the dry-method regenerated silk prepared in the method into a 95% (v/v) methanol solution, treating for 30min at room temperature, and then drying.

Example 8

(1) The silk fibroin concentrated solution prepared in the step (1) of example 1, having a mass fraction of 25 wt.%, was added to a 5mL syringe, the port of which was connected to a stainless steel needle having a diameter of 0.6 mm. The solution flow rate was controlled by a syringe pump at a rate of 40. mu.L/h. The diameter of the collecting device was 12mm and the rotational speed was 100 rpm. The distance between the needle tip and the collection means was 150 mm. The temperature during the experiment was 25 ℃ and the humidity was 45%. Finally, the dry-process regenerated silk is prepared, and the fiber diameter is 11.6 +/-1.1 mu m.

(2) And (3) placing the prepared regenerated silk by the dry method into an oven with the temperature of 80 ℃, standing for 30min, and then cooling to room temperature.

Example 9

(1) Placing silkworm cocoon in 0.02M Na₂CO₃Boiling in water solution at 60-100 deg.C for 30-180min to degum silkworm cocoon, and drying the obtained glue solution to obtain degummed silk.

The dried degummed silk was then dissolved in 9.3M LiBr aqueous solution, and the completely dissolved solution was then placed in a dialysis bag with a molecular weight cut-off of 3500 daltons (Da), wherein the dialysis solution was deionized water, the dialysis time was 3 days, the dialyzed solution was centrifuged at 9000rpm at normal pressure, the supernatant was aspirated and the precipitate was separated to give an initial concentration of 5.8 wt.% aqueous silk fibroin solution.

The solution was then placed under vacuum for centrifugal concentration to prepare a high-concentration silk fibroin aqueous solution. Wherein the concentration temperature is 45 deg.C, the centrifugal rotation speed is 5000rpm, the vacuum degree is 20mbar, and the concentration time is 15.5 h.

Centrifuging the concentrated solution at 4 deg.C and 9000rpm for 15 min. Finally, a concentrated silk fibroin solution with a mass fraction of 32 wt.% is obtained.

(2) Horseradish Peroxidase (HRP) was added at 1 wt% mass fraction based on silk protein in a 32 wt.% silk protein concentrated solution at room temperature. And fully mixing to obtain the composite spinning solution containing the HRP enzyme for later use.

(3) The same procedure as in step (1) of example 4 was repeated except that the above-mentioned silk protein concentrated solution and the prepared HRP enzyme-containing composite spinning solution were used in a mass fraction of 32 wt.%, respectively, to obtain two kinds of fibers, in which the HRP enzyme was not added to the spinning solution as a control.

(4) Two kinds of fibers having a length of 1cm were taken in a well plate, and then 50. mu.L of deionized water and 50. mu.L of 3,3 ', 5, 5' -Tetramethylbenzidine (TMB) color developing solution were sequentially added. After standing for a period of time, the solution with the HPR enzyme added to the fiber appeared blue, while the control was colorless and transparent. The reaction was then stopped by adding 100. mu.L of 1M HCl solution to the well plate. After the addition of the HCl solution, the solution changed from blue to bright yellow in the sample to which the HPR enzyme was added, whereas the control solution was always colorless and transparent.

From the above results, it can be confirmed that the spinning process of the present application allows HRP enzyme to maintain good biological activity in the fiber. This further reflects the great advantage of the silk protein based fibers of the present application compared to the silk protein based fibers of the prior art in the subsequent preparation of various biocompatible materials, in particular medical materials or medical devices, due to the absence of additional undesired chemicals.

Test example 1

The test method comprises the following steps: the surface structure and the cross-sectional structure of the fiber are observed by a Zeiss high-resolution analysis type field emission scanning electron microscope. Before testing, the fiber sample needs to be subjected to Pt spraying treatment on the surface of the fiber, and the thickness of the fiber sample is 5 nm. The test voltage is 3kV, and the height of the detector from the sample table is about 9 mm. In calculating the fiber diameter, the fiber diameter was counted by taking 5 SEM images of different portions of each sample, and the average value and standard deviation of the fiber diameter were calculated after measuring 20 times for each sample by using Image pro plus software.

And (3) testing results:

FIG. 1 is a scanning electron microscope image of the surface of the dry-process regenerated yarn obtained in step (2) of example 1. As can be seen from the figure, the prepared fiber has smooth surface and uniform diameter. The fiber diameter produced under the conditions of this example was 3.6. + -. 0.3 μm, close to the diameter of natural spider silk.

FIG. 2 is a scanning electron micrograph showing the cross section of the dry-laid regenerated yarn obtained in step (2) of example 2, and it is clear from the results that the fiber cross section is nearly circular.

Test example 2

The test method comprises the following steps: the FT-IR test results were obtained by Fourier transform microscopy infrared spectroscopy. The adopted test mode is ATR, and the instrument resolution is 4cm^-1Each line is superposed by 64 scans, and the spectrum range is 500-4000cm^-1. During the test, each sample was tested 3 times. In the obtained spectrogram, the region to the amide I (1600-^-1) And (5) performing peak-splitting fitting treatment, and quantitatively analyzing the secondary conformation of the silk protein.

And (4) analyzing results: the β -sheet content of each of the fibers obtained in examples 1-8 is shown in table 2 below.

TABLE 2

Example numbering	Beta-sheet content (%)
		1	21.7
2	25.4
		3	22.4
4	19.6
		5	26.8
6	29.7
		7	23.7
8	24.7

FIG. 3 is an infrared spectrum of the degummed silk, the dry-process regenerated silk and the dry-process regenerated silk after being soaked in methanol solution for 120min in example 2. As shown in FIG. 3, when the dry-process regenerated silk is immersed in the methanol solution for 120min, the infrared spectrogram of the dry-process regenerated silk which is not treated with the methanol solution is compared and analyzed, and the fiber treated with the methanol solution in the amide I area has the peak of 1643cm^-1Move to the right to 1622cm^-1Here, it is shown that the methanol solution treatment induced more beta-sheet structure formation in the fibroin structure, and the beta-sheet content increased from untreated 10.6% to 25.4%.

Fig. 5 is an infrared spectrum of the dry-process regenerated yarn of example 4 and its post-drawing treatment under acetic acid vapor (example 5) and ethanol vapor (example 4). As can be seen from FIG. 5, the IR spectra of the dry process regenerated filaments after the treatment with ethanol vapor and acetic acid vapor were compared with those of the untreated regenerated filaments, 1640cm^-1All peaks of the fiber are shifted to the right, wherein the peak of the fiber subjected to the post-drawing treatment under the acetic acid vapor condition is shifted to 1621cm^-1To (3). The condition that the beta-sheet content in the fiber can be improved to a greater extent by carrying out the post-drawing treatment under the condition of acetic acid vapor is shown.

Test example 3

The test method comprises the following steps: the mechanical properties of the fibers were tested and analyzed by using a biometrics testing instrument from Cell Scale. Before mechanical testing, the corresponding diameter of each test sample was measured by optical microscopy. The test conditions were: the clamping distance is 5mm, the stretching speed is 5mm/min, the temperature is 25 +/-3 ℃, and the humidity is 60 +/-5%. Each sample was tested 10 times.

And (4) analyzing results:

FIG. 4 is a tensile stress-strain curve of the degummed and dry-laid recycled filaments of example 3. As can be seen from the figure, the elongation at break of the dry-process regenerated yarn is larger than that of the degummed yarn, and can reach 58%, and the breaking strength is slightly lower than that of the degummed yarn and is 103 MPa.

Jin et al (Y.jin, Y.Zhang, Y.Handg, H.Shao, X.Hu, A simple process for dry spinning of generated silk fibroin aqueous solution, Journal of Materials Research 28(20) (2013)2897-₂Aqueous solution of Ca in the spinning solution²⁺The ion concentration was 0.3M. And then concentrating the spinning solution to 40-60 wt.% for dry spinning to obtain the silk protein-based fiber. The fiber obtained was post-drawn 4 times in 80v/v (%) aqueous ethanol and soaked under these conditions for 3 hours. Compared with the results obtained in Jin et al (10.6% elongation at break and 78.9MPa breaking strength), the spinning solution in the present application is pure silk protein based fiber, and no other metal ions or buffer solution for adjusting pH value are added, so that the prepared fiber shows more excellent breaking strength and elongation at break.

FIG. 6 is a tensile stress-strain curve of the degummed yarn, the dry process regenerated yarn, and the dry process regenerated yarn after 30min treatment with an aqueous methanol solution in example 7. As can be seen from the figure, the breaking strength of the dry-process regenerated silk is similar to that of the degummed silk and can reach 386 MPa. Meanwhile, the dry-process regenerated silk has excellent stretchability, and the elongation at break can reach 700%. After 30min of post-treatment of the dry-method regenerated silk by a methanol aqueous solution, according to FT-IR experimental results, the content of beta-sheet in the fiber is increased, the initial modulus is greatly improved, the elongation at break is reduced, and the change of the breaking strength is small.