Microneedle patch, preparation method thereof and method for colorimetrically detecting blood glucose

Abstract

The invention relates to a microneedle patch, a preparation method thereof and a blood glucose colorimetric detection method, and belongs to the technical field of biomedicine. The microneedle patch comprises a needle tip layer and a backing layer, the needle tip layer is a methacrylated natural biopolymer matrix in which immobilized enzyme is uniformly distributed, and the backing layer is a methacrylated natural biopolymer matrix in which a chromogenic substrate is uniformly distributed, the immobilized enzyme is composed of a zinc-based imidazoline metal organic framework MAF-7 carrier and a natural enzyme loaded in the framework. The immobilized enzyme with double-enzyme activity is loaded into the needle tip, the spatial distance of the cascade catalytic reaction is limited in the metal organic framework, the mass transfer efficiency is high, the reaction is rapid, the natural enzyme is encapsulated in the metal organic framework MAF-7, and the stability of the enzyme is enhanced; according to the method, color development is carried out by firstly extracting the interstitial fluid and then dropwise adding the color development substrate, the color development effect is obvious, and the time is shorter.

Description

Technical Field

[0001] The invention relates to a microneedle patch and a preparation method thereof and a method for colorimetrically detecting blood sugar, belonging to the technical field of biomedicine. Background Art

[0002] Using microneedle patches to extract skin interstitial fluid for related biomarker detection is a new sampling method that has become popular in recent years. After the hydrogel microneedle patch is inserted into the skin, corresponding micropores will appear on the skin. These micropores will leak interstitial fluid to swell the needle tip of the hydrogel microneedle retained on the skin, and then convert the concentration of the biomarker into a corresponding color change or electrical signal through a corresponding biochemical reaction with the reactants in the microneedle, and then recognize it through a smart device. Using microneedles to perform colorimetric analysis of biomarkers is a common method for microneedle detection. The most common method for using microneedle patches to detect blood sugar by colorimetric analysis is to load glucose oxidase, horseradish peroxidase, and a chromogenic substrate into a microneedle patch and react with glucose in the interstitial fluid to produce a cascade catalytic color reaction. However, this enzyme colorimetric analysis method usually has poor results due to the poor stability of natural enzymes, and often due to insufficient interstitial fluid extracted by the hydrogel microneedle, resulting in insufficient reaction, and poor in-situ color development results. In previous studies, the color development of microneedle patches was mainly achieved by the color substrate in the backing layer. The color development effect of this color development method is limited by the amount of color development substrate added to the backing layer. Since the color development substrate is usually prepared using organic solvents such as ethanol, when preparing the backing layer of the microneedle patch, adding too much volume will lead to poor formability of the microneedle patch. And when the concentration of the color development substrate is increased, it is easy to cause problems such as poor biocompatibility. Therefore, it is necessary to find a color development method that can achieve sufficient color development without affecting the formability of the microneedle. Summary of the invention

[0003] In view of the technical defects of the existing microneedle patch colorimetric blood sugar detection method, the present invention provides a microneedle patch and a preparation method thereof and a method for colorimetric blood sugar detection, the purpose of which is to first extract glucose in the interstitial fluid through the microneedle patch to produce a cascade catalytic reaction with an immobilized enzyme with dual enzyme activity loaded on the needle tip layer, and then add a chromogenic substrate ethanol solution to color it after taking it out, thereby converting the blood sugar concentration into a color change visible to the naked eye to achieve blood sugar detection, avoiding the continuous pain caused by puncture and blood sampling used in traditional blood sugar monitoring, and solving the problems of uneven color development, poor color development, and long color development time of the colorimetric microneedle patch.

[0004] A microneedle patch, comprising a needle tip layer and a backing layer, wherein the needle tip layer is a methacrylylated natural biopolymer matrix with an immobilized enzyme evenly distributed, and the backing layer is a methacrylylated natural biopolymer matrix with a chromogenic substrate evenly distributed, wherein:

[0005] The immobilized enzyme is composed of a zinc-based imidazoline metal organic framework MAF-7 carrier and a natural enzyme loaded in the framework, wherein:

[0006] The natural enzyme is one or more of glucose oxidase, lactose oxidase, maltase, horseradish peroxidase and catalase.

[0007] Furthermore, the natural enzyme is a combination of glucose oxidase and horseradish peroxidase in a mass ratio of 1 to 4:1.

[0008] Furthermore, the immobilized enzyme in the needle tip layer is evenly distributed inside and on the surface of the methacrylylated natural biopolymer matrix.

[0009] Furthermore, the color developing substrate in the backing layer is evenly distributed inside and on the surface of the methacrylylated natural biopolymer matrix.

[0010] The microneedle patch of the present invention, the immobilized enzyme is prepared by the following method:

[0011] Zinc acetate dihydrate, 3-methyl-1H-1,2,4-triazole and natural enzyme are dissolved in water, and ammonia water is added, and the mixture is stirred at 100-500 rpm for 6-24 hours, and then washed and freeze-dried to obtain an immobilized enzyme.

[0012] In the above technical solution, the molar ratio of zinc acetate dihydrate to 3-methyl-1H-1,2,4-triazole is 1:4-8.

[0013] In the above technical solution, the mass volume ratio of zinc acetate dihydrate to the reaction solution is 5-10 mg / mL.

[0014] In the above technical solution, the volume ratio of the ammonia water to the reaction solution is 1 to 5:100.

[0015] Furthermore, the volume fraction of the ammonia water is 10%.

[0016] In the above technical solution, the mass volume ratio of the natural enzyme to the reaction solution is 1 to 5:1 mg / mL.

[0017] The microneedle patch of the present invention, the methacrylated natural biopolymer is prepared by the following method:

[0018] After the natural biopolymer is dissolved in water, methacrylic anhydride is slowly added to carry out a grafting reaction, and the reaction is carried out at 30-60°C for 0.5-2.5 hours, followed by dialysis, and then freeze-drying to obtain the methacrylated natural biopolymer.

[0019] In the above technical solution, the mass volume ratio of the natural biopolymer to water is 50-150 mg / mL.

[0020] In the above technical solution, the mass volume ratio of the natural biopolymer to methacrylic anhydride is 1 to 1.5 g / mL.

[0021] In the microneedle patch of the present invention, the natural biopolymer is one or more of sodium alginate, cellulose, chitosan, gelatin, silk fibroin, and polypeptide.

[0022] Preferably, the methacrylated natural biopolymer is methacrylated gelatin.

[0023] In the microneedle patch of the present invention, the color developing substrate is one or more of 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt.

[0024] Preferably, the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine.

[0025] The needle tip layer of the microneedle patch of the present invention also includes a crosslinking agent and solvent water required for dissolving the modified natural biopolymer and the immobilized enzyme.

[0026] The backing layer of the microneedle patch of the present invention further comprises a crosslinking agent, water solvent required for dissolving the modified natural biopolymer, and ethanol solution solvent required for dissolving the color-developing substrate.

[0027] Another object of the present invention is to provide a method for preparing the above-mentioned microneedle patch.

[0028] A method for preparing a microneedle patch comprises the following steps:

[0029] S1: dissolving the immobilized enzyme in a mixed matrix solution consisting of a methacrylylated natural biopolymer matrix aqueous solution and a cross-linking agent to obtain a needle tip layer solution; mixing a color developing substrate ethanol solution with a solution consisting of a methacrylylated natural biopolymer matrix aqueous solution and a cross-linking agent to obtain a backing layer solution;

[0030] S2: Add the needle tip layer solution into the PDMS mold, centrifuge once to allow the needle tip to be filled with the needle tip solution, then add the backing layer solution on the needle tip layer, dry and demould it, place it under ultraviolet light for cross-linking, and obtain a microneedle patch.

[0031] In the above technical solution, the concentration of the methacrylated natural biopolymer in the needle tip layer is 10 to 40 wt %.

[0032] In the above technical solution, the concentration of the methacrylated natural biopolymer in the backing layer is 10-40 wt %.

[0033] Preferably, the concentration of the methacrylated natural biopolymer in the needle tip layer and the backing layer is 15-30 wt %.

[0034] In the above technical solution, the concentration of the immobilized enzyme in the needle tip layer is 0.5-3 mg / mL.

[0035] In the above technical solution, the concentration of the chromogenic substrate ethanol solution in the backing layer is 5 to 20 mmol / L.

[0036] Furthermore, the volume ratio of the color developing substrate ethanol solution to the solution consisting of the methacrylylated natural biopolymer matrix aqueous solution and the cross-linking agent is 0.5 to 2:100.

[0037] Furthermore, the concentration of the ethanol solution of the color developing substrate in the backing layer is within the above range so as not to cause the microneedle patch to develop color quickly.

[0038] In the above technical solution, the cross-linking agent is one or more of the photoinitiators TPO, I2959, and LAP.

[0039] Preferably, the crosslinking agent is photoinitiator I2959.

[0040] In the above technical solution, the concentration of the cross-linking agent in the needle tip layer and the backing layer solution is 1-5 mg / mL.

[0041] In the above technical solution, the ultraviolet light cross-linking time is 3 to 15 minutes.

[0042] Another object of the present invention is to provide a method for colorimetric detection of blood sugar.

[0043] A method for colorimetric detection of blood sugar comprises the following steps: dripping a colorimetric substrate ethanol solution onto the needle tip layer of a microneedle patch that has fully absorbed interstitial fluid, and the added colorimetric substrate ethanol solution enters the pores of the needle tip layer of the microneedle patch to trigger a color reaction.

[0044] Furthermore, the concentration of the added chromogenic substrate ethanol solution is 5 to 20 mmol / L, and the added amount is 10 to 100 μL.

[0045] Furthermore, the microneedle patch that fully absorbs interstitial fluid is a microneedle patch obtained by piercing the skin with the microneedle patch to generate interstitial fluid and cause the microneedle tips to swell.

[0046] The method for colorimetric detection of blood sugar by microneedle patch of the present invention, leaves micropores on the skin surface through the microneedle needle tip, uses the interstitial fluid that seeps out of the micropore to make the needle tip partially swell, the glucose in the interstitial fluid reacts with the immobilized enzyme loaded by the needle tip to generate hydrogen peroxide, and after dripping the chromogenic substrate ethanol solution, it and the small amount of chromogenic substrate in the original backing layer jointly produce a cascade catalytic reaction, showing the color change related to the glucose concentration, and the color development is obvious, which can be judged by the naked eye. Compared with dripping the chromogenic substrate ethanol solution after taking out the microneedle patch, adding the chromogenic substrate in the backing layer in advance and then adopting the above-mentioned color development scheme can speed up the color development process in time and space. Since the chromogenic substrate needs to enter through the swelling pores of the cross-linked microneedle patch and accumulate to an amount that can trigger the color development reaction, the process requires a certain amount of time. A small amount of chromogenic substrate is added in advance to the backing layer, and the color development reaction is triggered when the added chromogenic substrate just enters the pores of the microneedle patch, saving the time required for color development. In addition, after the microneedle patch prepared using the traditional in-situ colorimetric method is exposed to the interstitial fluid containing glucose and swells, the hydrogen peroxide generated in the first stage of the cascade catalysis needs to pass through the pores generated after the hydrogel microneedle patch swells, and then contact the chromogenic substrate of the backing layer and trigger the second stage of the cascade catalytic reaction. There will be a certain loss in this process, and the chromogenic substrate and hydrogen peroxide two substrates participating in the cascade catalytic reaction have a certain spatial distance. The microneedle patch prepared by the present invention is colored by adding a color developer, and the color developer enters the internal structure of the microneedle patch through the swollen pores, and directly contacts the hydrogen peroxide generated by the first stage reaction, thereby shortening the spatial distance required for the reaction and enhancing the color development effect.

[0047] The beneficial effects of the present invention are:

[0048] 1. The present invention provides a microneedle patch and a preparation method thereof and a method for colorimetric detection of blood sugar, which can be used for minimally invasive detection of blood sugar. The interstitial fluid generated by the microneedle patch piercing the skin causes the microneedle tip to partially swell, and the glucose in the interstitial fluid reacts with the natural enzyme active site in the immobilized enzyme loaded on the needle tip to generate hydrogen peroxide. Subsequently, in the presence of a small amount of chromogenic substrate in the backing layer, the chromogenic substrate ethanol solution is added again, and the hydrogen peroxide reacts with the natural enzyme active site in the immobilized enzyme to oxidize the chromogenic substrate and produce a color change. Compared with the detection mode of traditional commercial blood glucose meters, the pain caused by using a colorimetric microneedle patch to detect blood sugar is lower, and the detection results are accurate, intuitive, and easy to operate.

[0049] 2. The present invention loads the immobilized enzyme containing dual enzyme activity into the needle tip, limits the spatial distance of the cascade catalytic reaction within the metal organic framework, has high mass transfer efficiency, and reacts rapidly. Most of the existing colorimetric microneedle patches load the natural enzymes on the needle tip part and the backing layer part of the microneedle, respectively. In this mode, the mass transfer distance of glucose and the primary product hydrogen peroxide is long, and the loss is large, resulting in poor final color development. In addition, the present invention encapsulates the natural enzyme in the hydrophilic metal organic framework MAF-7, enhances the stability of the enzyme, and avoids the problem that the natural enzyme is easily inactivated and causes poor color development.

[0050] 3. The present invention adopts the method of extracting interstitial fluid first and then adding the chromogenic substrate to develop color, and the color development effect is obvious and the time is shorter. The existing colorimetric microneedle patch adds the chromogenic substrate to the microneedle matrix solution, but the color development effect is poor due to the addition of too little chromogenic substrate. In addition, after the chromogenic substrate is added again in the present invention, the cascade catalytic reaction is more sufficient and the color development effect is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a photo of the colorimetric microneedle patch obtained in Example 1.

[0052] Figure 2 This is a schematic diagram of RGB analysis of the colorimetric microneedle patch obtained in Example 1 after reacting in different glucose solutions.

[0053] Figure 3 This is the SEM image of the immobilized enzyme GOx / HRP@MAF-7 obtained in Example 1.

[0054] Figure 4 This is the SEM image of the colorimetric microneedle patch obtained in Example 1.

[0055] Figure 5 The color development of the colorimetric microneedle patch obtained in Example 2 after reacting in glucose solutions of different concentrations.

[0056] Figure 6 The color development of the immobilized enzyme GOx@ZIF-67 (left) obtained in Comparative Example 1 and the GOx / HRP@MAF-7 (right) obtained in Example 1 after reacting with 8 mmol / L of glucose.

[0057] Figure 7 The color development of the immobilized enzyme GOx / HRP@ZIF-8 (left) obtained in Comparative Example 2 and the GOx / HRP@MAF-7 (right) obtained in Example 1 after reacting with 8 mmol / L of glucose.

[0058] Figure 8 This is the swelling diagram of the colorimetric microneedle patch obtained in Comparative Example 3 under a shorter UV crosslinking time.

[0059] Fig. 9 The colorimetric microneedle patches with two needle tip lengths in Comparative Example 4 were used to detect the color development of a 5 mmol / L glucose solution.

[0060] Fig.10 These are the color images of the microneedle patch A in comparative example 5 in which no chromogenic substrate is added to the backing layer, the microneedle patch B in Example 1, and the in situ colorimetric microneedle patch C in comparative example 5 in which the backing layer contains a high concentration of chromogenic substrate. DETAILED DESCRIPTION

[0061] The following non-limiting embodiments may enable a person skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0062] The test methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0063] Example 1

[0064] A method for preparing a microneedle patch comprises the following steps:

[0065] (1) Zinc acetate dihydrate and 3-methyl-1H-1,2,4-triazole solids in a molar ratio of 1:4, glucose oxidase (GOx) and horseradish peroxidase (HRP) solids in a mass ratio of 2:1, and 60 μL of 10% ammonia water were added to 3 mL of water, stirred at 300 rpm for 24 h at room temperature, and then washed and freeze-dried to obtain immobilized enzyme GOx / HRP@MAF-7;

[0066] (2) After dissolving 10 wt % gelatin in water, 8 mL of methacrylic anhydride was slowly added to carry out grafting reaction, reacted at 60° C. for 2 h, and then dialyzed. After the dialysis, freeze-dried to obtain methacrylated gelatin;

[0067] (3) preparing a 20% aqueous solution of methacryloyl gelatin, adding a photoinitiator I2959 at a concentration of 2 mg / mL to obtain a matrix solution, mixing the GOx / HRP@MAF-7 prepared in step (1) with the matrix solution to make the concentration of GOx / HRP@MAF-7 2 mg / mL to prepare a needle tip layer solution; preparing a 20% aqueous solution of methacryloyl gelatin, adding a photoinitiator I2959 at a concentration of 2 mg / mL, mixing the solution with a 12 mmol / L 3,3',5,5'-tetramethylbenzidine ethanol solution at a volume ratio of 100:1 to obtain a backing layer solution;

[0068] (4) Add the needle tip layer solution into the PDMS mold, centrifuge once to allow the needle tip to be filled with the needle tip solution, then add the backing layer solution on the needle tip layer, wait for drying and demolding, and after demolding, place it under ultraviolet light for cross-linking for 7 minutes to obtain a microneedle patch.

[0069] Figure 1 This is a photo of the colorimetric microneedle patch obtained above, which was used to detect blood sugar in the interstitial fluid of the skin. The glucose contained in the interstitial fluid undergoes a cascade catalytic reaction with the immobilized enzyme and specific substrate in the microneedle tip, thereby producing a color change related to the glucose concentration. After absorbing the interstitial fluid, 50μL of 12mmol / L 3,3',5,5'-tetramethylbenzidine ethanol solution was dripped onto the microneedle patch, and the colored microneedle patch was subjected to RGB analysis. The schematic diagram of the RGB analysis of the color development of the colorimetric microneedle patch after reacting in glucose solutions of different concentrations is shown in Figure 2 , it can be seen that: as the glucose concentration increases, the microneedle patch shows a concentration-related color change; the higher the glucose concentration, the darker the color of the microneedle patch. By analyzing the RGB values ​​of the extracted microneedle patch, the relationship between the color of the microneedle patch and the glucose concentration can be more intuitively seen. It can be seen that the microneedle patch provided in this embodiment can be used for the analysis and detection of glucose, and the operation is simple and the effect is intuitive.

[0070] The immobilized enzyme GOx / HRP@MAF-7 obtained in this example was scanned by SEM. Figure 3 It can be seen that the immobilized enzyme GOx / HRP@MAF-7 obtained in this example has a typical regular dodecahedron structure and a particle size of about 1.5 μm.

[0071] The microneedle patch obtained in this example was scanned by SEM. Figure 4 It can be seen that the microneedle patch obtained in this embodiment has a good morphology, a complete pyramid-shaped needle tip, a needle tip length of 1000 μm, a needle bottom diameter of 500 μm, and evenly distributed GOx / HRP@MAF-7 particles can be seen on the surface of the microneedle tip layer.

[0072] Example 2

[0073] A method for preparing a microneedle patch comprises the following steps:

[0074] (1) Zinc acetate dihydrate and 3-methyl-1H-1,2,4-triazole solids in a molar ratio of 1:4, glucose oxidase (GOx) and horseradish peroxidase (HRP) solids in a mass ratio of 1:1, and 60 μL of 10% ammonia water were added to 3 mL of water, stirred at 300 rpm for 24 h at room temperature, and then washed and freeze-dried to obtain immobilized enzyme GOx / HRP@MAF-7;

[0075] (2) After dissolving 10 wt % gelatin in water, 10 mL of methacrylic anhydride was slowly added to carry out grafting reaction, reacted at 60° C. for 2 h, and then dialyzed. After the dialysis, freeze-dried to obtain methacrylated gelatin;

[0076] (3) preparing a 20% aqueous solution of methacryloyl gelatin, adding a photoinitiator I2959 at a concentration of 3 mg / mL, mixing the matrix solution with the GOx / HRP@MAF-7 prepared in step (1) to make the concentration of GOx / HRP@MAF-7 2 mg / mL, and preparing a needle tip layer solution; preparing a 20% aqueous solution of methacryloyl gelatin, adding a photoinitiator I2959 at a concentration of 3 mg / mL, and mixing the solution with a 12 mmol / L 3,3',5,5'-tetramethylbenzidine ethanol solution at a volume ratio of 150:1 to obtain a backing layer solution;

[0077] (4) Add the needle tip layer solution into the PDMS mold, centrifuge once to fill the needle tip with the needle tip solution, then add the backing layer solution on the needle tip layer, wait for drying and demoulding. After demoulding the microneedle patch, place it under ultraviolet light for crosslinking for 9 minutes to obtain a microneedle patch.

[0078] Microneedle patch detection of glucose: The microneedle patch was inserted into agarose gel containing different glucose concentrations, and then 50 μL of 12 mmol / L 3,3',5,5'-tetramethylbenzidine ethanol solution was added to the microneedle patch. The color state was shown in Figure 5 , it can be seen that the microneedle patch can produce concentration-related color changes in response to different glucose concentrations.

[0079] Example 3

[0080] A method for preparing a microneedle patch comprises the following steps:

[0081] (1) Zinc acetate dihydrate and 3-methyl-1H-1,2,4-triazole solids in a molar ratio of 1:4, glucose oxidase (GOx) and horseradish peroxidase (HRP) solids in a mass ratio of 3:1, and 60 μL of 10% ammonia water were added to 3 mL of water, stirred at 300 rpm for 24 h at room temperature, and then washed and freeze-dried to obtain immobilized enzyme GOx / HRP@MAF-7;

[0082] (2) After dissolving 10 wt % gelatin in water, 6 mL of methacrylic anhydride was slowly added to carry out grafting reaction, reacted at 60° C. for 2 h, and then dialyzed. After the dialysis, freeze-dried to obtain methacrylated gelatin;

[0083] (3) preparing a 20% aqueous solution of methacryloyl gelatin, adding a photoinitiator I2959 at a concentration of 5 mg / mL, mixing the matrix solution with the GOx / HRP@MAF-7 prepared in step (1) to make the concentration of GOx / HRP@MAF-7 2 mg / mL, and preparing a needle tip layer solution; preparing a 20% aqueous solution of methacryloyl gelatin, adding a photoinitiator I2959 at a concentration of 5 mg / mL, and mixing the solution with a 12 mmol / L 3,3',5,5'-tetramethylbenzidine ethanol solution at a volume ratio of 120:1 to obtain a backing layer solution;

[0084] (4) Add the needle tip layer solution into the PDMS mold, centrifuge once to fill the needle tip with the needle tip solution, then add the backing layer solution on the needle tip layer, wait for drying and demoulding. After demoulding the microneedle patch, place it under ultraviolet light for crosslinking for 8 minutes to obtain a microneedle patch.

[0085] Comparative Example 1

[0086] The difference between this example and Example 1 is that the immobilized enzyme metal organic framework used is ZIF-67, and only glucose oxidase is immobilized.

[0087] The synthesis method of immobilized enzyme GOx@ZIF-67 is as follows: take 3 mL of water into a sample bottle, first add 46.2 mg of 2-methylimidazole, shake well to completely dissolve it, then add 3 mg of glucose oxidase, shake well to completely dissolve it, and finally add 34.9 mg of cobalt nitrate hexahydrate (Co(NO3)2·6H2O), shake well, the solution immediately turns purple, and age in a refrigerator at 4°C for 24 hours. After washing, freeze-drying is performed to obtain immobilized enzyme GOx@ZIF-67.

[0088] Figure 6 The color development of GOx@ZIF-67 (left) and GOx / HRP@MAF-7 (right) after reacting with 8mmol / L glucose. It can be seen that GOx@ZIF-67 (left) has a low responsiveness to glucose and a lighter color. After ZIF-67 encapsulates glucose oxidase, it can have glucose oxidase activity and peroxidase activity. However, after glucose oxidase reacts with glucose, gluconic acid will lower the pH of the reaction system, which will lead to partial degradation of ZIF-67 and weaken the activity of peroxidase, resulting in poor color development.

[0089] Comparative Example 2

[0090] The difference between this example and Example 1 is that the metal organic framework used for immobilized enzyme is ZIF-8.

[0091] The synthesis method of immobilized enzyme GOx / HRP@ZIF-8 is as follows: take 3 mL of water in a sample bottle, first add 46.2 mg of imidazole-2-carboxaldehyde, shake well to dissolve it completely, then add 1 mg of GOx, shake well to dissolve it completely, and finally add 26.3 mg of zinc acetate (Zn(CH3COOH)2·2H2O), shake well, stir for 30 minutes, and then age in a refrigerator at 4°C for 24 hours, wash and freeze-dry to obtain immobilized enzyme GOx / HRP@ZIF-8.

[0092] Figure 7 The color development of GOx / HRP@ZIF-8 (left) and GOx / HRP@MAF-7 (right) after reacting with 8mmol / L glucose. It can be seen that GOx / HRP@ZIF-8 (left) has low responsiveness to glucose and the color is lighter, so it cannot be used for colorimetric analysis of glucose. ZIF-8 is less hydrophilic than MAF-7, so its affinity for enzymes is lower. The amount of encapsulated glucose oxidase and horseradish peroxidase during the synthesis process is small, resulting in poor final cascade catalysis and unclear color development.

[0093] Comparative Example 3

[0094] The difference between this example and Example 1 is that the ultraviolet cross-linking time is 2 minutes. The specific preparation method is the same as that of Example 1.

[0095] The duration of UV crosslinking affects the degree of crosslinking of the hydrogel matrix. The shorter the crosslinking time, the lower the mechanical strength of the hydrogel microneedle patch after swelling and the easier it is to break. After 2 minutes of UV crosslinking, the obtained microneedle patch is Figure 8 , it can be seen that: after the microneedle patch swells, the needle tip is broken and the interstitial fluid cannot be effectively extracted.

[0096] Comparative Example 4

[0097] The difference between this example and Example 1 is that the height of the microneedle patch is 600 μm. The specific preparation method is the same as that of Example 1.

[0098] Microneedle patches with needle tip lengths of 1000 μm and 600 μm were inserted into agarose gel containing the same concentration of glucose (5 mmol / L) to check the color development. Fig. 9The colorimetric microneedle patches with two different needle tip lengths were inserted into agarose gel containing 5mmol / L glucose for 10 minutes and then removed. The color development was observed after the TMB colorimetric substrate ethanol solution was added for 15 minutes. The left is a colorimetric microneedle patch with a needle tip length of 600μm, and the right is a colorimetric microneedle patch with a needle tip length of 1000μm. It can be seen that after the same extraction time and color development time, the microneedle patch with a needle tip length of 1000μm has a darker color, while the microneedle patch with a needle tip length of 600μm has a lighter color. This is because the tip of the microneedle will absorb the interstitial fluid in the skin after piercing the skin. If the needle tip is too short, too little interstitial fluid will be absorbed, and the cascade catalytic reaction in the microneedle will be insufficient, so the color development state is not good.

[0099] Comparative Example 5

[0100] The difference between this example and Example 1 is that no chromogenic substrate is added to the backing layer solution during the preparation of the microneedle patch, which is denoted as A; the microneedle patch prepared in Example 1 is denoted as B; and the concentration of the chromogenic substrate added to the backing layer is increased to 20 mmol / L during the preparation of the in situ colorimetric microneedle patch, which is denoted as C.

[0101] The A, B, and C microneedle patches were inserted into agarose gel containing the same concentration of glucose (10 mmol / L) and kept for 10 min. The color development at different time points was checked. The results are shown in Fig.10 , it can be seen that: the in-situ color-developing microneedle patch C has a lighter color after 10 minutes of extraction and swelling, and there is no obvious color change 15 minutes after the extraction. Therefore, the detection threshold of glucose in the microneedle patch of group C will increase due to the poor color development ability, which is not conducive to colorimetric analysis; after the microneedle patches A and B are extracted and swollen, they quickly develop color within the color development time of 15 minutes. Compared with the microneedle patch A in which the backing layer does not add the color development substrate when preparing the microneedle patch, the microneedle patch B in which the color development substrate is added to the backing layer quickly develops color after the color development substrate is added, and shows faster color changes and better color development effects in different time periods (1, 5, 10, 15 minutes). It can be seen that the microneedle patch prepared in Example 1 can improve the color development effect in terms of time and space.

Claims

1. A microneedle patch, characterized in that: It comprises a needle tip layer and a backing layer, wherein the needle tip layer is a methacrylylated natural biopolymer matrix with uniformly distributed immobilized enzymes, and the backing layer is a methacrylylated natural biopolymer matrix with uniformly distributed color development substrates, wherein: The immobilized enzyme is composed of a zinc-based imidazoline metal organic framework MAF-7 carrier and a natural enzyme loaded in the framework, wherein: The natural enzyme is one or more of glucose oxidase, lactose oxidase, maltase, horseradish peroxidase and catalase.

2. The microneedle patch according to claim 1, characterized in that: The natural enzyme is a combination of glucose oxidase and horseradish peroxidase in a mass ratio of 1 to 4:

1.

3. The microneedle patch according to claim 1, characterized in that: The immobilized enzyme is prepared by the following method: zinc acetate dihydrate, 3-methyl-1H-1,2,4-triazole and natural enzyme are dissolved in water, and ammonia water is added, and the mixture is stirred at 100-500 rpm for 6-24 hours, and then washed and freeze-dried to obtain the immobilized enzyme, wherein: The molar ratio of zinc acetate dihydrate to 3-methyl-1H-1,2,4-triazole is 1:4-8, the mass volume ratio of zinc acetate dihydrate to the reaction solution is 5-10 mg / mL, the volume ratio of ammonia water to the reaction solution is 1-5:100; and the mass volume ratio of the natural enzyme to the reaction solution is 1-5:1 mg / mL.

4. The microneedle patch according to claim 1, characterized in that: The methacrylic acid-acylated natural biopolymer is prepared by the following method: dissolving the natural biopolymer in water, slowly adding methacrylic anhydride to carry out grafting reaction, reacting at 30-60° C. for 0.5-2.5 hours, then dialyzing, and freeze-drying to obtain the methacrylic acid-acylated natural biopolymer, wherein: The mass volume ratio of the natural biopolymer to water is 50-150 mg / mL, and the mass volume ratio of the natural biopolymer to methacrylic anhydride is 1-1.5 g / mL.

5. The microneedle patch according to claim 1, characterized in that: The natural biopolymer is one or more of sodium alginate, cellulose, chitosan, gelatin, silk protein, and polypeptide; the color developing substrate is one or more of 3,3',5,5'-tetramethylbenzidine, o-phenylenediamine, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt.

6. The method for preparing the microneedle patch according to any one of claims 1 to 5, characterized in that: The steps include: S1: dissolving the immobilized enzyme in a mixed matrix solution consisting of a methacrylylated natural biopolymer matrix aqueous solution and a cross-linking agent to obtain a needle tip layer solution; mixing a color developing substrate ethanol solution with a solution consisting of a methacrylylated natural biopolymer matrix aqueous solution and a cross-linking agent to obtain a backing layer solution; S2: Add the needle tip layer solution into the PDMS mold, centrifuge once to allow the needle tip to be filled with the needle tip solution, then add the backing layer solution on the needle tip layer, dry and demould it, place it under ultraviolet light for cross-linking, and obtain a microneedle patch.

7. The method for preparing the microneedle patch according to claim 6, characterized in that: The concentration of the methacrylylated natural biopolymer in the needle tip layer is 10-40wt%, and the concentration of the immobilized enzyme is 0.5-3mg / mL; the concentration of the methacrylylated natural biopolymer in the backing layer is 10-40wt%, and the concentration of the chromogenic substrate ethanol solution is 5-20mmol / L, and the volume ratio of the chromogenic substrate ethanol solution to the solution composed of the methacrylylated natural biopolymer matrix aqueous solution and the cross-linking agent is 0.5-2:

100.

8. The method for preparing a microneedle patch according to claim 6, characterized in that: The crosslinking agent is one of the photoinitiators TPO, I2959 and LAP, and the concentrations in the needle tip layer and the backing layer solution are both 1-5 mg / mL.

9. The method for preparing a microneedle patch according to claim 6, characterized in that: The UV cross-linking time is 3 to 15 minutes.

10. A method for colorimetric detection of blood sugar using a microneedle patch, characterized in that: A colorimetric substrate ethanol solution is dripped onto the needle tip layer of the microneedle patch that has fully absorbed the interstitial fluid. The added colorimetric substrate ethanol solution enters the pores of the needle tip layer of the microneedle patch to trigger a color reaction, wherein the concentration of the dripped colorimetric substrate ethanol solution is 5 to 20 mmol / L, and the dripping amount is 10 to 100 μL.

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