CN114736332B

CN114736332B - Preparation method and application of enzymatic biogenic salt gel

Info

Publication number: CN114736332B
Application number: CN202210314932.8A
Authority: CN
Inventors: 丁凤霞; 尚英辉; 王启刚
Original assignee: Tongji University
Current assignee: Tongji University
Priority date: 2022-03-28
Filing date: 2022-03-28
Publication date: 2023-07-04
Anticipated expiration: 2042-03-28
Also published as: CN114736332A

Abstract

The invention relates to an enzymatic biogenic salt gel and a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) Mixing biological salt and water to prepare biological salt solutions with different concentrations; (2) Modifying the chondroitin sulfate to prepare acrylated modified chondroitin sulfate with double bonds as a cross-linking agent; (3) Mixing the cross-linking agents prepared in the step (1) and the step (2), adding a monomer and a cascading enzyme initiation system to prepare uniform precursor liquid, and forming gel by the obtained gel precursor liquid to prepare the biological salt gel. Compared with the prior art, the preparation process is mild and efficient, the material is environment-friendly, and the prepared biological salt gel has enzymatic electron transfer and ion transfer capabilities and can be used for glucose sensing.

Description

Preparation method and application of enzymatic biogenic salt gel

Technical Field

The invention relates to the technical field of gel materials, in particular to an enzymatic biogenic salt gel and a preparation method and application thereof.

Background

Bioelectronics is a rapidly growing and popular interdisciplinary, and its research purpose is mainly to identify, transmit and convert biological signals into electronic signals that can be identified by man-made machines. In recent years, wearable bioelectronics have played an important role in human health detection, diagnosis and protection. For example, the method can be applied to the fields of human motion sensing, biological molecule detection, skin tissue protection, flexible self-energy supply, minimally invasive glucose monitoring and the like. In particular, in the field of glucose detection, the global prevalence of diabetes and impaired glucose tolerance has increased significantly over the last decades, and thus it is of great importance to study glucose sensors that can continuously detect blood glucose levels.

The difficulty in the research of the bioelectronics at present is the incompatibility problem of the interfaces of the biological tissues and the artificial devices. It is well known that in nature biological electrical signal transmission is conducted mainly by ions, while artificial electronic materials are conducted mainly by electrons. Biological tissue is mostly soft and moist material, whereas artificial electronic materials are mainly hard and inflexible materials. Thus, the substantial difference between the two renders the interface problem difficult to solve, thereby restricting the development of the whole bioelectronics.

The gel material is a biological material with soft and wet texture, has stable chemical property, and particularly has better biocompatibility when being doped with the biological material. The gel also has a three-dimensional network structure similar to a biological tissue structure, has a through network channel, and can provide guarantee for material transmission. In addition, the gel has good stretching, compressing and adhering properties, and can simulate the flexibility of a similar biological tissue structure. Therefore, the gel can be used as a bridge material for communicating biological tissues and artificial electronic devices, and is an excellent bioelectronic material. In addition, in order to achieve detection of biological signals, components must be introduced into the system to achieve identification and transmission of biological signals. The enzyme is a specific biocatalyst, can catalyze biological reaction in organisms, and can gently and efficiently initiate free radical polymerization to realize gelation in material synthesis. The method of adopting enzyme to promote the gel not only can realize the gentle construction of a three-dimensional network structure, but also can realize the immobilization of the enzyme in situ so as to be used for detecting biological signals. The dual enzyme mediated priming system has the advantage of being more efficient and rapid.

Conventional conductive hydrogels require the addition of high concentrations of electrolyte solutions, such as inorganic salts, strong acids or bases, that induce irreversible denaturation of proteins and enzymes in the living body in order to achieve ionic conduction. In addition, hydrogels inevitably freeze below zero and dry and harden due to moisture evaporation, losing flexibility and ionic conductivity. These problems present new challenges for the synthesis of materials.

Patent CN112210041a discloses a hydrated ionic liquid gel, and a preparation method and application thereof, the hydrated ionic liquid gel is prepared by the following method: when the enzyme-driven gel is prepared, the hydrated ionic liquid is used as a solvent, the hydrated ionic liquid contains organic matters of anions and cations, the hydrated ionic liquid follows the Huffman ion effect, can be used as a protective agent to stabilize a hydration layer of the enzyme, and has good biocompatibility. However, the ionic liquid used by the hydrated ionic liquid gel is complex in preparation and purification, high in price and not suitable for large-scale production. In addition, the hydrated ion liquid belongs to exogenous molecules, is easy to trigger tissue rejection reaction, and cannot be applied to an implantable sensing device. The biological salt related by the invention is a biological metabolite, belongs to endogenous substances, and has excellent biocompatibility. And the material is cheap and easy to obtain, and is suitable for mass production.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide an enzymatic biogenic salt gel as well as a preparation method and application thereof.

The aim of the invention is achieved by the following technical scheme:

a method for preparing an enzymatic biogenic gel, comprising the steps of:

(1) Mixing biological salt with water in different proportions to prepare uniform biological salt solution;

(2) Grafting and modifying the chondroitin sulfate to prepare an acrylic chondroitin sulfate biological crosslinking agent;

(3) Adding the cross-linking agent obtained in the step (2), a monomer and an enzyme initiation system into the biological salt solution obtained in the step (1), stirring until the solution is completely dissolved into a uniform gel precursor solution, and performing enzymatic initiation to form gel to prepare the biological salt gel with the in-situ loaded enzyme.

And (3) preparing the glucose sensor by using the composite electrode material on the basis of the biological salt gel prepared in the step (3).

Preferably, in step (1): the gelling solvent is a biological salt solution, the biological salt is D-fructose-1, 6-trisodium diphosphate, and the addition concentration of the biological salt is not higher than 0.8g/ml.

Preferably, in step (2): the cross-linking agent is a modified biological cross-linking agent, the modified cross-linking agent is an acrylic chondroitin sulfate cross-linking agent, and the adding amount of the cross-linking agent is 4-12mg/ml.

Preferably, in step (3): the monomer is an acrylamide monomer, and the addition amount of the monomer is 20-30% of the total weight of the solvent.

Preferably, the monomer is one or more of acrylamide (AAm), methacrylamide (MMAA), N-methylolacrylamide (N-MMAA).

Preferably, in step (3): the enzyme priming system is a double-enzyme mediated cascade enzyme system, comprising glucose oxidase, glucose, horseradish peroxidase and acetylacetone.

Preferably, the addition amount of the glucose oxidase is 1.0mg/ml, the addition amount of glucose is 0.36mg/ml, the addition amount of horseradish peroxidase is 0.2mg/ml, and the addition amount of acetylacetone is 10 μl/ml.

Preferably, the enzymatic initiation of the gel forming mode in the step (3) is as follows: incubating at 37deg.C for 30min.

An application of an enzymatic biogenic salt gel composite electrode material in a glucose sensor.

The process conditions in the present invention are the most suitable preparation conditions. The solvent is a biological salt liquid to conduct ions and protect the enzyme protein from denaturation and provide additional intermolecular interactions, improving the mechanical and freeze resistance properties of the gel. The addition of the biogenic salt affects the ionic conductivity of the final gel, the activity of the enzyme and the water retention of the gel, too low a biogenic salt content results in a low ionic conductivity of the gel, while too high a biogenic salt content results in a reduced ionic conductivity and in a poor mechanical property of the gel. The amount of enzyme added will affect the gel formation time and the enzyme concentration on the surface of the final biogenic salt gel, the higher the enzyme concentration the shorter the gel formation time and the higher the enzyme concentration on the surface of the final gel. The biological crosslinking agent, namely the acrylic chondroitin sulfate, is a modified polysaccharide, has a folding structure and can form various hydrogen bonds with a solvent and a polymer in the gel so as to enhance mechanical properties. The addition of monomers and crosslinking agents can affect the properties of the final gel, especially its mechanical properties, such as too low a level of monomers or crosslinking agents can reduce the mechanical strength of the gel and even lead to non-gelling.

According to the method, the chondroitin sulfate is grafted and modified to prepare the acrylic chondroitin sulfate serving as the cross-linking agent, so that the problem that the conventional gel material is weak in stretching capacity is solved. The chondroitin sulfate is a natural polysaccharide, the modified chondroitin sulfate maintains good biocompatibility, double bonds are introduced to form covalent crosslinking points, the acrylic chondroitin sulfate serves as functional crosslinking points, covalent crosslinking points can be provided, and a large number of non-covalent crosslinking points are provided through molecular folding action and hydrogen bonds of the acrylic chondroitin sulfate, so that a gel network can buffer larger external force, and the acrylic chondroitin sulfate has excellent mechanical properties.

The invention solves the difficulty of enzyme denaturation in the traditional gel high-concentration acid, alkali and salt solvents by introducing the biological salt solvents, and realizes the combination of ionic conduction and enzymatic electron transfer. The adopted organic biological salt D-fructose-1, 6-trisodium diphosphate (FDP) not only can protect a hydration layer of enzyme, so that the hydration layer can keep high activity to realize long-term stable enzymatic electron transfer, but also can provide free moving ions to realize ion conduction. In addition, the biological salt has good biocompatibility, is a cell metabolite existing in human body, is an important intracellular glycolysis intermediate product, and can regulate the activities of various enzyme systems in glucose metabolism. The gel prepared by the biological salt solution system can well ensure the activity and stability of enzyme so as to be used for glucose sensing application.

Compared with the traditional hydrogel, the biological salt gel can retain water for a long time, does not crystallize in a low-temperature environment, and can reduce the volatilization of water at a high temperature, so that the activity of enzyme can be maintained in the storage process at a low temperature and a high temperature for a long time.

According to the invention, the gel is formed through an enzymatic initiation system, on one hand, the enzyme is used for preparing a material by initiating the gel in the early stage, and on the other hand, the enzyme is loaded in the gel in situ for the later-stage glucose sensing application, so that the synthesis of the gel, the loading of the enzyme and the subsequent enzymatic application are realized by a one-step method, and the method is simple and efficient.

The invention relates to a novel preparation method of enzymatic biogenic salt gel, which is simple, convenient, efficient, green and environment-friendly. Compared with the defect that enzyme denaturation is caused by most ion conductive gels, the invention can preserve more than eighty percent of enzyme activity, thereby realizing the organic combination of ion conduction and enzymatic electron conduction of the gel and having wide application prospect. The enzymatic biogenic gel can be used as a composite electrode material for a glucose sensor and realizes wearable detection.

Compared with the prior art, the invention has the following beneficial effects:

(1) Compared with the traditional gel material, the biological salt gel prepared by the invention adopts the biological salt solution as a solvent, adopts organic ion conduction, and has more biocompatibility.

(2) The biological salt solution system adopted by the invention can protect the hydration layer around the protein, and can be used as a biological protective agent to maintain the flexible structure of the protein, so that the protein has biological catalytic activity.

(3) Compared with aqueous solution, the ionic conductivity of the biological salt gel is obviously improved, and the ionic conductivity of the biological salt gel prepared by the method is higher than that of the traditional hydrogel.

(4) The biological salt solution can effectively reduce saturated vapor pressure of the whole system, can form a large number of hydrogen bonds with water molecules, and has a good protective effect on the water molecules, so that the biological salt gel prepared by the invention is not easy to volatilize, can be stored for a long time, has freezing resistance, and can still work normally below the freezing point.

(5) The biological salt solution and the polymer chains in the gel network generate a plurality of weak interactions, and can absorb energy when external force acts, thereby playing a role of buffering. Therefore, the biogenic salt gel has more excellent comprehensive performance.

(6) Different from traditional photoinitiation and thermal initiation gel forming, the biological salt gel prepared by the invention adopts a milder enzyme-driven gel method, can load zymogen sites on a gel network during polymerization, and has milder and more efficient double-enzyme-mediated cascade enzyme system.

(7) Compared with the traditional paper glucose test paper, the enzymatic bioglass gel prepared by the invention can be recycled and can be stored at room temperature for a long time and still be normally used.

(8) The preparation method is simple and convenient to operate, mild in condition and environment-friendly.

Drawings

FIG. 1 is a graph showing the tensile properties and 3D printability of the enzymatic biogenic gel of example 1;

FIG. 2 is an electron paramagnetic resonance spectrum of the enzymatic biogenic gel initiation system of example 1;

FIG. 3 is a dynamic time-scanning experimental plot of the enzymatic biogenic gel of example 1;

FIG. 4 is a dynamic frequency sweep experimental plot of the enzymatic biogenic gel of example 1;

FIG. 5 is a graph showing the compression curve of the enzymatic biogenic gel of example 1;

FIG. 6 is a graph of compressive modulus and compressive strength of the enzymatic biogenic gel of example 1;

FIG. 7 is a plot of a tensile test of the enzymatic biogenic gel of example 1;

FIG. 8 is a plot of tensile modulus and elongation at break for the enzymatic biogenic gel of example 1;

FIG. 9 is a graph of a puncture experiment of an enzymatic biogenic gel of example 1;

FIG. 10 is a graph of a peel test of the enzymatic biogenic gel of example 1;

FIG. 11 is a Lineweaver-B μrk plot of glucose oxidase and free glucose oxidase loaded in an enzymatic bioglass gel of example 1;

FIG. 12 is a graph of the long-term use activity of glucose oxidase supported in the enzymatic biogasite gel of example 1;

FIG. 13 is an ion conductance graph of the enzymatic biogenic gel of example 1 at various biogenic salt concentrations;

FIG. 14 is an ionic conductance diagram of the enzymatic biogenic gel of example 1 at various temperatures;

FIG. 15 is a physical diagram of a glucose sensor prepared based on the enzymatic bioglass gel of example 1;

FIG. 16 is a CV plot of the enzymatic bioglass gel of example 1 as a glucose sensor for glucose concentration detection;

FIG. 17 is a graph of glucose concentration versus current magnitude for the enzymatic bioglass gel of example 1 as applied to a glucose sensor;

FIG. 18 is an anti-interference graph of the enzymatic bioglass gel of example 1 as applied to a glucose sensor.

Detailed Description

The invention will now be described in detail with reference to the drawings and specific examples.

Example 1

300mg of acrylamide (AAm), 10mg of double bond Chondroitin Sulfate (CSMA), 10. Mu.l of horseradish peroxidase (20 mg/ml), 10. Mu.l of acetylacetone, 50. Mu.l of glucose (40 mM) were added to 830. Mu.l of a biological salt solution having a concentration of 0.4g/ml, and stirred uniformly until the solution became homogeneous and transparent. Finally, 100. Mu.L of glucose oxidase solution (10 mg/mL) was added to obtain a precursor solution. And (3) placing the precursor solution in a constant-temperature water bath, and incubating at 37 ℃ for 30min to obtain the enzymatic biogenic gel. The gel forming time is less than 800s; the compression strength is 1.63MPa, the compression modulus is 46.61kPa, and the deformation of the steel can be resisted by 90 percent; tensile modulus 46.11kPa and maximum tensile deformation 2211.85%; piercing energy of 7.21MJm ^-3 Can bear the penetration deformation of 6000% of the thickness of the plastic; adhesion to carbon cloth of 100Nm ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Can bear low temperature of minus 20 ℃ and ion conductivity of 18.42mScm under room temperature condition ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Can preserve 88% of the enzyme activity; the sensor has good sensitivity, stability and anti-interference capability when used for glucose sensing.

As shown in FIG. 1, the enzymatic biogenic gel of the embodiment has good mechanical properties, can be stretched in all directions, has a moldable property, can print complex patterns in 3D, and can support shapes well.

FIG. 2 is an electron paramagnetic resonance image of the enzyme initiation system of a biogenic salt gel, which is a distinct characteristic peak of carbon radicals, illustrating that the entire enzymatic initiation process is successful and efficient.

Fig. 3 is a flow chart of the gelling process of an enzymatic biogenic gel with a gelling time of less than 800s and a good mechanical stability after gelling of the biogenic gel as can be seen from the dynamic frequency sweep of fig. 4.

In addition, the biogenic gel has excellent compression performance, as shown in fig. 5, which can be compressed to 90% of its own volume and restored to its original shape, as shown in fig. 6, the biogenic gel has a compression strength of 1.63MPa and a compression modulus of 46.61kPa.

The biogenic salt gel of this example has excellent tensile properties, and fig. 7 shows the tensile properties of the biogenic salt gel, which has a maximum elongation of 2211.85% and a tensile modulus of 46.11kPa, and a higher tensile length than conventional hydrogels, as shown in fig. 8. FIG. 9 is a puncture test of a biological salt gel, intended to measure the omnibearing flexibility and puncture resistance of the gel, the puncture energy of the sample of this example being 7.21MJm ^-3 Can bear the penetration deformation of 6000% of the thickness of the hydrogel, and is far higher than the traditional hydrogel.

FIG. 10 is a graph showing the adhesion performance test of biogenic salt gel, the peeling performance test using hydrophobic carbon cloth and gel, the adhesion force of which is 100Nm ^-1 Has stronger adhesion performance. FIG. 11 shows the activity of glucose oxidase carried in a biogel salt gel, and the results show that the activity of glucose oxidase carried in the gel can still be kept 88% compared with the activity of free enzyme, and FIG. 12 shows the activity of enzyme stored for a long time in the sample of this example, and the activity of enzyme carried in the biogel salt gel can be kept above 60% after 30 days of storage at room temperature.

FIG. 13 shows ionic conductivity of biogenic salt gels up to 18.42mScm ^-1 . Fig. 14 shows ion conductivity at various temperatures of the biogenic salt gel, which can withstand low temperatures of-20 c. FIG. 15 is a schematic representation of a glucose sensor prepared on the basis of this example, which can be realized by glucose oxidase supported on the surfaceSensing of glucose. FIG. 16 is a CV diagram of glucose sensor for detecting glucose prepared by the present example, which demonstrates that the signal output is stable and reliable when the present example is used for glucose sensing. Fig. 17 is a graph showing the correspondence between the glucose concentration and the current signal when the present example was used for glucose measurement, and it can be seen that the present example has good detection sensitivity. FIG. 18 is an anti-interference graph of the present example for glucose test, which demonstrates excellent anti-interference performance.

Example 2

300mg of acrylamide (AAm), 10mg of double bond Chondroitin Sulfate (CSMA), 10. Mu.L of horseradish peroxidase (20 mg/mL), 10. Mu.L of acetylacetone, 50. Mu.L of glucose (40 mM) were added to 830. Mu.L of a biological salt solution having a concentration of 0.2g/mL, and stirred uniformly until the solution became homogeneous and transparent. Finally, 100. Mu.L of glucose oxidase solution (10 mg/mL) was added to obtain a precursor solution. And (3) placing the precursor solution in a constant-temperature water bath, and incubating at 37 ℃ for 30min to obtain the enzymatic biogenic gel.

Example 3

300mg of acrylamide (AAm), 10mg of double bond Chondroitin Sulfate (CSMA), 10. Mu.L of horseradish peroxidase (20 mg/mL), 10. Mu.L of acetylacetone, 50. Mu.L of glucose (40 mM) were added to 830. Mu.L of a biological salt solution having a concentration of 0.6g/mL, and stirred uniformly until the solution became homogeneous and transparent. Finally, 100. Mu.L of glucose oxidase solution (10 mg/mL) was added to obtain a precursor solution. And (3) placing the precursor solution in a constant-temperature water bath, and incubating at 37 ℃ for 30min to obtain the enzymatic biogenic gel.

Example 4

300mg of acrylamide (AAm), 10mg of double bond Chondroitin Sulfate (CSMA), 10. Mu.L of horseradish peroxidase (20 mg/mL), 10. Mu.L of acetylacetone, 50. Mu.L of glucose (40 mM) were added to 830. Mu.L of a biological salt solution having a concentration of 0.8g/mL, and stirred uniformly until the solution became homogeneous and transparent. Finally, 100. Mu.L of glucose oxidase solution (10 mg/mL) was added to obtain a precursor solution. And (3) placing the precursor solution in a constant-temperature water bath, and incubating at 37 ℃ for 30min to obtain the enzymatic biogenic gel.

Example 5

300Mg of Methacrylamide (MMAA), 10mg of double bond Chondroitin Sulfate (CSMA), 10. Mu.L of horseradish peroxidase (20 mg/mL), 10. Mu.L of acetylacetone and 50. Mu.L of glucose (40 mM) were added to 830. Mu.L of a biological salt solution with a concentration of 0.4g/mL, and stirred uniformly until the solution became homogeneous and transparent. Finally, 100. Mu.L of glucose oxidase solution (10 mg/mL) was added to obtain a precursor solution. And (3) placing the precursor solution in a constant-temperature water bath, and incubating at 37 ℃ for 30min to obtain the enzymatic biogenic gel.

Example 6

300 mgN-methylolacrylamide (N-MMAA), 10mg double bond Chondroitin Sulfate (CSMA), 10. Mu.L horseradish peroxidase (20 mg/mL), 10. Mu.L acetylacetone, 50. Mu.L glucose (40 mM) were added to 830. Mu.L of a biological salt solution at a concentration of 0.4g/mL, and stirred uniformly until the solution became homogeneous and transparent. Finally, 100. Mu.L of glucose oxidase solution (10 mg/mL) was added to obtain a precursor solution. And (3) placing the precursor solution in a constant-temperature water bath, and incubating at 37 ℃ for 30min to obtain the enzymatic biogenic gel.

Example 7

300mg of acrylamide (AAm), 0.5mg of N, N' -Methylenebisacrylamide (MBAA), 10. Mu.L of horseradish peroxidase (20 mg/mL), 10. Mu.L of acetylacetone, 50. Mu.L of glucose (40 mM) were added to 830. Mu.L of a biological salt solution having a concentration of 0.4g/mL, and stirred uniformly until the solution became homogeneous and transparent. Finally, 100. Mu.L of glucose oxidase solution (10 mg/mL) was added to obtain a precursor solution. And (3) placing the precursor solution in a constant-temperature water bath, and incubating at 37 ℃ for 30min to obtain the enzymatic biogenic gel.

Example 8

300Mg of Methacrylamide (MMAA), 0.5mg of N, N' -Methylenebisacrylamide (MBAA), 10. Mu.L of horseradish peroxidase (20 mg/mL), 10. Mu.L of acetylacetone, 50. Mu.L of glucose (40 mM) were added to 830. Mu.L of a biological salt solution with a concentration of 0.4g/mL, and stirred uniformly until the solution became homogeneous and transparent. Finally, 100. Mu.L of glucose oxidase solution (10 mg/mL) was added to obtain a precursor solution. And (3) placing the precursor solution in a constant-temperature water bath, and incubating at 37 ℃ for 30min to obtain the enzymatic biogenic gel.

Example 9

300 mgN-methylolacrylamide (N-MMAA), 0.5mg N, N' -Methylenebisacrylamide (MBAA), 10. Mu.L horseradish peroxidase (20 mg/mL), 10. Mu.L acetylacetone, 50. Mu.L glucose (40 mM) were added to 830. Mu.L of the biological salt solution at a concentration of 0.4g/mL, and stirred uniformly until the solution became homogeneous and transparent. Finally, 100. Mu.L of glucose oxidase solution (10 mg/mL) was added to obtain a precursor solution. And (3) placing the precursor solution in a constant-temperature water bath, and incubating at 37 ℃ for 30min to obtain the enzymatic biogenic gel.

Example 10

300mg of acrylamide (AAm), 5mg of polyethylene glycol diacrylate (PEGDA-200), 10. Mu.L of horseradish peroxidase (20 mg/mL), 10. Mu.L of acetylacetone, 50. Mu.L of glucose (40 mM) were added to 830. Mu.L of a biological salt solution having a concentration of 0.4g/mL, and stirred uniformly until the solution became homogeneous and transparent. Finally, 100. Mu.L of glucose oxidase solution (10 mg/mL) was added to obtain a precursor solution. And (3) placing the precursor solution in a constant-temperature water bath, and incubating at 37 ℃ for 30min to obtain the enzymatic biogenic gel.

Example 11

300Mg of Methacrylamide (MMAA), 5mg of polyethylene glycol diacrylate (PEGDA-200), 10. Mu.L of horseradish peroxidase (20 mg/mL), 10. Mu.L of acetylacetone, 50. Mu.L of glucose (40 mM) were added to 830. Mu.L of a biological salt solution at a concentration of 0.4g/mL, and stirred uniformly until the solution became homogeneous and transparent. Finally, 100. Mu.L of glucose oxidase solution (10 mg/mL) was added to obtain a precursor solution. And (3) placing the precursor solution in a constant-temperature water bath, and incubating at 37 ℃ for 30min to obtain the enzymatic biogenic gel.

Example 12

300 mgN-methylolacrylamide (N-MMAA), 5mg polyethylene glycol diacrylate (PEGDA-200), 10. Mu.L horseradish peroxidase (20 mg/mL), 10. Mu.L acetylacetone, 50. Mu.L glucose (40 mM) were added to 830. Mu.L of the biological salt solution at a concentration of 0.4g/mL, and stirred uniformly until the solution became homogeneous and transparent. Finally, 100. Mu.L of glucose oxidase solution (10 mg/mL) was added to obtain a precursor solution. And (3) placing the precursor solution in a constant-temperature water bath, and incubating at 37 ℃ for 30min to obtain the enzymatic biogenic gel.

The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.

Claims

1. A method for preparing an enzymatic biogenic gel, comprising the steps of:

(1) Mixing biological salt with water to prepare uniform biological salt solution;

(2) Grafting and modifying the chondroitin sulfate to prepare an acrylated chondroitin sulfate crosslinking agent;

(3) Mixing the biological salt solution prepared in the step (1) with the acrylic chondroitin sulfate cross-linking agent prepared in the step (2) and a monomer, adding an enzyme initiation system to obtain a precursor solution, and initiating gel formation to obtain biological salt gel;

the biological salt is D-fructose-1, 6-trisodium diphosphate;

the biogenic gel is used for an implantable sensing device.

2. The method for preparing an enzymatic biogenic gel according to claim 1, wherein said biogenic gel has an added concentration of no more than 0.8g/ml.

3. The method for preparing an enzymatic biogenic gel according to claim 1, wherein the amount of said acrylated chondroitin sulfate cross-linker added in step (3) is 4-12mg/ml.

4. The method of claim 1, wherein the monomer in step (3) comprises one or more of acrylamide, methacrylamide, and N-methylolacrylamide.

5. The method for preparing the enzymatic bioglass gel according to claim 4, wherein the addition amount of the monomer is 20% -30% of the total weight of the solvent.

6. The method for preparing an enzymatic biogenic gel according to claim 1, wherein said enzyme priming system of step (3) is a double enzyme mediated cascade enzyme system.

7. The method for preparing an enzymatic biogenic gel according to claim 6, wherein said cascade enzyme system comprises glucose oxidase, glucose, horseradish peroxidase or acetylacetone;

the addition amount of the glucose oxidase is 1.0mg/ml, the addition amount of glucose is 0.36mg/ml, the addition amount of horseradish peroxidase is 0.2mg/ml, and the addition amount of acetylacetone is 10 μl/ml.

8. The method for preparing an enzymatic biogenic gel according to claim 1, wherein the gelling means in step (3) is: incubating at 37deg.C for 30min.

9. The use of the enzymatic biogenic gel prepared by the preparation method according to claim 1 in a glucose sensor.