阅读说明：本技术 一种利用酵母菌发酵制备多功能水凝胶的方法 (Method for preparing multifunctional hydrogel by yeast fermentation ) 是由 汪少芸 程静 游力军 熊蔡华 蔡茜茜 陈选 于 2021-09-06 设计创作，主要内容包括：本发明涉及一种利用酵母菌发酵制备多功能水凝胶的方法。本发明首先利用聚多巴胺还原氧化石墨烯得到还原氧化石墨烯溶液,然后配制一定浓度的明胶PCA葡萄糖混合溶液以及活化的酵母菌溶液；通过一锅反应法将三者混合,搅拌均匀,倒入模具中,在30℃水浴中发酵一定的时间,即得Gel-PrGO-PCA-酵母多功能水凝胶材料。该方法简单方便、快速高效,所得水凝胶具有良好的透气性、超强的机械性能、导电性、生物相容性,并且可通过贴敷于不同的皮肤位置检测心电和肌电。本发明为制备多孔透气导电水凝胶电极提供一种新的思路及方法,有助于导电水凝胶材料的开发利用,以便其应用在生物传感器领域。(The invention relates to a method for preparing multifunctional hydrogel by yeast fermentation. Firstly, reducing graphene oxide by polydopamine to obtain a reduced graphene oxide solution, and then preparing a gelatin PCA glucose mixed solution with a certain concentration and an activated yeast solution; mixing the three materials by a one-pot reaction method, stirring uniformly, pouring into a mould, and fermenting in a water bath at 30 ℃ for a certain time to obtain the Gel-PrGO-PCA-yeast multifunctional hydrogel material. The method is simple, convenient, rapid and efficient, and the obtained hydrogel has good air permeability, super mechanical property, electric conductivity and biocompatibility, and can be applied to different skin positions to detect electrocardio and myoelectricity. The invention provides a new thought and a new method for preparing the porous breathable conductive hydrogel electrode, and is beneficial to the development and utilization of the conductive hydrogel material so as to be applied to the field of biosensors.)

1. A method for preparing multifunctional hydrogel by yeast fermentation is characterized by comprising the following steps:

(1) activating yeast in hot water of 30 ℃ to obtain yeast liquid;

(2) weighing plate count agar PCA, dissolving in deionized water at 100 deg.C, boiling for 20 min, and cooling to 50 deg.C to obtain PCA solution; adding gelatin Gel into the PCA solution, stirring in a water bath at 50 ℃ for 30 min, then adding glucose, continuously stirring and dissolving to obtain a Gel-PCA-glucose mixed solution, and cooling to 30 ℃;

(3) and (3) uniformly stirring and mixing the yeast liquid obtained in the step (1) and the Gel-PCA-glucose mixed solution obtained in the step (2), pouring the mixture into a mold, fermenting the mixture in a water bath at 30 ℃ for 30 min, and then placing the mixture at 4 ℃ for 10 min to obtain the Gel-PCA-yeast multifunctional hydrogel.

2. The method for preparing multifunctional hydrogel by yeast fermentation according to claim 1, wherein the method comprises the following steps: the concentration of the yeast liquid in the step (1) is 0.2 g/mL-0.45 g/mL.

3. The method for preparing multifunctional hydrogel by yeast fermentation according to claim 1, wherein the method comprises the following steps: the concentration of the PCA solution in the step (2) is 0.0235 g/mL; the addition amount of the gelatin is 5 to 35 weight percent; the addition amount of the glucose is 0.01-0.06 g/mL.

4. The method for preparing multifunctional hydrogel by yeast fermentation according to claim 1, wherein the method comprises the following steps: adding reduced graphene oxide PrGO with the concentration of 1-4 mg/mL into the PCA solution in the step (2); obtaining Gel-PrGO-PCA-glucose mixed solution; and (2) uniformly stirring and mixing the yeast liquid obtained in the step (1) and the Gel-PrGO-PCA-glucose mixed solution, pouring the mixture into a mold, fermenting the mixture in a water bath at 30 ℃ for 30 min, and then placing the mixture at 4 ℃ for 10 min to obtain the Gel-PrGO-PCA-yeast multifunctional hydrogel.

5. The method for preparing multifunctional hydrogel by yeast fermentation according to claim 4, wherein the method comprises the following steps: the preparation method of the reduced graphene oxide comprises the following steps:

(1) preparing graphene oxide: weighing 1.2 g of graphite, adding 50 mL of concentrated sulfuric acid, uniformly stirring, placing into an ice bath, adding 1.5 g of sodium nitrate under a stirring state, slowly adding 6 g of potassium permanganate, stirring overnight at 35 ℃, slowly adding 100 mL of deionized water, controlling the temperature to be 90 ℃, reacting for 1 h, diluting 30 vol% of hydrogen peroxide by 5 times, slowly adding into the solution until no bubbles are generated, stopping adding, continuing to react for 3 h, cooling to room temperature, washing to be neutral, performing ultrasonic dispersion for 20 min, and freeze-drying to obtain graphene oxide powder;

(2) weighing 20 mg of GO powder, adding 2.5 mL of deionized water, and performing ultrasonic treatment until the GO powder is completely dissolved to obtain a GO dispersion liquid; weighing 50 mg of dopamine hydrochloride powder, and dissolving the dopamine hydrochloride powder in 2.5 mL of 10 mM Tris-HCl solution with pH =8.5 to obtain dopamine hydrochloride dispersion liquid; and then adding the dopamine hydrochloride dispersion liquid into the GO dispersion liquid, ultrasonically dispersing for 2 hours in an ice bath, and stirring for 12 hours in a water bath at the temperature of 60 ℃ to obtain a reduced graphene oxide PrGO solution.

6. The method for preparing multifunctional hydrogel by yeast fermentation according to claim 1, wherein the method comprises the following steps: and (4) freezing the Gel-PCA-yeast hydrogel obtained in the step (3) at-80 ℃ for 20 min, cutting into slices with the thickness of 1 mm or cutting into any shape, and soaking in a mixed solution of a salt solution and glycerol for 12 h.

7. The method of claim 6, wherein the method comprises the steps of: the salt solution is ammonium sulfate or sodium citrate solution; the concentration of the salt solution is 10-30 wt%; the volume ratio of the saline solution to the glycerol is 2: 1. 1: 1 or 1: 2.

8. a multifunctional hydrogel prepared by the method of any one of claims 1 to 7.

9. The use of the multifunctional hydrogel of claim 8, wherein: the multifunctional hydrogel is used as a conductive material in a biosensor or in a drug-loaded and antibacterial wound dressing.

Technical Field

The invention belongs to the field of preparation of conductive hydrogel, and particularly relates to a method for preparing multifunctional hydrogel by yeast fermentation.

Background

The hydrogel is a material with a three-dimensional polymer or supermolecule polymer network structure, has good flexibility, and can be pulled, pressed, bent and the like, wherein the conductive hydrogel is prepared by adding a conductive high molecular polymer to carry out physical crosslinking. Their unique properties (e.g., flexibility, high water content, biocompatibility, electrical conductivity, etc.) have prompted their widespread use in various biomedical applications, including the detection of physiological signals in humans, regenerative medicine, nerve repair, etc. However, the conductive hydrogel has poor air permeability, mechanical property and water retention property, which greatly limits the application of the conductive hydrogel in the field of biomedical materials. Therefore, the method has important significance for improving the air permeability, the mechanical property and the water retention of the hydrogel.

The biosensor is an important device for detecting and tracking physiological signals of a human body, and the hydrogel is a novel biosensor. The hydrogel has higher comfort, shape controllability and sensitivity for detecting and tracking electrocardio, myoelectricity, electroencephalogram and nerve signals of a human body, most of the existing hydrogels have the problems of air impermeability, poor mechanical strength and the like, so the porous air-permeable high-strength conductive hydrogel prepared by the method has broad development and application prospects. And the preparation of the conductive hydrogel reported at present contains a synthetic polymer which is incompatible with biology, a toxic cross-linking agent and a complex operation flow, so that the development of a simple, rapid, safe, efficient and breathable method has important significance.

Disclosure of Invention

The invention aims to provide a method for preparing multifunctional hydrogel by yeast fermentation aiming at the defects of the research in the field. The method is simple, rapid and efficient to operate, and the obtained hydrogel has good air permeability, water retention, flexibility and electrical conductivity.

In order to realize the purpose, the following technical scheme is adopted:

a method for preparing multifunctional hydrogel by yeast fermentation comprises the following steps:

(1) activating yeast in hot water of 30 ℃ to obtain yeast liquid;

(2) weighing Plate Count Agar (PCA), dissolving in deionized water at 100 deg.C, boiling for 20 min, and cooling to 50 deg.C to obtain PCA solution; adding gelatin (Gel) into the PCA solution, stirring in 50 deg.C water bath for 30 min, adding glucose, stirring for dissolving to obtain Gel-PCA-glucose mixed solution, and cooling to 30 deg.C;

(3) and (3) uniformly stirring and mixing the yeast liquid obtained in the step (1) and the Gel-PCA-glucose mixed solution obtained in the step (2), pouring the mixture into a mold, fermenting the mixture in a water bath at 30 ℃ for 30 min, and then placing the mixture at 4 ℃ for 10 min to obtain the Gel-PCA-yeast multifunctional hydrogel.

The concentration of the yeast liquid in the step (1) is 0.2 g/mL-0.45 g/mL.

The concentration of the PCA solution in the step (2) is 0.0235 g/mL; the addition amount of the gelatin is 5 to 35 weight percent; the addition amount of the glucose is 0.01-0.06 g/mL.

Further, in the step (2), reduced graphene oxide (PrGO) with the concentration of 1-4 mg/mL is added into the PCA solution; obtaining Gel-PrGO-PCA-glucose mixed solution; and (2) uniformly stirring and mixing the yeast liquid obtained in the step (1) and the Gel-PrGO-PCA-glucose mixed solution, pouring the mixture into a mold, fermenting the mixture in a water bath at 30 ℃ for 30 min, and then placing the mixture at 4 ℃ for 10 min to obtain the Gel-PrGO-PCA-yeast multifunctional hydrogel.

Further, the preparation method of the reduced graphene oxide (PrGO) comprises the following steps:

(1) preparation of Graphene Oxide (GO): weighing 1.2 g of graphite, adding 50 mL of concentrated sulfuric acid, uniformly stirring, placing into an ice bath, adding 1.5 g of sodium nitrate under a stirring state, slowly adding 6 g of potassium permanganate, stirring overnight at 35 ℃, slowly adding 100 mL of deionized water, controlling the temperature to be 90 ℃, reacting for 1 h, diluting 30 vol% of hydrogen peroxide by 5 times, slowly adding into the solution until no bubbles are generated, stopping adding, continuing to react for 3 h, cooling to room temperature, washing to be neutral, performing ultrasonic dispersion for 20 min, and freeze-drying to obtain Graphene Oxide (GO) powder;

(2) weighing 20 mg of GO powder, adding 2.5 mL of deionized water, and performing ultrasonic treatment until the GO powder is completely dissolved to obtain a GO dispersion liquid; weighing 50 mg dopamine hydrochloride (DA) powder, and dissolving in 2.5 mL 10 mM Tris-HCl solution (pH = 8.5) to obtain dopamine hydrochloride dispersion; and then adding the dopamine hydrochloride dispersion liquid into the GO dispersion liquid, ultrasonically dispersing for 2 hours in an ice bath, and stirring for 12 hours in a water bath at the temperature of 60 ℃ to obtain a reduced graphene oxide PrGO solution.

Further, the Gel-PCA-yeast hydrogel obtained in the step (3) is frozen at-80 ℃ for 20 min, cut into slices with the thickness of 1 mm or cut into any shape, and soaked in a mixed solution of a salt solution and glycerol for 12 h. The salt solution is ammonium sulfate or sodium citrate solution; the concentration of the salt solution is 10-30 wt%; the volume ratio of the saline solution to the glycerol is 2: 1. 1: 1 or 1: 2.

the multifunctional hydrogel prepared by any one of the methods above.

The multifunctional hydrogel is used as a conductive material in a biosensor or in a drug-loaded and antibacterial wound dressing.

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

(1) the invention is prepared by mixing gelatin, reduced graphene oxide, PCA, yeast and glucose and then fermenting, and then soaking the mixture in a salt solution or a mixed solution of the salt solution and glycerol.

(2) The multifunctional hydrogel prepared by yeast fermentation has multiple functions, wherein the yeast endows the multifunctional hydrogel with porous air permeability, and the gelatin, PrGO and salt solution endow the multifunctional hydrogel with conductivity and mechanical properties.

(3) The hydrogel has fatigue resistance and super-strong tensile property, the tensile property can reach 1000%, and the hydrogel can sensitively detect electrocardio and myoelectric signals, and is expected to be applied to the fields of biosensors or wearable equipment and the like.

Drawings

Figure 1 effect of adding different amounts of gelatin on pore size.

Figure 2 hydrogel topography. a, a topography of hydrogel without yeast added under a common camera; b, a hydrogel topography map without yeast under an optical microscope; c, adding a shape graph of hydrogel of yeast under a common camera; and d, adding a hydrogel topography of the yeast under an optical microscope.

FIG. 3 tensile stress-strain curves of Gel-PCA-Yeast-ammonium sulfate hydrogel and Gel-PCA-Yeast-sodium citrate hydrogel after soaking in different salt solutions.

FIG. 4 is a graph of Young's modulus.

FIG. 5 is a graph of morphology, conductivity, and detection of electrocardiographic and myoelectrical signals for Gel-PrGO-PCA-yeast hydrogels. a, the shape of Gel-PrGO-PCA-yeast hydrogel; b, detecting the conductivity; c, detecting electrocardiosignals; and d, detecting the electromyographic signals.

FIG. 6 Water retention Performance of Gel-PCA-Yeast and Gel-PCA-Yeast-ammonium sulfate-Glycerol hydrogels.

Figure 7 rheological properties of Gel-PCA-yeast and Gel-PCA-yeast-ammonium sulfate-glycerol hydrogels.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be illustrative only and not limiting.

Example 1

Weighing 7 parts of 0.235g Plate Count Agar (PCA), respectively dissolving in 9 mL of deionized water at 100 ℃, boiling for 20 min, cooling to 50 ℃, respectively adding 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt% and 35 wt% gelatin (Gel), stirring in a water bath at 50 ℃ for 30 min, then adding 0.15 g glucose, continuously stirring and dissolving to obtain a mixed solution of Gel-PCA and glucose, and cooling to 30 ℃; weighing 0.45 g of yeast powder, dissolving in 1 mL of 30 ℃ deionized water, then uniformly stirring and mixing yeast liquid and the mixed solution of the Gel-PCA-glucose, pouring into a mold, placing in a water bath at 30 ℃ for fermentation for 30 min, and then placing the mold at 4 ℃ for 10 min to obtain the Gel-PCA-yeast hydrogel. And finally, freezing the mixture at the temperature of minus 80 ℃ for 20 min, cutting the mixture into slices with the thickness of 1 mm or cutting the slices into any shape, soaking the slices in a mixed solution of 20 wt% of ammonium sulfate and glycerol (the volume ratio is 1: 1) for 12 h, and then detecting the slices. As shown in FIG. 1, the pore size of the obtained Gel-PCA-yeast hydrogel first decreased and then increased with the increase of the gelatin content.

Example 2

Dissolving 0.235g of PCA in 9 mL of 100 ℃ deionized water, boiling for 20 min, cooling to 50 ℃, adding 20 wt% of gelatin, stirring in a 50 ℃ water bath for 30 min, then adding 0.15 g of glucose, continuously stirring and dissolving to obtain a mixed solution of Gel-PCA and glucose, and cooling to 30 ℃; weighing 0.45 g of yeast powder, dissolving in 1 mL of 30 ℃ deionized water (no yeast is added in a control group), then uniformly stirring and mixing yeast liquid and the mixed solution of the Gel-PCA-glucose, pouring into a mold, placing in a water bath at 30 ℃ for fermentation for 30 min, and then placing at 4 ℃ for 10 min to obtain the Gel-PCA-yeast hydrogel. Finally, the product is frozen at-80 ℃ for 20 min and then cut into slices with the thickness of 1 mm or cut into any shape for detection. As shown in FIG. 2, the obtained Gel-PCA-yeast hydrogel has a better network porous structure.

Example 3

Dissolving 0.235g of PCA in 9 mL of 100 ℃ deionized water, boiling for 20 min, cooling to 50 ℃, adding 20 wt% of gelatin, stirring in a 50 ℃ water bath for 30 min, then adding 0.15 g of glucose, continuously stirring and dissolving to obtain a mixed solution of Gel-PCA and glucose, and cooling to 40 ℃; weighing 0.45 g of yeast powder and dissolving in 1 mL of 30 ℃ deionized water; and then uniformly stirring and mixing the yeast liquid and the mixed solution of the Gel-PCA and the glucose, pouring the mixture into a mold, placing the mold in a water bath at 30 ℃ for fermentation for 30 min, and then placing the mold at 4 ℃ for 10 min to obtain the Gel-PCA-yeast hydrogel. After cutting into a desired shape, it was immersed in 10 wt%, 20 wt%, and 30 wt% ammonium sulfate or sodium citrate salt solutions for 12 hours, respectively, and then tested for tensile properties. As shown in fig. 3, Gel-PCA-yeast-ammonium sulfate hydrogel and Gel-PCA-yeast-sodium citrate hydrogel obtained by soaking group with 20% salt solution have super-strong tensile properties, wherein the maximum tensile strain of the Gel-PCA-yeast-sodium citrate hydrogel reaches 1000%, and the maximum tensile stress is 0.28 MPa; the Gel-PCA-yeast-ammonium sulfate hydrogel has a maximum tensile strain of 850% and a maximum tensile stress of 0.14 MPa, and although the tensile properties of the sodium citrate group are greater than those of the ammonium sulfate group, the Young's modulus of the ammonium sulfate group is less than that of the sodium citrate group (FIG. 4), indicating that the flexibility or elasticity is better. The mechanical property of the hydrogel soaked in the 10% salt solution is poor, and the tensile property is less than 100%.

Example 4

A method for preparing multifunctional hydrogel by yeast fermentation comprises the following steps:

(1) preparation of Graphene Oxide (GO): weighing 1.2 g of graphite, adding 50 mL of concentrated sulfuric acid, uniformly stirring, placing into an ice bath, adding 1.5 g of sodium nitrate under a stirring state, slowly adding 6 g of potassium permanganate, stirring overnight at 35 ℃, slowly adding 100 mL of deionized water, controlling the temperature to be 90 ℃, reacting for 1 h, diluting 30 vol% hydrogen peroxide by 5 times, slowly adding into the solution until no bubbles are generated, stopping adding, continuing to react for 3 h, cooling to room temperature, washing to be neutral, performing ultrasonic dispersion for 20 min, and freeze-drying to obtain GO powder.

(2) Weighing 20 mg of GO powder, adding 2.5 mL of deionized water, and performing ultrasonic treatment until the GO powder is completely dissolved to obtain a GO dispersion liquid; weighing 50 mg dopamine hydrochloride (DA) powder, and dissolving in 2.5 mL 10 mM Tris-HCl solution (pH = 8.5) to obtain dopamine hydrochloride dispersion; and then adding the dopamine hydrochloride dispersion liquid into the GO dispersion liquid, ultrasonically dispersing for 2 hours in an ice bath, and stirring for 12 hours in a water bath at the temperature of 60 ℃ to obtain a reduced graphene oxide PrGO solution.

(3) Dissolving 0.235g of PCA in 4 mL of 100 ℃ deionized water, boiling for 20 min, cooling to 50 ℃, uniformly mixing with the PrGO solution prepared in the step (2), adding 20 wt% of gelatin, stirring in a water bath at 50 ℃ for 30 min, then adding 0.15 g of glucose, continuously stirring and dissolving to obtain a Gel-PrGO-PCA-glucose mixed solution, and cooling to 30 ℃. 0.45 g of yeast powder is weighed and dissolved in 1 mL of 30 ℃ deionized water to obtain yeast liquid. And then uniformly stirring and mixing the yeast liquid and the mixed solution of the Gel-PrGO-PCA-glucose, pouring the mixture into a mold, placing the mold in a water bath at 30 ℃ for fermentation for 30 min, and then placing the mold at 4 ℃ for 10 min to obtain the Gel-PrGO-PCA-yeast hydrogel. As shown in figure 5, the obtained Gel-PrGO-PCA-yeast hydrogel has good conductivity which is 0.015S/m, has a good network porous structure, and can well detect electrocardio and myo-electric signals.

Example 5

Dissolving 0.235g PCA in 9 mL of deionized water at 100 ℃, boiling for 20 min, cooling to 50 ℃, adding 20 wt% gelatin, stirring in a water bath at 50 ℃ for 30 min, then adding 0.15 g glucose, continuously stirring and dissolving to obtain a mixed solution of Gel-PCA and glucose, and cooling to 30 ℃. 0.45 g of yeast powder was weighed and dissolved in 1 mL of deionized water at 30 ℃. And then uniformly stirring and mixing the yeast liquid and the mixed solution of the Gel-PCA and the glucose, pouring the mixture into a mold, placing the mold in a water bath at 30 ℃ for fermentation for 30 min, and then placing the mold at 4 ℃ for 10 min to obtain the Gel-PCA-yeast hydrogel. Finally, the sample was immersed in a mixed solution of 20 wt% ammonium sulfate and glycerol (1: 2, 1: 1, 2: 1 (v/v)) for 12 hours, and then examined. As shown in fig. 6 and 7, the obtained Gel-PCA-yeast-ammonium sulfate-glycerol hydrogel has good water retention and rheological properties, and the ratio of the soaked ammonium sulfate to glycerol is 1: 1 and 1: 2 hours, the mass was almost unchanged after three days at room temperature, indicating less water loss. The rheological detection result shows that the ratio of the soaked ammonium sulfate to the glycerol is 1: the storage modulus of the hydrogel is increased from 100 Pa to 1000 Pa at 1, which is 2 orders of magnitude higher than that of the non-soaked group, and the strength of the hydrogel after soaking is higher than that of the non-soaked group.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.