CN115840040A

CN115840040A - Preparation method and application of micro-nano enzyme-loaded capsule

Info

Publication number: CN115840040A
Application number: CN202211318884.6A
Authority: CN
Inventors: 崔基炜; 崔小苗; 高至亮; 郝京诚
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2022-10-26
Filing date: 2022-10-26
Publication date: 2023-03-24
Anticipated expiration: 2042-10-26
Also published as: CN115840040B

Abstract

The invention provides a preparation method and application of a micro-nano enzyme-loaded capsule, and belongs to the technical field of biological medicine and biochemical sensing. The invention utilizes the inherent amphiphilic property of protein to make protein be adsorbed on oil-water interface based on ultrasonic emulsification. The micro-nano capsule is obtained by using volatile oil phase perfluorohexane as a sacrificial template through solvent evaporation under the conditions of not damaging a protein membrane and not using strong acid and strong alkali. The method can load a plurality of enzymes to realize enzyme cascade reaction. The enzyme capsule has high catalytic efficiency and can realize quick and sensitive visual detection of glucose. The preparation method is simple and convenient in preparation process, the product has high catalytic efficiency and high sensitivity, and has good value of practical application, low cost and safety and no toxic or side effect, and batch production can be realized, so that the preparation method has good value of practical application.

Description

Preparation method and application of micro-nano enzyme-loaded capsule

Technical Field

The invention belongs to the technical field of biomedicine and biochemical sensing, and particularly relates to a preparation method and application of a micro-nano enzyme-loaded capsule.

Background

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Enzymes exist in various life forms, and are proteins produced by living cells with high specificity and high catalytic efficiency for their substrates. In biology, enzymes are important substances for many functions in the organism. Enzymes are indispensable in life activities, and provide necessary power and catalysts for biochemical reactions of life. Enzymes, which reduce the energy required for the conversion of a substrate into a product, are ideal catalysts in green chemistry, are commonly used as catalysts in organic synthesis, as processing tools in the food industry, as environmental water pollution treatments and detergents in environmental chemistry, and importantly have wide applications in biomedical tumor therapy.

Given the multiple functions of natural enzymes (high efficiency, specificity and selectivity) and the inherent complexity of their chemical properties (three-dimensional structure), the embedded enzyme strategy mimics the enzymatic reactions of natural enzymes, offering a wide range of applications. The enzyme can be immobilized on different substrates such as porous film surfaces, nanoparticles, hydrogels, metal Organic Frameworks (MOFs) and the like by covalent or non-covalent modification. However, in most cases, the enzyme is immobilized on a nanoparticle or modified in a cross-linked network by covalent bonds. Covalent bond modifications, however, may disrupt the tertiary structure of the enzyme, and the coating of the enzyme in different materials may increase the mass transfer distance between the substrate and the enzyme, which not only affects the activity of the enzyme, but also decreases the catalytic efficiency of the enzyme. In order to satisfy the requirements of maintaining and even enhancing the activity of the enzyme, improving the long-term stability of the enzyme to various environments and improving the enzyme catalysis efficiency, a new and generalizable method for forming the ideal immobilized enzyme needs to be established.

Inspired by the unique microenvironment of eukaryotic cells, capsules were used to immobilize enzymes. The micro-nano capsule has the characteristic of unique biochemical microenvironment, so the micro-nano capsule is widely concerned in the fields of drug delivery, micro-nano reactors and the like. The natural enzyme encapsulated in the capsule cavity can protect the enzyme from external interference, thereby improving the stability of the enzyme. However, the preparation of functional micro-nano capsules also faces serious challenges at present, such as less template types and harsh template removal conditions, for example, strong acid and strong base are needed to etch the template for preparing the capsules by using the templates such as silicon dioxide, calcium carbonate and the like. In addition to encapsulating the enzyme inside the capsule, mass transfer of the substrate depends only on the clearance of the capsule surface, which inevitably increases not only the mass transfer distance but also the diffusion resistance between the catalyst and the substrate.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a preparation method and application of a micro-nano enzyme-loaded capsule. The enzyme carrier capsule is prepared by adopting an ultrasonic emulsification technology, the regulation and control of the capsule particle size are realized by controlling the ultrasonic power, and the micro-nano capsule is obtained by solvent evaporation under the conditions of not damaging a protein membrane and not using strong acid and strong alkali by using the volatile Perfluorohexane (PFH) as a template. The capsule has high catalytic efficiency and can realize quick and sensitive visual detection of glucose.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in the first aspect of the present invention, the preparation method is simple by preparing the emulsion using probe-type ultrasound. In addition, the ultrasonic emulsification technology can realize the homogenization of components of other oils such as lithospermum oil, squalene and the like to obtain the micro-nano emulsion.

In the second aspect of the invention, the preparation method of the micro-nano enzyme-loaded capsule comprises the steps of preparing required materials including protein, tannic acid and perfluorohexane.

The protein is one of casein, bovine serum albumin, human serum albumin, lysozyme, glucose oxidase or horse radish peroxidase or a mixture of glucose oxidase or horse radish peroxidase.

The preparation method comprises the following steps:

dissolving protein in phosphate buffer solution, slowly adding perfluorohexane, ultrasonically preparing micro-nano emulsion, adding tannic acid solution, and heating.

In a third aspect of the invention, the micro-nano enzyme-loaded capsule is prepared by the method.

In a fourth aspect of the invention, the micro-nano enzyme-loaded capsule is applied to rapid and sensitive detection of glucose.

The invention has the beneficial effects that:

1. the invention adopts the ultrasonic emulsification technology to prepare the micro-nano enzyme-loaded capsule, and utilizes the volatile Perfluorohexane (PFH) as a template to obtain the micro-nano capsule by solvent evaporation under the conditions of not damaging a protein membrane and not using strong acid and strong alkali.

2. The invention adopts the ultrasonic emulsification technology to prepare the micro-nano enzyme-loaded capsule, and can realize the regulation and control of emulsion and capsule particle size by changing the ultrasonic power so as to obtain the nano-scale and micron-scale capsule.

3. The invention adopts the ultrasonic emulsification technology to prepare the micro-nano enzyme-loaded capsule, and utilizes the emulsification technology to increase the interface area, so that the enzymatic reaction can occur faster than a static two-phase plane interface, and the invention is beneficial to a two-phase reaction system or a multi-phase catalytic reaction. The enzyme is immobilized at the liquid-liquid interface, allowing the enzyme to contact the substrate, giving the capsule high catalytic efficiency.

4. The capsule prepared by the invention has higher catalytic efficiency and can realize quick and sensitive visual detection of glucose.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to illustrate an exemplary embodiment of the invention and not to limit the invention.

FIG. 1 is a photograph of a CaS capsule prepared in example 1 of the present invention;

FIG. 2 is a photograph of a transmission electron microscope of CaS capsules prepared in example 1 of the present invention;

FIG. 3 is a particle size characterization of CaS capsules prepared in example 1 of the present invention;

FIG. 4 is a photomicrograph of capsules prepared according to examples 2-6 of the present invention, wherein FIG. 4 (a) corresponds to BSA capsules of example 2, FIG. 4 (b) corresponds to HSA capsules of example 3, FIG. 4 (c) corresponds to LYZ capsules of example 4, FIG. 4 (d) corresponds to GOx capsules of example 5, and FIG. 4 (e) corresponds to HRP capsules of example 6;

FIG. 5 is a TEM micrograph of capsules prepared according to examples 2-6 of the present invention, wherein FIG. 5 (a) corresponds to BSA capsule of example 2, FIG. 5 (b) corresponds to HSA capsule of example 3, FIG. 5 (c) corresponds to LYZ capsule of example 4, FIG. 5 (d) corresponds to GOx capsule of example 5, and FIG. 5 (e) corresponds to HRP capsule of example 6;

FIG. 6 is a characterization of the particle size of capsules prepared in examples 2-6 of the present invention;

FIG. 7 is a potential characterization of capsules prepared in examples 2-6 of the present invention;

FIG. 8 is a photomicrograph of GOx-HRP capsules of example 7 of the present invention;

FIG. 9 is a TEM photograph of GOx-HRP capsules in example 7 of the present invention;

FIG. 10 is a graph showing the ultraviolet absorption of GOx capsules in example 5 of the present invention to demonstrate the occurrence of a cascade reaction;

FIG. 11 is a graph showing the ultraviolet absorption of GOx-HRP capsules in example 7 of the present invention to demonstrate the cascade reaction;

FIG. 12 is a kinetic characterization of GOx-HRP capsules of example 7 of the present invention;

FIG. 13 shows the kinetic characterization of GOx capsules according to example 5 of the present invention;

FIG. 14 shows a comparison of the enzymatic activities of GOx-HRP capsules of example 7 and of GOx capsules of example 5 and of HRP capsules of example 6 loaded with two enzymes, respectively, according to the present invention;

FIG. 15 is a graph of the UV absorption color chart of GOx-HRP capsules incubated for different periods of time with different concentrations of glucose in example 7 of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The technical scheme of the invention is as follows:

provided is a preparation method of a micro-nano enzyme-loaded capsule, wherein the material comprises casein (CaS), bovine Serum Albumin (BSA), human Serum Albumin (HSA), lysozyme (LYZ), glucose oxidase (GOx), horseradish peroxidase (HRP), tannic Acid (TA) and Perfluorohexane (PFH). First we studied the preparation of ultrasound mediated protein capsules using casein as a model protein. The invention can reduce the size of the protein capsule from micron to nanometer by changing the power of the ultrasonic wave, thereby realizing the controllable adjustment of the capsule grain size. In addition, the ultrasonic emulsification method can realize the batch preparation of the capsules, thereby being beneficial to industrial production and application. Tannic acid is added into the system, and strong interaction between protein and polyphenol is utilized to stabilize the capsule. By utilizing the strategy, functional protein such as enzyme is prepared into the capsule, and the nano-scale capsule prepared by ultrasonic increases the interface area of enzyme catalysis, thereby ensuring higher catalytic efficiency. In addition, the enzyme is fixed on the interface, so that the mass transfer distance between the substrate and the enzyme is reduced, and the high-efficiency proceeding of two-step enzyme cascade reaction is facilitated. The method is used for preparing the multienzyme load capsule for detecting the glucose by using the glucose oxidase and the horseradish peroxidase, so that the rapid and sensitive detection of the glucose is realized.

According to the technical scheme, the amphiphilic protein solution is used as a water phase, the perfluorohexane is used as a dispersion phase to prepare the emulsion, a complex surfactant is not needed, the emulsion can be obtained, then the polyphenol is added for stabilization, in addition, the micro-nano capsule obtained by removing a liquid drop template through solvent evaporation can be obtained under the conditions that a protein film is not damaged and strong acid and strong base are not used, and a simple and convenient method is provided for further preparing ultrasonic technology capsules in batches. The capsule prepared by the technical scheme has simple preparation process, good biocompatibility, capacity of simply realizing regulation and control of the capsule particle size and universality.

The above protocol utilizes functional proteins, such as enzymes, to prepare enzyme-loaded capsules. The multi-enzyme catalytic cascade represents a major chemical reaction that plays a key role in biological signal transduction and metabolic pathways. The enzyme not only acts as a surfactant emulsifier, but also acts as a biocatalytic site with the multi-enzyme loaded capsule for glucose detection with GOx and HRP. The mass transfer distance between the substrate glucose and the enzyme is reduced, and the visual monitoring of the glucose is realized.

The invention provides a method for preparing capsules by using volatile perfluorohexane as a sacrificial template through an ultrasonic emulsification technology, and the capsule shows good application potential in the aspects of enhancing the stability and the enzyme activity of the enzyme. First, emulsification techniques increase the interfacial area, so that enzymatic reactions occur faster than static two-phase planar interfaces, facilitating two-phase reaction systems or heterogeneous catalytic reactions. The enzyme is immobilized at the liquid-liquid interface, allowing the enzyme to contact the substrate, giving the capsule high catalytic efficiency.

In a specific embodiment of the present invention, a method for preparing a micro-nano enzyme-loaded capsule is provided, wherein the preparation method comprises the steps of preparing a protein, tannic acid and perfluorohexane.

The preparation method comprises the following steps:

The preparation method is a universal method for preparing the micro-nano enzyme-loaded capsule. The bovine serum albumin, the human serum albumin, the lysozyme, the glucose oxidase and the horseradish peroxidase capsules prepared by the method prove that the method has universality.

The preparation method of the ultrasonic-mediated enzyme-loaded capsule is simple, so that the ultrasonic-mediated enzyme-loaded capsule is favorable for batch production, for example, the enzyme-loaded capsule glucose sensor can be obtained by heating to remove the template.

In still another embodiment of the present invention, when the protein is one of casein, bovine serum albumin, human serum albumin, lysozyme, glucose oxidase and horseradish peroxidase, the concentration of the protein solution is controlled to be 0.1 to 100mg/mL, more preferably 1 to 50mg/mL, and still more preferably 2 to 10mg/mL.

In another embodiment of the present invention, when the protein is glucose oxidase or a mixture of horseradish peroxidase, the concentration of glucose oxidase is controlled to be 0.1 to 100mg/mL, more preferably 1 to 50mg/mL, and still more preferably 2 to 10mg/mL; the concentration of horseradish peroxidase is controlled to be 0.1 to 100mg/mL, more preferably 1 to 50mg/mL, and still more preferably 2 to 10mg/mL. The two enzyme solutions were mixed. The mass ratio of the horseradish peroxidase to the glucose oxidase is 1:1-5, preferably 1:1.

In yet another embodiment of the present invention, the phosphate buffer is used in an amount of 4mL and the pH is 7.4.

In still another embodiment of the present invention, the amount of perfluorohexane added is controlled to 5 to 1000. Mu.L, more preferably 10 to 500. Mu.L, and still more preferably 50 to 200. Mu.L.

In yet another embodiment of the present invention, the volume ratio of the protein solution to perfluorohexane is 1:4 to 40, preferably 1.

In still another embodiment of the present invention, the concentration of the tannic acid solution is controlled to be 1 to 150mg/mL, more preferably 20 to 100mg/mL, and still more preferably 30 to 50mg/mL.

In another embodiment of the present invention, the specific conditions of ultrasound are: ultrasonic treatment is carried out for 45s to 10min under the ultrasonic condition of 30 to 250W, and ultrasonic treatment for 2min at 150W is preferred.

In another embodiment of the present invention, the heating is performed under the following conditions: heating for 2-10 min under the condition of rotary steaming at 30-70 ℃, preferably heating for 10min at 65 ℃.

In another embodiment of the present invention, the centrifugation conditions are as follows: centrifuging at 1000-8000 rpm for 1-10 min, preferably 2500rpm for 5min.

In another specific embodiment of the invention, the micro-nano enzyme-loaded capsule is prepared by the method.

In another specific embodiment of the invention, the micro-nano enzyme-loaded capsule is applied to rapid and sensitive detection of glucose.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1

(1) Casein (10 mg) was accurately weighed, and 4mL of a phosphate buffer solution (pH 7.4) was added thereto to obtain a solution A.

(2) Tannic acid 40mg was accurately weighed, and 1mL of ultrapure water was added thereto to obtain a solution B.

(3) 2mL of solution A was placed in a 5mL centrifuge tube, and 100. Mu.L of perfluorohexane was added to solution A.

(4) And (3) using probe type ultrasound, placing a probe at the interface of the protein solution and the perfluorohexane solution, performing ultrasound for 2min at the power of 150W, performing ice bath, and adding the ice bath to obtain emulsion C.

(5) And (3) dropwise adding the solution B into the emulsion C under a vortex mixer by taking 10mL of the solution B, uniformly mixing by vortex, and carrying out rotary evaporation on the emulsion at 65 ℃ for 10min to obtain a final product capsule, namely a CaS capsule.

The picture of the CaS capsule is shown in fig. 1, the photograph of the transmission electron microscope is shown in fig. 2 and 4 (a), and the particle size characterization is shown in fig. 3 and 5.

Example 2

(1) 10mg of bovine serum albumin was accurately weighed, and 4mL of a phosphate buffer solution (pH 7.4) was added thereto to obtain solution A.

(4) The probe was placed at the interface of the protein solution and the perfluorohexane solution using probe-type ultrasound, and ultrasound was performed at a power of 50W for 2min. An additional ice bath afforded emulsion C.

(5) And (3) taking 10 mu L of the solution B, dropwise adding the solution B into the emulsion C under a vortex mixer, uniformly mixing by vortex, and carrying out rotary evaporation on the emulsion at 65 ℃ for 10min to obtain a final product, namely an enzyme-loaded capsule and a BSA capsule.

The micrograph of the BSA capsules is shown in fig. 4 (a), the micrograph of the BSA capsules is shown in fig. 5 (a), the particle size characterization is shown in fig. 6, and the potential characterization is shown in fig. 7.

Example 3

(1) Human serum albumin was accurately weighed at 10mg, and 4mL of a phosphate buffer solution (pH 7.4) was added thereto to obtain solution A.

(5) And (3) dropwise adding the solution B into the emulsion C under a vortex mixer by taking 10 mu L of the solution B, uniformly mixing by vortex, and carrying out rotary evaporation on the emulsion at 65 ℃ for 10min to obtain a final product, namely the enzyme-loaded capsule and the HSA capsule.

The micrograph of this HSA capsule is shown in fig. 4 (b), the photograph of the HSA capsule is shown in fig. 5 (b), the particle size characterization is shown in fig. 6, and the potential characterization is shown in fig. 7.

Example 4

(1) 10mg of lysozyme was accurately weighed, and 4mL of a phosphate buffer solution (pH 7.4) was added thereto to obtain a solution A.

(4) The probe was placed at the interface of the protein solution and the perfluorohexane solution using probe-type ultrasound, and ultrasound was performed at a power of 50W for 2min. An ice bath was added to give emulsion C.

(5) And (3) dripping 10 mu L of the solution B into the emulsion C under a vortex mixer, uniformly mixing by vortex, and carrying out rotary evaporation on the emulsion at 65 ℃ for 10min to obtain a final product, namely the enzyme-loaded capsule and the LYZ capsule.

The LYZ capsules are shown in FIG. 4 (c) in a photomicrograph, FIG. 5 (c) in a transmission electron microscope, FIG. 6 in a particle size characterization, and FIG. 7 in a potential characterization.

Example 5

(1) Glucose oxidase was accurately weighed at 10mg, and 4mL of a phosphate buffer solution (pH 7.4) was added thereto to obtain solution A.

(4) The probe type ultrasound is used, the probe is placed at the interface of the protein solution and the perfluorohexane solution, and ultrasound is carried out for 2min at the power of 50W. An ice bath was added to give emulsion C.

(5) And (3) dropwise adding the solution B into the emulsion C under a vortex mixer by taking 10 mu L of the solution B, uniformly mixing by vortex, and carrying out rotary evaporation on the emulsion at 65 ℃ for 10min to obtain a final product, namely the enzyme-loaded capsule and the GOx capsule.

The micrograph of the GOx capsule is shown in FIG. 4 (d), the micrograph of the transmission electron microscope is shown in FIG. 5 (d), the particle size characterization is shown in FIG. 6, and the potential characterization is shown in FIG. 7.

Example 6

(1) Horseradish peroxidase (10 mg) was accurately weighed, and 4mL of a phosphate buffer solution (pH 7.4) was added thereto to obtain a solution a.

(5) And (3) taking 10 mu L of the solution B, dropwise adding the solution B into the emulsion C under a vortex mixer, uniformly mixing by vortex, and carrying out rotary evaporation on the emulsion at 65 ℃ for 10min to obtain a final product, namely an enzyme-loaded capsule and an HRP capsule.

The micrograph of the HRP capsule is shown in fig. 4 (e), the micrograph of the HRP capsule is shown in fig. 5 (e), the particle size characterization is shown in fig. 6, and the potential characterization is shown in fig. 7.

Example 7

(1) Glucose oxidase was accurately weighed at 10mg, and 4mL of a phosphate buffer solution (pH 7.4) was added thereto to obtain solution A. Horseradish peroxidase (10 mg) was accurately weighed, and 4mL of a phosphate buffer solution (pH 7.4) was added thereto to obtain a solution B.

(3) Taking 1mL of the solution A, taking 1mL of the solution B in a 5mL centrifuge tube, uniformly mixing by using a vortex mixer to obtain a solution C, and adding 100 mu L of perfluorohexane into the solution C.

(4) The probe was placed at the interface of the protein solution and the perfluorohexane solution using probe-type ultrasound, and ultrasound was performed at a power of 50W for 2min. Emulsion D was obtained.

(5) And (3) taking 10 mu L of the solution B, dropwise adding the solution B into the emulsion D under a vortex mixer, uniformly mixing by vortex, and carrying out rotary evaporation on the emulsion at 65 ℃ for 10min to obtain a final product, namely an enzyme-loaded capsule and a GOx-HRP capsule.

The microphotograph of the GOx-HRP capsule is shown in FIG. 8, and the photomicrograph of the transmission electron microscope is shown in FIG. 9.

Hydrogel Performance testing

(1)H ₂ O ₂ Detection of (2)

mu.L of glucose oxidase capsules (225. Mu.g/mL) were dispersed in water to 5mL, followed by addition of 1.5mL of a glucose solution thereto, incubation for 60min followed by addition of 300. Mu.L of an ammonium titanyl oxalate solution to the mixed solution, and absorbance of the solution at 405nm was measured by UV, and the kinetics was monitored. The solution appeared yellow, and fig. 10 demonstrated that the solution absorbed at 405nm, demonstrating the production of hydrogen peroxide.

(2) GOx-HRP Capsule catalytic Activity test

100 μ L of glucose oxidase-horseradish peroxidase capsules were dispersed in water to 5mL, and then 100 μ L of a glucose solution and 3,3',5,5' -Tetramethylbenzidine (TMB) were added thereto, and 3mL was buffered with citric acid-disodium hydrogenphosphate, incubated for 20min, and absorbance of the solution at 652nm was measured with ultraviolet light, and the solution appeared blue as the reaction proceeded, FIG. 11 demonstrated that a characteristic absorption peak appeared at 652nm in the presence of glucose, which demonstrated the occurrence of a cascade reaction, and the kinetics were monitored as in FIGS. 12 and 13, and the solution color deepened and the absorbance increased as the reaction proceeded in a certain period of time.

Enzyme capsule suspensions (25. Mu.L), glucose solutions (100. Mu.L, 25 mM) and 3,3',5,5' -tetramethylbenzidine solutions (100. Mu.L, 25. Mu.g/mL, DMSO) were prepared at the same concentrations in citrate-disodium hydrogen phosphate buffer solution, and the catalytic activities of the multi-enzyme capsules (GOx-HRP capsules) and the single-enzyme capsules (GOx, HRP capsules) were quantitatively compared using the method described above. The enzyme concentration was quantified by fluorescence spectroscopy. As can be seen from fig. 14, the catalytic activity of the capsules loaded with the two enzymes is much higher than that of the capsules loaded with the two enzymes, i.e., the capsules loaded with the two enzymes fix the enzymes on the interface, and the mass transfer distance between the substrate and the enzymes is shortened on the uniformly dispersed interface of the two enzymes, so that the catalytic activity of the enzymes is improved.

(3) GOx-HRP capsule detection of glucose with different concentrations

Glucose oxidase-horseradish peroxidase capsules are taken and dispersed in water, glucose solutions with different concentrations and 3,3',5,5' -tetramethylbenzidine solutions are added into the water, the mixed solutions are incubated for different times, and then the absorbance of the solutions at 652nm is detected by an enzyme labeling instrument, as shown in fig. 15, the glucose with different concentrations and the capsules are incubated for different times, the absorbance is increased along with the increase of the glucose concentration or the extension of the incubation time, and the color which can be distinguished by naked eyes can be shown within 3 min.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing micro-nano enzyme-loaded capsules is characterized in that materials required in preparation comprise protein, tannic acid and perfluorohexane;

the preparation method comprises the following steps: dissolving protein in phosphate buffer solution, slowly adding perfluorohexane into the phosphate buffer solution, and ultrasonically preparing micro-nano emulsion;

preparing micro-nano emulsion by an ultrasonic emulsification method, wherein oil phase components of the micro-nano emulsion can be components of other oils such as lithospermum oil, squalene and the like, so that micro-nano emulsion can be obtained;

performing ultrasonic emulsification to obtain a perfluorohexane emulsion, adding a tannic acid solution, and heating to obtain the emulsion;

the protein is selected from one of casein, bovine serum albumin, human serum albumin, lysozyme, glucose oxidase or horse radish peroxidase or the mixture of the glucose oxidase and the horse radish peroxidase.

2. The preparation method of the micro-nano enzyme-loaded capsule according to claim 1, wherein when the protein is one of casein, bovine serum albumin, human serum albumin, lysozyme, glucose oxidase or horseradish peroxidase, the concentration of the protein solution is controlled to be 0.1-100 mg/mL, more preferably 1-50 mg/mL, still more preferably 2-10 mg/mL;

or, when the protein is glucose oxidase or a mixture of horseradish peroxidase, the concentration of the glucose oxidase is controlled to be 0.1-100 mg/mL, more preferably 1-50 mg/mL, and still more preferably 2-10 mg/mL; the concentration of the horseradish peroxidase is controlled to be 0.1-100 mg/mL, more preferably 1-50 mg/mL, and still more preferably 2-10 mg/mL; mixing the two enzyme solutions; the mass ratio of the horseradish peroxidase to the glucose oxidase is 1:1-5, preferably 1:1.

3. The preparation method of the micro-nano enzyme-loaded capsule according to claim 1, wherein the dosage of the phosphate buffer solution is 4mL, the pH value is 7.4;

further, the amount of the perfluorohexane added is controlled to 5 to 1000. Mu.L, preferably 10 to 500. Mu.L, and more preferably 50 to 200. Mu.L.

4. The preparation method of the micro-nano enzyme-loaded capsule according to claim 1, wherein the volume ratio of the protein solution to the perfluorohexane is 1:4-40, preferably 1.

5. The preparation method of the micro-nano enzyme-loaded capsule according to claim 1, wherein the concentration of the tannic acid solution is controlled to be 1-150 mg/mL, preferably 20-100 mg/mL, and more preferably 30-50 mg/mL.

6. The preparation method of the micro-nano enzyme-loaded capsule according to claim 1, wherein the specific conditions of the ultrasound are as follows: ultrasonic treatment is carried out for 45s to 10min under the ultrasonic condition of 30 to 250W, and ultrasonic treatment for 2min at 150W is preferred.

7. The preparation method of the micro-nano enzyme-loaded capsule according to claim 1, wherein the specific conditions of heating are as follows: heating for 2-10 min under the condition of rotary steaming at 30-70 ℃, preferably heating for 10min at 65 ℃.

8. The preparation method of the micro-nano enzyme-loaded capsule according to claim 1, wherein the specific centrifugation conditions are as follows: centrifuging at 1000-8000 rpm for 1-10 min, preferably 2500rpm for 5min.

9. A micro-nano enzyme-loaded capsule, which is characterized by being prepared by the method of any one of claims 1 to 8.

10. The use of the micro-nano enzyme-loaded capsule according to claim 9 in rapid and sensitive detection of glucose.