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

Abstract

The invention provides a preparation method and application of micronano enzyme-loaded capsules, and the invention belongs to the technical fields of biomedicine and biochemical sensing. The invention utilizes the inherent amphipathic property of protein, and makes the protein adsorb on the oil-water interface based on ultrasonic emulsification. Using volatile oil-phase perfluorohexane as a sacrificial template, the micro-nanocapsules can be obtained by solvent evaporation without destroying the protein film and without using strong acid and strong alkali. The method can load multiple enzymes to realize enzyme cascade reactions. The enzyme capsule has high catalytic efficiency and can realize rapid and sensitive visual detection of glucose. The preparation process of the invention is simple, the product has high catalytic efficiency and high sensitivity, has good practical application value, is low in cost, can realize mass production, and the product is safe and has no toxic and side effects, so it has good practical application value.

Description

A preparation method and application of micronano enzyme-loaded capsules

technical field

The invention belongs to the technical field of biomedicine and biochemical sensing, and specifically relates to a preparation method and application of micronano enzyme-loaded capsules.

Background technique

The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

Enzymes, found in various life forms, are proteins produced by living cells that are highly specific for their substrates and highly catalytically efficient. In biology, enzymes are important substances for many functions in living organisms. Enzymes are indispensable in life activities, which provide the necessary power and catalysts for the biochemical reactions of life. Enzymes can reduce the energy required to convert substrates into products, and are ideal catalysts in green chemistry. Enzymes are usually used as catalysts in organic synthesis, as processing tools in the food industry, and as environmental water pollution in environmental chemistry. Handling and detergents are important in biomedical oncology treatment with a wide range of applications.

Given the diverse functions of natural enzymes (high efficiency, specificity, and selectivity) and the inherent complexity of their chemical properties (three-dimensional structure), the strategy of embedding enzymes to mimic the enzymatic reactions of natural enzymes offers broad application prospects. Enzymes can be immobilized on different substrates, such as porous film surfaces, nanoparticles, hydrogels, and metal-organic frameworks (MOFs), through covalent or non-covalent modifications. However, in most cases, enzymes are immobilized on nanoparticles or modified by covalent bonds in cross-linked networks. However, the covalent bond modification may destroy the tertiary structure of the enzyme, and the encapsulation 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 reduces the catalytic efficiency of the enzyme. In order to meet the needs of maintaining or even enhancing enzyme activity, improving long-term stability of enzymes to various environments, and high enzyme catalytic efficiency, a new and scalable method for forming ideal immobilized enzymes needs to be established urgently.

Inspired by the unique microenvironment of eukaryotic cells, capsules are used to immobilize enzymes. Micro-nanocapsules have received extensive attention in the fields of drug delivery and micro-nano reactors due to their unique biochemical microenvironment characteristics. By encapsulating the natural enzyme in the cavity of the capsule, the researchers can protect the enzyme from external interference, thereby improving its stability. However, the preparation of functional micro-nanocapsules is currently facing severe challenges, such as fewer types of templates, and harsh template removal conditions. For example, using templates such as silica and calcium carbonate to prepare capsules requires the use of strong acids and alkalis to etch the templates. . In addition, the enzyme is encapsulated inside the capsule, and the mass transfer of the substrate only depends on the gap on the surface of the capsule, which not only inevitably increases the mass transfer distance, but also increases the diffusion resistance between the catalyst and the substrate.

Contents of the invention

In order to solve the deficiencies of the prior art, the object of the present invention is to provide a preparation method and application of micronano enzyme-loaded capsules. The enzyme carrier capsules were prepared by ultrasonic emulsification technology, and the particle size of the capsules could be regulated by controlling the ultrasonic power. The function of using volatile perfluorohexane (PFH) as a template can be used without destroying the protein film and without using strong acid and strong alkali. , micronanocapsules obtained by solvent evaporation. The capsule has high catalytic efficiency and can realize fast and sensitive visual detection of glucose.

In order to achieve the above object, the technical solution of the present invention is:

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

The second aspect of the present invention is a method for preparing micronano enzyme-loaded capsules. The materials required for the preparation include protein, tannic acid, and perfluorohexane.

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

The specific preparation methods include:

The protein is dissolved in phosphate buffer, and then perfluorohexane is slowly added therein, the micro-nano emulsion is prepared by ultrasonication, and then tannic acid solution is added, and heated.

In the third aspect of the present invention, a micronano enzyme-loaded capsule is prepared by the above method.

The fourth aspect of the present invention is an application of the above-mentioned micronano enzyme-loaded capsules in rapid and sensitive detection of glucose.

The beneficial effects of the present invention are:

1. The present invention adopts ultrasonic emulsification technology to prepare micro-nano enzyme-loaded capsules, which can be obtained by solvent evaporation without destroying the protein film and without using strong acid and strong alkali by using volatile perfluorohexane (PFH) as a template micronanocapsules.

2. The present invention adopts ultrasonic emulsification technology to prepare micro-nano enzyme-loaded capsules. By changing the ultrasonic power, the particle size of the emulsion and capsules can be adjusted to obtain nano- and micron-sized capsules.

3. The present invention adopts ultrasonic emulsification technology to prepare micro-nano enzyme-loaded capsules, and the emulsification technology increases the interface area, so the enzymatic reaction occurs faster than the static two-phase plane interface, which is beneficial to the two-phase reaction system or heterogeneous catalytic reaction. The immobilization of the enzyme at the liquid-liquid interface allows the enzyme to contact with the substrate, endowing the capsule with high catalytic efficiency.

4. The capsules prepared by the present invention have high catalytic efficiency and can realize rapid and sensitive visual detection of glucose.

Description of drawings

The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations to the present invention.

Fig. 1 is the picture of the prepared CaS capsule of the embodiment of the present invention 1;

Fig. 2 is the photograph of the transmission electron microscope of the CaS capsule prepared by the embodiment of the present invention 1;

Fig. 3 is the particle size characterization of the CaS capsule prepared in Example 1 of the present invention;

Fig. 4 is the micrograph of the capsule prepared in the embodiment of the present invention 2-6, wherein, Fig. 4 (a) corresponds to the BSA capsule in the embodiment 2, Fig. 4 (b) corresponds to the HSA capsule in the embodiment 3, Fig. 4 ( c) corresponding to the LYZ capsule in Example 4, Figure 4 (d) corresponding to the GOx capsule in Example 5, and Figure 4 (e) corresponding to the HRP capsule in Example 6;

Fig. 5 is the transmission electron micrograph of the capsule prepared in the embodiment of the present invention 2-6, wherein, Fig. 5 (a) corresponds to the BSA capsule in the embodiment 2, Fig. 5 (b) corresponds to the HSA capsule in the embodiment 3, Fig. 5 (c) corresponds to the LYZ capsule in Example 4, Figure 5 (d) corresponds to the GOx capsule in Example 5, and Figure 5 (e) corresponds to the HRP capsule in Example 6;

Fig. 6 is the particle size characterization of the capsules prepared in Examples 2-6 of the present invention;

Figure 7 is the potential characterization of the capsules prepared in Examples 2-6 of the present invention;

Fig. 8 is a micrograph of the GOx-HRP capsule in Example 7 of the present invention;

Figure 9 is a transmission electron micrograph of the GOx-HRP capsule in Example 7 of the present invention;

Figure 10 is the ultraviolet absorption of the GOx capsules in Example 5 of the present invention to prove the occurrence of cascade reactions;

Figure 11 is the ultraviolet absorption of GOx-HRP capsules in Example 7 of the present invention to prove the occurrence of cascade reactions;

Fig. 12 is the kinetic characterization of the GOx-HRP capsule of Example 7 of the present invention;

Fig. 13 is the kinetic characterization of the 5GOx capsule of the embodiment of the present invention;

Figure 14 is a comparison of the enzymatic activity of the GOx-HRP capsule of Example 7 of the present invention, the GOx capsule of Example 5 and the HRP capsule of Example 6 loaded with two enzymes respectively;

Fig. 15 is the ultraviolet absorption color chart of Example 7 of the present invention in which GOx-HRP capsules were incubated with different concentrations of glucose for different times.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the present invention. 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 should be noted that the terminology used here is only for describing specific embodiments, and is not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural, and it should also be understood that when the terms "comprising" and/or "comprising" are used in this specification, they mean There are features, steps, operations, means, components and/or combinations thereof.

Technical scheme of the present invention is as follows:

Provide a kind of preparation method of micronano-loaded enzyme capsule, described material comprises casein (CaS), bovine serum albumin (BSA), human serum albumin (HSA), lysozyme (LYZ), glucose oxidase (GOx) , horseradish peroxidase (HRP), tannic acid (TA), perfluorohexane (PFH). First, we used casein as a model protein to study the preparation of ultrasound-mediated protein capsules. The invention can reduce the size of the protein capsule from the micron level to the nano level by changing the ultrasonic power, so as to realize the controllable adjustment of the particle size of the capsule. In addition, capsules can be prepared in batches by ultrasonic emulsification, which is beneficial to industrial production and application. Tannic acid is added to the system to utilize the strong interaction between protein and polyphenols to stabilize the capsules. Using this strategy, functional proteins such as enzymes are used to prepare capsules, and nanoscale capsules prepared by ultrasound increase the interface area of enzyme catalysis and ensure higher catalytic efficiency. In addition, immobilizing the enzyme on the interface reduces the mass transfer distance between the substrate and the enzyme, which facilitates the efficient progress of the two-step enzyme cascade reaction. The above method is used to prepare a multi-enzyme-loaded capsule that uses glucose oxidase and horseradish peroxidase to detect glucose, so as to realize rapid and sensitive detection of glucose.

The above technical solution uses the amphiphilic protein solution as the water phase and perfluorohexane as the dispersed phase to prepare the emulsion. The emulsion can be obtained without complex surfactants, and then polyphenols are added to stabilize it. In addition, the protein film can be And without the use of strong acid and strong base, the micro-nanocapsules obtained by removing the droplet template by solvent evaporation provide a convenient method for the further preparation of ultrasonic technology to prepare capsules in batches. The capsule prepared by the above technical solution has a simple preparation process and good biocompatibility, and can easily realize the regulation of the particle size of the capsule, and has universal applicability.

Enzyme-loaded capsules are prepared using functional proteins such as enzymes in the above protocol. Multienzyme-catalyzed cascades represent a major class of chemical reactions that play key roles in biological signal transduction and metabolic pathways. A multi-enzyme-loaded capsule for glucose detection using GOx and HRP, the enzyme not only acts as an interface-active emulsifier but also serves as a biocatalytic site. The mass transfer distance between the substrate glucose and the enzyme is reduced, and the visual monitoring of glucose is realized.

The present invention proposes to use volatile perfluorohexane as a sacrificial template to prepare capsules through ultrasonic emulsification technology, which shows good application potential in terms of enhancing enzyme stability and enzyme activity. First, the emulsification technique increases the interface area, so the enzymatic reaction occurs faster than the static two-phase planar interface, which is beneficial to the two-phase reaction system or heterogeneous catalytic reaction. The immobilization of the enzyme at the liquid-liquid interface allows the enzyme to contact with the substrate, endowing the capsule with high catalytic efficiency.

In a specific embodiment of the present invention, a method for preparing micronano enzyme-loaded capsules is provided, and the materials required for the preparation include protein, tannic acid, and perfluorohexane.

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

The specific preparation methods include:

The protein is dissolved in phosphate buffer, and then perfluorohexane is slowly added therein, the micro-nano emulsion is prepared by ultrasonication, and then tannic acid solution is added, and heated.

The above preparation method is a universal method for preparing micronano enzyme-loaded capsules in the present invention. The preparation of bovine serum albumin, human serum albumin, lysozyme, glucose oxidase and horseradish peroxidase capsules by the above method proves that the method has universal applicability.

The preparation method of the ultrasound-mediated enzyme-loaded capsule is simple, which is conducive to mass production. For example, ultrasound is used for mass production, and the template is removed by heating to obtain an enzyme-loaded capsule glucose sensor.

In yet another embodiment of the present invention, 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 0.1-100 mg/mL, more preferably 1-50 mg/mL, still more preferably 2-10 mg/mL.

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

In yet another specific embodiment of the present invention, the dosage of the phosphate buffer is 4 mL, and the pH is 7.4.

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

In yet another specific embodiment of the present invention, the volume ratio of protein solution to perfluorohexane is 1:4-40, preferably 1:20.

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

In yet another specific embodiment of the present invention, the specific conditions of ultrasound are: under the condition of 30-250W ultrasound, ultrasound for 45s-10min, preferably 150W ultrasound for 2min.

In yet another specific embodiment of the present invention, the specific heating conditions are: heating at 30-70° C. for 2-10 minutes, preferably 65° C. for 10 minutes under the condition of rotary steaming.

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

In yet another specific embodiment of the present invention, a micronano enzyme-loaded capsule is provided, which is prepared by the above method.

In yet another specific embodiment of the present invention, an application of the above-mentioned micronano enzyme-loaded capsules in rapid and sensitive detection of glucose.

In order to enable those skilled in the art to understand the technical solution of the present invention more clearly, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

Example 1

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

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

(3) Put 2 mL of solution A in a 5 mL centrifuge tube, and add 100 μL of perfluorohexane into solution A.

(4) Using a probe-type ultrasound, place the probe at the interface between the protein solution and the perfluorohexane solution, ultrasonicate at a power of 150 W for 2 minutes, and ice-bath, and add an ice-bath to obtain emulsion C.

(5) Take 10mL of solution B, add the solution B dropwise to the emulsion C under the vortex mixer, and vortex to mix, the emulsion is steamed at 65°C for 10min to obtain the final product capsule, CaS capsule.

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

Example 2

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

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

(3) Put 2 mL of solution A in a 5 mL centrifuge tube, and add 100 μL of perfluorohexane into solution A.

(4) Using a probe-type ultrasound, place the probe at the interface between the protein solution and the perfluorohexane solution, and sonicate for 2 minutes with a power of 50W. An ice bath was added to obtain emulsion C.

(5) Take 10 μL of solution B, add solution B dropwise to emulsion C under a vortex mixer, and vortex to mix evenly, and spin-steam the emulsion at 65°C for 10 minutes to obtain the final product, enzyme-loaded capsules, BSA capsules.

The microscope photo of the BSA capsule is shown in Figure 4(a), the transmission electron microscope photo is shown in Figure 5(a), the particle size characterization is shown in Figure 6, and the potential characterization is shown in Figure 7.

Example 3

(1) Accurately weigh 10 mg of human serum albumin, and add 4 mL of phosphate buffered saline solution (pH 7.4) therein to obtain solution A.

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

(3) Put 2 mL of solution A in a 5 mL centrifuge tube, and add 100 μL of perfluorohexane into solution A.

(4) Using a probe-type ultrasound, place the probe at the interface between the protein solution and the perfluorohexane solution, and sonicate for 2 minutes with a power of 50W. An ice bath was added to obtain emulsion C.

(5) Take 10 μL of solution B, add solution B dropwise to emulsion C under a vortex mixer, and vortex to mix evenly, and spin-steam the emulsion at 65°C for 10 minutes to obtain the final product, enzyme-loaded capsules, HSA capsules.

The microscope photo of the HSA capsule is shown in Figure 4(b), the transmission electron microscope photo is shown in Figure 5(b), the particle size characterization is shown in Figure 6, and the potential characterization is shown in Figure 7.

Example 4

(1) Accurately weigh 10 mg of lysozyme, and add 4 mL of phosphate buffer solution (pH 7.4) to it to obtain solution A.

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

(3) Put 2 mL of solution A in a 5 mL centrifuge tube, and add 100 μL of perfluorohexane into solution A.

(4) Using a probe-type ultrasound, place the probe at the interface between the protein solution and the perfluorohexane solution, and sonicate for 2 minutes with a power of 50W. An ice bath was added to obtain emulsion C.

(5) Take 10 μL of solution B, add solution B dropwise to emulsion C under a vortex mixer, and vortex to mix well, and spin-steam the emulsion at 65°C for 10 minutes to obtain the final product, enzyme-loaded capsules, LYZ capsules.

The microscope photo of the LYZ capsule is shown in Figure 4(c), the transmission electron microscope photo is shown in Figure 5(c), the particle size characterization is shown in Figure 6, and the potential characterization is shown in Figure 7.

Example 5

(1) Accurately weigh 10 mg of glucose oxidase, and add 4 mL of phosphate buffered saline solution (pH 7.4) therein to obtain solution A.

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

(3) Put 2 mL of solution A in a 5 mL centrifuge tube, and add 100 μL of perfluorohexane into solution A.

(4) Using a probe-type ultrasound, place the probe at the interface between the protein solution and the perfluorohexane solution, and sonicate for 2 minutes with a power of 50W. An ice bath was added to obtain emulsion C.

(5) Take 10 μL of solution B, add solution B dropwise to emulsion C under a vortex mixer, and vortex to mix well, and spin-steam the emulsion at 65°C for 10 minutes to obtain the final product, enzyme-loaded capsules, GOx capsules.

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

Example 6

(1) Accurately weigh 10 mg of horseradish peroxidase, and add 4 mL of phosphate buffer solution (pH 7.4) therein to obtain solution A.

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

(3) Put 2 mL of solution A in a 5 mL centrifuge tube, and add 100 μL of perfluorohexane into solution A.

(4) Using a probe-type ultrasound, place the probe at the interface between the protein solution and the perfluorohexane solution, and sonicate for 2 minutes with a power of 50W. An ice bath was added to obtain emulsion C.

(5) Take 10 μL of solution B, add solution B dropwise to emulsion C under a vortex mixer, and vortex to mix evenly, and spin-steam the emulsion at 65°C for 10 minutes to obtain the final product, enzyme-loaded capsules, HRP capsules.

The microscope photo of the HRP capsule is shown in Figure 4(e), the transmission electron microscope photo is shown in Figure 5(e), the particle size characterization is shown in Figure 6, and the potential characterization is shown in Figure 7.

Example 7

(1) Accurately weigh 10 mg of glucose oxidase, and add 4 mL of phosphate buffered saline solution (pH 7.4) therein to obtain solution A. 10 mg of horseradish peroxidase was accurately weighed, and 4 mL of phosphate buffered saline solution (pH 7.4) was added thereto to obtain solution B.

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

(3) Take 1mL of solution A, take 1mL of solution B in a 5mL centrifuge tube and vortex with a vortex mixer to obtain solution C, take 100 μL of perfluorohexane and add to solution C.

(4) Using a probe-type ultrasound, place the probe at the interface between the protein solution and the perfluorohexane solution, and sonicate for 2 minutes with a power of 50W. Emulsion D is obtained.

(5) Take 10 μL of solution B, add solution B dropwise to emulsion D under a vortex mixer, and vortex to mix, and spin-steam the emulsion at 65°C for 10 minutes to obtain the final product, enzyme-loaded capsules, GOx-HRP capsules.

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

Hydrogel performance testing

(1) Detection of H ₂ O ₂

Take 300 μL of glucose oxidase capsules (225 μg/mL) and disperse them in water to 5 mL, then add 1.5 mL of glucose solution, incubate for 60 min, add 300 μL of titanyl ammonium oxalate solution to the mixed solution, and detect the absorbance of the solution at 405 nm with ultraviolet light, and monitor the kinetics. The solution is yellow, and Figure 10 proves that the solution has absorption at 405nm, which proves the generation of hydrogen peroxide.

(2) Catalytic activity test of GOx-HRP capsules

Take 100 μL of glucose oxidase-horseradish peroxidase capsules and disperse them in water to 5 mL, then add 100 μL of glucose solution and 3,3',5,5'-tetramethylbenzidine (TMB) to it, and use citric acid- Add disodium hydrogen phosphate buffer solution to 3mL, incubate for 20min, and use ultraviolet light to detect the absorbance of the solution at 652nm. As the reaction progresses, the solution turns blue. Figure 11 proves that a characteristic absorption peak appears at 652nm in the presence of glucose, proving the cascade The occurrence of the reaction and the monitoring kinetics are shown in Figure 12 and Figure 13. Within a certain period of time, as the reaction progresses, the color of the solution deepens and the absorbance increases.

Prepare the same concentrations of enzyme vesicle suspension (25 μL), glucose solution (100 μL, 25 mM) and 3,3′,5,5′-tetramethylbenzidine solution (100 μL, 25 μg/mL, DMSO), the catalytic activities of multi-enzyme-loaded capsules (GOx-HRP capsules) and single-enzyme-loaded capsules (GOx, HRP capsules) were quantitatively compared by the above method. Enzyme concentration was quantified by fluorescence spectroscopy. It can be seen from Figure 14 that the catalytic activity of the capsules loaded with two enzymes is much higher than that of GOx capsules and HRP capsules loaded with two enzymes respectively. The reason is that the capsules loaded with two enzymes immobilize the enzymes on the interface, and the two The evenly dispersed interface of the enzyme shortens the mass transfer distance between the substrate and the enzyme and improves the catalytic activity of the enzyme.

(3) GOx-HRP capsules detect different concentrations of glucose

Take glucose oxidase-horseradish peroxidase capsules and disperse them in water, then add glucose solutions of different concentrations and 3,3',5,5'-tetramethylbenzidine solutions to incubate for different times and then mix the solution , and use a microplate reader to detect the absorbance of the solution at 652nm, as shown in Figure 15, different concentrations of glucose and capsules were co-incubated for different times, with the increase of glucose concentration or the extension of incubation time, the absorbance increased, within 3min Colors that can be distinguished by naked eyes can be displayed within a short period of time, which proves that the invention realizes the visual monitoring of glucose in a short period of time.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. A preparation method for micro-nano enzyme-carrying capsules, characterized in that the materials required for the preparation include protein, tannic acid, and perfluorohexane; The specific preparation method is: dissolving protein in phosphate buffer, then slowly adding perfluorohexane to it, and ultrasonically preparing micro-nano emulsion; Ultrasonic emulsification method is used to prepare micro-nano-emulsion emulsion, and its oil phase components can be comfrey oil, squalene and other oil components, and micro-nano-emulsion can be obtained; After ultrasonic emulsification to obtain perfluorohexane emulsion, add tannic acid solution and heat to obtain it; The protein is selected from casein, bovine serum albumin, human serum albumin, lysozyme, glucose oxidase or horseradish peroxidase or a mixture of glucose oxidase and horseradish peroxidase. 2. the preparation method of a kind of micronano enzyme-carrying capsule as claimed in claim 1, is characterized in that, when described protein is casein, bovine serum albumin, human serum albumin, lysozyme, glucose oxidase or spicy In the case of one of the root peroxidases, the concentration of the protein solution is controlled to be 0.1-100 mg/mL, more preferably 1-50 mg/mL, even more preferably 2-10 mg/mL; Or, when the protein is a mixture of glucose oxidase or horseradish peroxidase, the concentration of glucose oxidase is controlled to be 0.1-100 mg/mL, more preferably 1-50 mg/mL, even more preferably 2-10 mg /mL; the concentration of horseradish peroxidase is controlled to be 0.1～100mg/mL, more preferably 1～50mg/mL, more preferably 2～10mg/mL; two kinds of enzyme solutions are mixed; The mass ratio of oxidase to glucose oxidase is 1:1-5, preferably 1:1. 3. the preparation method of a kind of micronano enzyme-carrying capsule as claimed in claim 1, is characterized in that, the consumption of described phosphate buffer is 4mL, and pH is 7.4; Further, the addition amount of the perfluorohexane is controlled to be 5-1000 μL, preferably 10-500 μL, more preferably 50-200 μL. 4. The method for preparing micronano enzyme-loaded capsules according to claim 1, wherein the volume ratio of the protein solution to perfluorohexane is 1:4-40, preferably 1:20. 5. The preparation method of a kind of micronano enzyme-carrying capsule as claimed in claim 1, is characterized in that, the concentration control of described tannic acid solution is 1～150mg/mL, is preferably 20～100mg/mL, more preferably 30~50mg/mL. 6. The preparation method of a micro-nano enzyme-loaded capsule as claimed in claim 1, wherein the specific conditions of the ultrasound are: under the condition of 30-250W ultrasound, ultrasound for 45s-10min, preferably 150W ultrasound for 2min . 7. The preparation method of a micro-nano enzyme-loaded capsule as claimed in claim 1, characterized in that, the specific conditions for heating are: under the condition of rotary steaming at 30-70°C, heating for 2-10min, preferably 65°C ℃ heating for 10min. 8 . The method for preparing micronano enzyme-loaded capsules according to claim 1 , wherein the specific centrifugation conditions are: centrifugation at 1000-8000 rpm for 1-10 minutes, preferably 2500 rpm for 5 minutes. 9. A micronano enzyme-loaded capsule, characterized in that it is prepared by the method according to any one of claims 1-8. 10. the application of a kind of micronano enzyme-carrying capsule described in claim 9 in the rapid and sensitive detection of glucose.

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