A high-enzyme-loading porous catalytic hydrogel, and a preparation method and application thereof

Abstract

The application provides a porous catalytic hydrogel with high enzyme loading and a preparation method and application thereof, the nanoparticle immobilized enzyme contained in the porous hydrogel enhances the enzyme immobilization effect, plays a certain leakage prevention effect, and prolongs the service life; meanwhile, the hydrogel has the catalytic property and oxygen carrying property of the nanoparticle immobilized enzyme and certain viscoelasticity, has good biocompatibility, and is more convenient to adhere and replace with skin and other tissues; the porous catalytic hydrogel is gelled in a thermal polymerization mode, and has the advantages of simplicity and rapidness; the prepared hydrogel has a reticular structure inside, has a high specific surface area, and the preparation method can adjust the pore size of the hydrogel by adjusting the use ratio of the carboxymethylated cellulose nanofiber and betaine, so that the hydrogel matched with the higher enzyme loading is obtained, the hydrogel has higher catalytic capacity, and has wide application prospects in electrochemical sensors.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biocatalytic materials, in particular to a porous catalytic hydrogel with high enzyme loading and a preparation method and application thereof. BACKGROUND

[0002] Enzymes are nanoscale protein catalysts in nature, and have been applied to many fields such as biology and chemistry as a common oxidation method. Nowadays, the size of enzyme-based catalytic materials can be reduced to nanoscale, and the efficiency of enzyme catalysis is also improved. However, the reduced size also makes it more difficult to reuse enzyme catalytic materials. Therefore, the establishment of a catalytic system with high activity, durability, operability and life compatibility has attracted considerable attention.

[0003] With the progress of new biomaterials in highly controllable manufacturing and processability, enzyme composite materials based on polymer hydrogels have become a common strategy due to their swelling, nanomicro-porous and aqueous 3D structure, which can immobilize catalytic enzymes, electroactive substances, whole cells and complex tissue models. And due to its environmentally friendly process and designed catalytic reaction, it can improve the practicability and economy of enzyme catalysts, and at the same time maintain adjustable mechanical properties in wearable and implantable devices, so the synthesis of hydrogel composite enzyme catalytic materials has been widely studied at this stage.

[0004] However, most of these hydrogel composite enzyme catalytic materials have the problems of low enzyme loading and even enzyme leakage, which leads to low catalytic performance and short service life of the hydrogel composite enzyme catalytic material. Based on this, how to develop a high-performance catalytic system for biological and chemical sensing applications has become a technical problem to be solved at this stage. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a porous catalytic hydrogel with high enzyme loading.

[0006] Another technical problem to be solved by the present application is to provide a preparation method of the porous catalytic hydrogel with high enzyme loading.

[0007] Another technical problem to be solved by the present application is to provide an application of the porous catalytic hydrogel with high enzyme loading.

[0008] The technical solution adopted by the present application is:

[0009] The porous catalytic hydrogel with high enzyme loading is a copolymer porous hydrogel with a reticular structure inside, wherein nano-particle immobilized enzymes are dispersed, the nano-particle immobilized enzymes are immobilized on Al2O3 nano-particles as immobilized carriers and modified with enzymes on the surface.

[0010] Preferably, the high-enzyme-loading porous catalytic hydrogel is prepared by incorporating the nanoparticle-immobilized enzyme into the porous hydrogel.

[0011] The nanoparticle-immobilized enzyme in the high-enzyme-loading porous catalytic hydrogel is modified on the surface of Al2O3 nanoparticles, and better enzyme coating effect is achieved by selecting the particle size and stability of the nanoparticles. In addition, the stability and concentration of the nanoparticle-immobilized enzyme in the hydrogel are ensured by selecting the raw material ratio and preparation method.

[0012] Preferably, the high-enzyme-loading porous catalytic hydrogel has an Al2O3 nanoparticle diameter of 10-20 nm, and the nanoparticle-immobilized enzyme has a size of 15-25 nm.

[0013] Preferably, the high-enzyme-loading porous catalytic hydrogel has an Al2O3 nanoparticle diameter of 10-20 nm, and the nanoparticle-immobilized enzyme has a size of 15-25 nm.

[0014] Preferably, the high-enzyme-loading porous catalytic hydrogel has an Al2O3 nanoparticle diameter of 10-20 nm, and the nanoparticle-immobilized enzyme has a size of 15-25 nm.

[0015] The preparation method of the high-enzyme-loading porous catalytic hydrogel is obtained by constructing carboxylated cellulose nanofiber and betaine through electrostatic binding mechanism, and the specific steps are as follows:

[0016] (1) Using Al2O3 nanoparticle emulsion as emulsifier, adding enzyme solution, and preparing nanoparticle-immobilized enzyme solution loaded with enzyme by ice water bath method and oscillation;

[0017] (2) Dissolve betaine monomer in carboxylated cellulose nanofiber solution, add crosslinking agent, and then perform ultrasonic treatment under ice water bath condition. Then, add crosslinking catalyst and thermal initiator into the solution, and stir in ice water bath to obtain zwitterionic polymer solution;

[0018] (3) Add nanoparticle-immobilized enzyme solution into the zwitterionic polymer solution, stir and mix in ice water bath to obtain hydrogel solution. Then, inject the hydrogel solution into a mold, and polymerize under hot water bath condition to obtain high-enzyme-loading porous catalytic hydrogel.

[0019] Preferably, in the preparation method of the high-enzyme-loading porous catalytic hydrogel, the concentration of Al2O3 nanoparticle emulsion in step (1) is 0.1-5 mg / ml, and the concentration of enzyme solution is 10-50 mg / ml. The ice water bath temperature is-10-0℃, the shaking table oscillation speed is 100-200 r / min, and the oscillation time is 15-45 min.

[0020] Preferably, in the preparation method of the high-enzyme-loading porous catalytic hydrogel, the volume ratio of the Al2O3 nanoparticle emulsion and the enzyme solution in step (1) is 1:1.

[0021] Preferably, in the preparation method of the high-enzyme-loading porous catalytic hydrogel, the carboxylated cellulose nanofiber solution in step (2) is a 1% to 10% carboxylated cellulose nanofiber solution by mass fraction, the betaine monomer is a betaine monomer powder with an active content of 94% to 98%, the crosslinking agent is methylene bisacrylamide, the catalytic crosslinking agent is tetramethyl ethylenediamine, and the thermal initiator is ammonium persulfate; the ultrasonic time is 30 to 60 min, the ice water bath temperature is -10 to 0 DEG C, the stirring speed is 300 to 450 rpm, and the stirring time is 3 to 15 min.

[0022] Preferably, in the preparation method of the high-enzyme-loading porous catalytic hydrogel, the betaine monomer is sulfobetaine or phosphobetaine.

[0023] Preferably, in the preparation method of the high-enzyme-loading porous catalytic hydrogel, the volume ratio of the nanoparticle immobilized enzyme solution and the zwitterionic polymer solution in step (3) is 0.1 to 10:1, the ice water bath temperature is -10 to 0 DEG C, the stirring speed is 300 to 450 rpm, and the stirring time is 3 to 15 min; the hot water bath temperature is 30 to 40 DEG C, and the polymerization time is 24 to 36 h.

[0024] The high-enzyme-loading porous catalytic hydrogel is applied to an electrochemical sensor.

[0025] Preferably, the high-enzyme-loading porous catalytic hydrogel is applied as a sensing material in an electrochemical sensor.

[0026] The high-enzyme-loading porous catalytic hydrogel has the following beneficial effects:

[0027] The high-enzyme-loading porous catalytic hydrogel has the following beneficial effects: BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a structural schematic diagram of the high-enzyme-loading porous catalytic hydrogel of the present application;

[0029] Figure 2 is a structural schematic diagram of the high-enzyme-loading nanoparticle-immobilized enzyme of the present application. DETAILED DESCRIPTION

[0030] To further illustrate the present application, the following examples are provided:

[0031] Example 1

[0032] A high-enzyme-loading porous catalytic hydrogel, the preparation method thereof comprising the following steps:

[0033] (1) Synthesis of nanoparticle-immobilized enzyme solution

[0034] Take 10 ml of 1 mg / ml Al2O3 nanoparticle emulsion with a diameter of 10-20 nm in a beaker, add 10 ml of 30 mg / ml glucose oxidase solution to the beaker, place the beaker in ice water at -5°C, and use a shaker for 30 min of shaking to obtain a uniformly mixed nanoparticle-immobilized enzyme solution, as shown in Figure 2 .

[0035] (2) Preparation of hydrogel zwitterionic polymer solution

[0036] Take 50 ml of 2% carboxylated cellulose nanofiber solution in a beaker, dissolve 10 mg of sulfobetaine monomer in the carboxylated cellulose nanofiber solution to obtain a zwitterionic monomer solution; prepare a 1% methylene bisacrylamide solution, add 6 ml of 1% methylene bisacrylamide solution to the monomer solution, place the beaker in an ultrasonic machine under ice water conditions at -5°C, and ultrasonic for 30 min; after ultrasonic, add 6 ml of 2% ammonium persulfate and 10 ul of 1% tetramethyl ethylenediamine to the above mixed solution, place the beaker in ice water at -5°C, and use a stirrer to stir at a speed of 300 rpm for 10 min to obtain a hydrogel zwitterionic polymer solution.

[0037] (3) Preparation of high-enzyme-loading porous catalytic hydrogel

[0038] Mix the prepared nanoparticle-immobilized enzyme solution and the hydrogel zwitterionic polymer solution in a beaker at a ratio of 1:1, place the beaker in ice water at -5°C, use a stirrer to stir at a speed of 300 rpm for 10 min to obtain a hydrogel mixed solution, inject the hydrogel mixed solution into a mold, and polymerize for 24 h at a temperature of 37°C in a hot water bath to obtain a high-catalytic-performance porous catalytic hydrogel, as shown in Figure 1 .

[0039] In the method for preparing the high-enzyme-loading porous catalytic hydrogel, first, the zwitterionic hydrogel is synthesized by using the carboxylated cellulose nanofiber and the sulfobetaine, the zwitterionic hydrogel can provide a high specific surface area, and by changing the proportion of the carboxylated cellulose nanofiber, the pore size of the hydrogel can be adjusted to match the maximum enzyme adsorption capacity, thereby improving the enzyme loading capacity. Further, in order to enhance the enzyme immobilization effect, the enzyme-Al2O3 particles are synthesized by modifying the enzyme on the surface of Al2O3, and the particles are incorporated into the zwitterionic hydrogel, thereby obtaining the high-enzyme-loading porous catalytic hydrogel. The high-enzyme-loading porous catalytic hydrogel can avoid the leakage of the enzyme, and by adhering to the surrounding environment, a good catalytic environment is established, and the high-enzyme-loading porous catalytic hydrogel has the value of clinical transformation.

[0040] Example 2

[0041] The sensor electrode is placed in a hydrogel vessel, and the hydrogel mixed solution prepared in step (3) of Example 1 is added dropwise to the sensor electrode until the hydrogel is filled to the electrode, and the vessel is placed in a water bath for polymerization at a temperature of 37℃ for 24h, thereby encapsulating the biosensor modified with the high-enzyme-loading porous catalytic hydrogel on the sensor electrode.

[0042] The above-described examples only describe the preferred embodiments of the present application, and do not limit the scope of the present application, and various modifications and improvements to the technical solutions of the present application made by the ordinary engineering technical personnel without departing from the design spirit of the present application shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for preparing a porous catalytic hydrogel with high enzyme loading capacity, characterized in that: The method involves constructing carboxylated cellulose nanofibers and betaine via an electrostatic binding mechanism, with the specific steps outlined below: (1) Using Al2O3 nanoparticle emulsion as emulsifier, add enzyme solution, and prepare enzyme-loaded nanoparticle immobilized enzyme solution by shaking in an ice-water bath. (2) Dissolve betaine monomer in carboxylated cellulose nanofiber solution, add crosslinking agent and sonicate the solution under ice-water bath conditions, then add crosslinking catalyst and thermal initiator to the solution and stir in ice-water bath to obtain zwitterionic polymer solution, wherein the betaine monomer is sulfonate betaine monomer; (3) The nanoparticle immobilized enzyme solution is added to the zwitterionic polymer solution and stirred in an ice-water bath to obtain a hydrogel solution. The hydrogel solution is injected into a mold and polymerized under hot water bath conditions to obtain a porous catalytic hydrogel with high enzyme loading. The enzyme is glucose oxidase.

2. The method for preparing a porous catalytic hydrogel with high enzyme loading capacity according to claim 1, characterized in that: In step (1), the concentration of the Al2O3 nanoparticle emulsion is 0.1–5 mg / ml, and the concentration of the enzyme solution is 10–50 mg / ml; the ice-water bath temperature is -10–0℃, the shaking speed is 100–200 r / min, and the shaking time is 15–45 min; in step (2), the carboxylated cellulose nanofiber solution is a 1%–10% carboxylated cellulose nanofiber solution, and the betaine monomer is a powder with an active betaine monomer content of 94%–98%; the crosslinking agent is methylenebisacrylamide, the catalytic crosslinking agent is tetramethylethylenediamine, and the thermal initiator is ammonium persulfate; the ultrasonic time is 30–60 min, the ice-water bath temperature is -10–0℃, the stirring speed is 300–450 rpm, and the stirring time is 3–15 min. min; the volume ratio of the nanoparticle immobilized enzyme solution and the zwitterionic polymer solution in step (3) is 0.1-10:1, the ice-water bath temperature is -10 to 0℃, the stirring speed is 300 to 450 rpm, the stirring time is 3 to 15 min; the hot water bath temperature is 30 to 40℃, and the polymerization time is 24 to 36 h.

3. The method for preparing a porous catalytic hydrogel with high enzyme loading capacity according to claim 1 or 2, characterized in that: In step (1), the volume ratio of Al2O3 nanoparticle emulsion to enzyme solution is 1:

1.

4. The method for preparing a porous catalytic hydrogel with high enzyme loading capacity according to claim 1 or 2, characterized in that: The Al2O3 nanoparticles have a diameter of 10-20 nm, and the enzyme immobilized in the nanoparticles has a size of 15-25 nm.

5. A porous catalytic hydrogel with high enzyme loading capacity, characterized in that: It is prepared by the preparation method described in any one of claims 1-4.

6. The application of the porous catalytic hydrogel with high enzyme loading capacity as described in claim 5 in electrochemical sensors.

7. The application of the porous catalytic hydrogel with high enzyme loading capacity according to claim 6, characterized in that: The porous catalytic hydrogel is used as a sensing material in an electrochemical sensor.

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