A composite enzyme material, a preparation method and application thereof

Abstract

The application provides a composite enzyme material, a preparation method and application thereof, and relates to the technical field of medical materials. The composite enzyme material is a composition of chemically cross-linked GOX / HRP, which is obtained by polymerization reaction, dialysis and drying of reaction raw materials including GOX containing a chemical bonded carbon-carbon double bond, HRP containing a chemical bonded carbon-carbon double bond and a water-soluble multifunctional polymerizable monomer; preferably, the composite enzyme material can also contain a water-soluble monofunctional polymerizable monomer. The composite enzyme material can be used for glucose concentration detection, and good detection results can be achieved in a wide concentration range.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, and relates to a composite enzyme material, its preparation method and its application. Background Technology

[0002] Blood glucose levels are a crucial indicator for diagnosing diabetes. While blood glucose meters and test strips offer convenient and quick testing, the need to prick the finger with a lancet to collect capillary blood can cause significant discomfort, especially with frequent testing, potentially leading to psychological stress. Studies have found a positive correlation between saliva glucose concentration and blood glucose concentration, making saliva glucose concentration a viable indicator of overall blood glucose levels. Saliva testing is also less or no longer uncomfortable for patients. However, saliva glucose concentration is 1-2 orders of magnitude lower than blood glucose concentration, necessitating improvements in the limitations of glucose detection methods and their accuracy and precision at low glucose concentrations. Chinese patent CN115343277A discloses a rapid test strip for detecting glucose in saliva. The test strip includes glucose oxidase, peroxidase, 2,4,6-tribromo-3-hydroxybenzoic acid, 4-aminoantipyrine, a colorimetric enhancer, and a pH adjuster. However, the detection limit for glucose using this method is not low enough, only at the mg / L level, which translates to approximately 100 μmol / L.

[0003] Therefore, there is an urgent need to establish a method for detecting glucose concentration that can provide accurate results even at low concentrations. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a composite enzyme material, its preparation method, and its application.

[0005] The technical solution of the present invention is as follows:

[0006] A composite enzyme material, wherein the composite enzyme material is a chemically cross-linked GOX / HRP composition.

[0007] A method for preparing a composite enzyme material, wherein the composite enzyme material is a chemically cross-linked GOX / HRP composition, obtained by polymerization, dialysis and drying of reaction raw materials comprising the following materials (i)-(iii);

[0008] Material (i), GOX with chemically bonded carbon-carbon double bonds;

[0009] Material (ii), HRP with chemically bonded carbon-carbon double bonds;

[0010] And, materials (iii), water-soluble multifunctional polymerizable monomers.

[0011] Preferably, the chemically bonded carbon-carbon double bond GOX is acrylated GOX or acrylamide GOX.

[0012] Preferably, the chemically bonded carbon-carbon double bond HRP is acrylated HRP or acrylamided HRP.

[0013] Preferably, the water-soluble multifunctional polymerizable monomer is selected from one or a combination of two or more of acrylamide monomers and polyethylene glycol polyacrylates.

[0014] Preferably, the weight ratio of the chemically bonded carbon-carbon double bond GOX, the chemically bonded carbon-carbon double bond HRP, and the water-soluble multifunctional polymerizable monomer is 20:1:10-1:1:0.1.

[0015] Preferably, the reaction raw materials further include material (iv) a water-soluble monofunctional polymerizable monomer.

[0016] More preferably, the water-soluble monofunctional polymerizable monomer is selected from one or a combination of two or more of amino or quaternary ammonium salt monomers, acrylamide monomers, hydroxyalkyl acrylates, (meth)acrylic acid and its salts, vinyl benzoic acid and its salts, and polyethylene glycol monoacrylates. Preferably, the water-soluble monofunctional polymerizable monomer accounts for 20-80% of the weight of the reactants.

[0017] Preferably, the reaction medium for the polymerization reaction is selected from a buffer solution, and the reaction temperature for the polymerization reaction is 5-35°C.

[0018] The application of a composite enzyme material as described in the above embodiments or a composite enzyme material prepared by the method described in any of the above embodiments, as a detection material for body fluid glucose.

[0019] The beneficial effects of this invention are as follows: This invention uses a combination of glucose oxidase (GOX) and horseradish peroxidase (HRP) as the detection material for glucose concentration in body fluids (especially saliva). By chemically grafting carbon-carbon double bonds onto GOX and HRP respectively, and then combining GOX and HRP through free radical polymerization, the binding and interaction of GOX and HRP are improved, so that even at low glucose concentrations, relatively accurate detection results can be achieved. Attached Figure Description

[0020] Figure 1 This is the logarithmic relationship curve between grayscale value and glucose concentration in Example 1. Detailed Implementation

[0021] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0022] On the one hand, the present invention proposes a composite enzyme material, which is a chemically cross-linked GOX / HRP composition.

[0023] Co-immobilization of GOX and HRP is a common co-immobilization system. GOX and HRP have similar optimal pH ranges, are both glycoproteins, and have similar sugar contents. However, the binding affinity (including binding tightness and binding force) between GOX and HRP significantly affects the performance of the GOX / HRP immobilized complex enzyme system. Better binding affinity between GOX and HRP is more beneficial to the performance of the GOX / HRP immobilized complex enzyme system. To improve the binding affinity between GOX and HRP, this invention uses a chemically cross-linked GOX / HRP composition, where GOX and HRP are linked together by chemical bonds. This results in a tighter and more stable binding affinity compared to physical methods (such as electrostatic adsorption).

[0024] On the other hand, the present invention proposes a method for preparing a composite enzyme material, wherein the composite enzyme material is a chemically cross-linked GOX / HRP composition, which is obtained by polymerization, dialysis and drying of reaction raw materials containing the following materials (i)-(iii);

[0025] Material (i), GOX with chemically bonded carbon-carbon double bonds;

[0026] Material (ii), HRP with chemically bonded carbon-carbon double bonds;

[0027] And, materials (iii), water-soluble multifunctional polymerizable monomers.

[0028] This invention employs a chemically cross-linked GOX / HRP complex enzyme system. By chemically bonding carbon-carbon double bonds onto GOX and HRP respectively, and then polymerizing them together with a water-soluble multifunctional polymerizable monomer, a chemically cross-linked GOX / HRP composition can be obtained. Since GOX and HRP are nanoscale in size, the size of the chemically bonded carbon-carbon double bonds on their surfaces is relatively small. Using a water-soluble multifunctional polymerizable monomer can effectively cross-link GOX and HRP through chemical bonding.

[0029] In some embodiments, the chemically bonded carbon-carbon double bond GOX is acrylated GOX or acrylamide GOX. Acrylated GOX or acrylamide GOX has the characteristics of high polymerization reactivity and convenient bonding grafting. For example, acrylated GOX can be obtained by reacting N-acryloyloxysuccinimide (NAS) with GOX.

[0030] In some embodiments, the chemically bonded carbon-carbon double bond HRP is acrylated HRP or acrylamide HRP. Acrylated HRP or acrylamide HRP has the characteristics of high polymerization reactivity and convenient bonding grafting. For example, acrylated HRP can be obtained by reacting NAS with HRP.

[0031] The reaction of NAS with GOX or HRP can be performed as follows: the weight ratio of GOX or HRP to NSA is 1:0.2-1. Dissolve GOX or HRP in a buffer solution with pH 6.0-10.0 (concentration can be 1-20 mg / ml), then add NSA solution (e.g., DMSO solution, concentration can be 10-50 mg / ml), stir at room temperature for 1-10 hours, then dialyze against a neutral buffer solution, and finally freeze-dry under vacuum to obtain the final product.

[0032] In some embodiments, the water-soluble multifunctional polymerizable monomer is selected from one or more combinations of diallyl monomers and polyethylene glycol polyacrylates. The functionality of a water-soluble multifunctional polymerizable monomer refers to the average number of carbon-carbon double bonds in its molecular structure that can participate in the polymerization reaction; multifunctionality means having at least two functions and acting as a crosslinking agent. For example, diallyl monomers can be selected from N,N'-methylenediacrylamide, N,N'-ethylenediacrylamide, N,N'-propylenediacrylamide, etc. More preferably, the diallyl monomer can be N,N'-methylenediacrylamide. For example, polyethylene glycol polyacrylates can be difunctional, such as polyethylene glycol (200) diacrylate, polyethylene glycol (400) diacrylate, polyethylene glycol (200) dimethacrylate, etc., or trifunctional or more, such as four-arm polyethylene glycol tetraacrylate, six-arm polyethylene glycol hexaacrylate, etc.

[0033] In some embodiments, the weight ratio of chemically bonded carbon-carbon double-bonded GOX, chemically bonded carbon-carbon double-bonded HRP, and water-soluble multifunctional polymerizable monomer is 20:1:10 to 1:1:0.1. For example, the weight ratio can be 20:1:10, 20:1:5, 20:1:2, 20:1:1, 20:1:0.5, 20:1:0.3, 20:1:0.1, 15:1:10, 10:1:10, 8:1:10, or 5:1. The value can be any one of the following: :10, 3:1:10, 1:1:10, 15:1:5, 15:1:3, 10:1:8, 10:1:5, 10:1:3, 10:1:1, 8:1:5, 8:1:3, 5:1:5, 5:1:6, 5:1:1, 5:1:0.5, 5:1:0.3, 3:1:1, 3:1:0.5, 3:1:0.3, 3:1:0.0.2, 3:1:0.1, etc., without any special restrictions.

[0034] In some embodiments, the reaction raw materials further include material (iv) a water-soluble monofunctional polymerizable monomer. The water-soluble monofunctional polymerizable monomer has only one carbon-carbon double bond in its molecular structure, which can participate in the polymerization reaction and act as a chain extender, further improving the effect of the cross-linking polymerization reaction.

[0035] In some embodiments, the water-soluble monofunctional polymerizable monomer is selected from one or a combination of two or more of amino- or quaternary ammonium salt monomers, acrylamide monomers, hydroxyalkyl acrylates, (meth)acrylic acid and its salts, vinyl benzoic acid and its salts, and polyethylene glycol monoacrylates. Amino- or quaternary ammonium salt monomers refer to monomers whose molecular structure simultaneously contains polymerizable carbon-carbon double bonds and amino groups (or quaternary ammonium salts). Amino- or quaternary ammonium salt monomers carry a positive charge, which can promote the binding of GOX and HRP before the polymerization reaction, thus enhancing the crosslinking and binding effect between GOX and HRP. For example, amino-containing monomers can be dimethylaminoethyl (meth)acrylate, dimethylaminobutyl (meth)acrylate, dimethylaminopropyl acrylate, aminoethyl acrylate, aminoethyl methacrylate, aminopropyl acrylate, aminopropyl methacrylate, N-methylaminoethyl acrylate, N-methylaminoethyl methacrylate, N-hydroxyethylaminoethyl acrylate, N-hydroxyethylaminoethyl methacrylate, N-ethylaminoethyl acrylate, etc.; quaternary ammonium salt-containing monomers can be acryloyloxyethyltrimethylammonium chloride, methacryloyloxyethyltrimethylammonium chloride, methacryloyloxypropyltrimethylammonium chloride, methacryloyloxybutyltrimethylammonium chloride, etc.; acrylamide monomers can be acrylamide, N,N-dimethylacrylamide, 2- Methacrylamide, N-methacrylamide, N-ethylacrylamide, N,N-diethylacrylamide, N-hydroxyethylacrylamide, N-methyl-2-methylacrylamide, etc.; hydroxyalkyl acrylates can be hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxypropyl methacrylate, hydroxypropyl acrylate, etc.; (meth)acrylic acid and its salts can be acrylic acid, sodium acrylate, ammonium acrylate, sodium methacrylate, etc.; vinyl benzoic acid and its salts can be vinyl benzoic acid, sodium vinyl benzoate, potassium vinyl benzoate, etc.; polyethylene glycol monoacrylates can be polyethylene glycol (200) monoacrylate, polyethylene glycol (200) monomethacrylate, polyethylene glycol (400) monoacrylate, triethylene glycol monoacrylate, triethylene glycol monomethyl ether acrylate, etc.

[0036] Preferably, the water-soluble monofunctional polymerizable monomer accounts for 20-80% of the weight of the reactants. For example, the weight percentage can be any value from 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, and 80%, without particular limitation. Further, the water-soluble monofunctional polymerizable monomer can account for 20-50% of the weight of the reactants.

[0037] In some embodiments, the reaction medium for the polymerization reaction is selected from buffer solutions, and the reaction temperature is 5-35°C. For example, the buffer solution as the reaction medium can be a PBS buffer solution with pH 7.0, a 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution with pH 7.0, a Tris-HCl buffer solution with pH 7.0, a Tris-HCl buffer solution with pH 7.5, a sodium carbonate buffer solution with pH 9.3, a borax-boric acid buffer solution with pH 8.6, or a borate buffer solution with pH 9.18, etc. A reaction temperature of 5-35°C for free radical polymerization ensures the reactivity of GOX, HRP, and the product complex enzyme material, avoiding deactivation. The free radical initiator used in the free radical polymerization reaction of this invention is not particularly limited. Preferably, it can be obtained by combining ammonium persulfate (APS) and tetramethylethylenediamine (TEMED). APS is the initiator, and TEMED can catalyze the generation of free radicals from APS at room temperature and accelerate the polymerization reaction of carbon-carbon double bonds, thus achieving the polymerization reaction at room temperature and obtaining the cross-linked GOX / HRP complex enzyme material. In this invention, an initiator composed of APS and TEMED is used. The polymerization reaction time at room temperature can be 2 hours, 3 hours, etc., which can be adjusted according to the amount of initiator and the ratio of APS / TEMED.

[0038] Furthermore, this invention proposes the application of a composite enzyme material as described in the above embodiments, or a composite enzyme material prepared by the method described in any of the above embodiments, as a detection material for glucose in body fluids. The composite enzyme material obtained by this invention can be used for the detection of glucose concentration in solution, especially in body fluids. Because the composite enzyme material of this invention has a wide detection range for glucose concentration and can detect lower concentrations of glucose, it can be used for the detection of glucose concentration in body fluids such as saliva, sweat, urine, and blood. Moreover, the detection method is convenient and can achieve non-invasive detection.

[0039] The technical solution of the present invention will be further described and illustrated below with reference to various embodiments. Unless otherwise specified, the parts mentioned in the following embodiments are parts by weight.

[0040] Preparation Example 1: Preparation of Acrylate-based GOX

[0041] Preparation Example 1

[0042] 50 mg GOX was dissolved in 10 ml of pH 7.0 PBS buffer solution, and then 25 mg NAS was slowly added to DMSO solution. The mixture was stirred at room temperature for 3 hours, then transferred to a dialysis bag with a molecular weight cutoff of 3000 and dialyzed in pH 7.0 phosphate buffer solution in PBS buffer solution at pH 7.0 for 24 hours, with the water changed every 2 hours. The solid was collected by centrifugation and then freeze-dried under vacuum to obtain acrylated GOX.

[0043] Preparation Example 2: Preparation of Acrylated HRP

[0044] Preparation Example 2

[0045] 40 mg GOX was dissolved in 10 ml of pH 7.5 Tris-HCl buffer solution, and then 20 mg NAS was slowly added to DMSO solution. The mixture was stirred at room temperature for 2.5 hours, then transferred to a dialysis bag with a molecular weight cutoff of 3000 and dialyzed in pH 7.0 phosphate buffer solution and then in pH 7.0 PBS buffer solution for 24 hours, changing the water every 2 hours. After centrifugation, the solid was collected and freeze-dried to obtain acrylated HRP.

[0046] Example 1

[0047] 8 mg of acrylated GOX from Preparation Example 1 and 1 mg of acrylated HRP from Preparation Example 2 were dissolved in 10 ml of PBS buffer (20 mM, pH 7.0) and stirred until homogeneous. Then, 1 ml of DMSO solution containing 5 mg of N,N'-methylenediacrylamide was added and stirred until homogeneous. The reaction system was purged with nitrogen for 30 min. Immediately after adding 0.5 ml of APS aqueous solution (12 mg / ml), 0.5 ml of TEMED aqueous solution (30 μL / ml) was added, and the polymerization reaction was carried out at room temperature for 2.5 h. The reaction product was dialyzed for 18 h in PBS buffer solution at pH 7.0 using a dialysis bag with a molecular weight cutoff of 3500, with the water changed every 2 h. After dialysis, the product was freeze-dried under vacuum to obtain the composite enzyme material.

[0048] Glucose concentration detection method

[0049] The above-mentioned composite enzyme material was dispersed in water to prepare a composite enzyme dispersion with a concentration of 2 mg / ml.

[0050] Prepare aqueous solutions of glucose at different concentrations: 10 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 90 μmol / L, 110 μmol / L, 130 μmol / L, 160 μmol / L, 250 μmol / L, 350 μmol / L, 500 μmol / L, 700 μmol / L, 1000 μmol / L, 1500 μmol / L, 3000 μmol / L, 5000 μmol / L, and 8000 μmol / L.

[0051] Take 20 μL of the above-mentioned complex enzyme dispersion and drop it onto the surface of a 10 mm × 10 mm filter paper. Then, drop 20 μL of a tetramethylbenzidine ethanol solution (concentration 15 mmol / ml) onto the paper and dry it at room temperature for 24 h. Take 20 μL of the above-mentioned aqueous solutions of different glucose concentrations and add them onto the above-mentioned test paper. After 3 min, record the color using a digital camera. The RGB images (R value, G value, and B value) of the colors recorded by the digital camera are converted into grayscale values ​​and analyzed.

[0052] The grayscale value is calculated as follows: Grayscale value = 0.299 × R value + 0.587 × G value + 0.114 × B value. Different glucose concentrations result in different RGB images, which are then converted to different grayscale values. A curve is plotted to establish the relationship between glucose concentration and grayscale value.

[0053] Appendix Figure 1 To find the logarithmic relationship between grayscale values ​​and glucose concentrations at concentrations of 60 μmol / L, 130 μmol / L, 250 μmol / L, 500 μmol / L, 1000 μmol / L, 3000 μmol / L, 5000 μmol / L, and 8000 μmol / L, the R-squared value of the line is... 2 =0.9938, indicating a good linear relationship. The fitted equation is y = -0.3974x + 4.3082.

[0054] Example 2

[0055] 10 mg of acrylated GOX from Preparation Example 1 and 1 mg of HRP from Preparation Example 1 were dissolved in 10 ml of PBS buffer (20 mM, pH = 7.0) and stirred until homogeneous. Then, 1 ml of DMSO solution containing 3 mg of N,N'-methylenediacrylamide was added and stirred until homogeneous. The reaction system was purged with nitrogen for 30 min. Immediately after adding 0.5 ml of APS aqueous solution (12 mg / ml), 0.5 ml of TEMED aqueous solution (30 μL / ml) was added, and the polymerization reaction was carried out at room temperature for 2.5 h. The reaction product was dialyzed for 12 h using a dialysis bag with a molecular weight cutoff of 3500, with the water changed every 2 h. After dialysis, the product was freeze-dried under vacuum to obtain the composite enzyme material.

[0056] Following the detection method described in Example 1 above, the R-squared value of the logarithmic relationship between grayscale value and glucose concentration was measured. 2 =0.9945, indicating a good linear relationship. The fitted equation is y = -0.4012x + 4.4307.

[0057] Example 3

[0058] 10 mg of acrylated GOX from Preparation Example 1, 1 mg of HRP from Preparation Example 1, and 14 mg of acrylamide were dissolved in 10 ml of PBS buffer (20 mM, pH 7.0) and stirred until homogeneous. Then, 1 ml of DMSO solution containing 3 mg of N,N'-methylenediacrylamide was added and stirred until homogeneous. The reaction system was purged with nitrogen for 30 min. Immediately after adding 0.5 ml of APS aqueous solution (12 mg / ml), 0.5 ml of TEMED aqueous solution (30 μL / ml) was added, and the polymerization reaction was carried out at room temperature for 2.5 h. The reaction product was dialyzed for 12 h using a dialysis bag with a molecular weight cutoff of 3500, with water changed every 2 h. After dialysis, the product was freeze-dried under vacuum to obtain the composite enzyme material.

[0059] Following the detection method described in Example 1 above, the R-squared value of the logarithmic relationship between grayscale value and glucose concentration was measured. 2 =0.9972, indicating a good linear relationship. The fitted equation is y = -0.4007x + 4.3750.

[0060] Example 4

[0061] The difference between Example 4 and Example 3 is that in Example 3, 14 mg of acrylamide was replaced with a combination of 4 mg of dimethylaminoethyl methacrylate and 10 mg of acrylamide. The remaining steps remained unchanged.

[0062] Following the detection method described in Example 1 above, the R-squared value of the logarithmic relationship between grayscale value and glucose concentration was measured. 2 =0.9992, indicating a good linear relationship. The fitted equation is y = -0.4186x + 4.4061.

[0063] Example 5

[0064] The difference between Example 5 and Example 3 is that in Example 3, 14 mg of acrylamide was replaced with a combination of 5 mg N-methylaminoethyl acrylate and 9 mg of acrylamide. The remaining steps remained unchanged.

[0065] Following the detection method described in Example 1 above, the R-squared value of the logarithmic relationship between grayscale value and glucose concentration was measured. 2 =0.9980, indicating a good linear relationship. The fitted equation is y = -0.4133x + 4.3892.

[0066] Example 6

[0067] The difference between Example 6 and Example 3 is that in Example 3, 14 mg of acrylamide was replaced with 14 mg of dimethylaminoethyl methacrylate. The remaining steps remained unchanged.

[0068] Following the detection method described in Example 1 above, the R-squared value of the logarithmic relationship between grayscale value and glucose concentration was measured. 2 =0.9976, indicating a good linear relationship. The fitted equation is y = -0.4117x + 4.4472.

[0069] Comparative Example 1

[0070] The difference between Comparative Example 1 and Example 2 is that N,N'-methylenediacrylamide was not added in Example 2. The remaining steps remained unchanged.

[0071] Following the detection method described in Example 1 above, the R-squared value of the logarithmic relationship between grayscale value and glucose concentration was measured. 2 =0.9586, indicating a poor linear relationship.

[0072] Comparative Example 2

[0073] The difference between Comparative Example 2 and Example 2 is that in Example 2, N,N'-methylenediacrylamide was replaced with an equal weight of acrylamide. The remaining steps remained unchanged.

[0074] Following the detection method described in Example 1 above, the R-squared value of the logarithmic relationship between grayscale value and glucose concentration was measured. 2 =0.9762, indicating a poor linear relationship.

[0075] Comparative Example 3

[0076] The difference between Comparative Example 3 and Example 2 is that in Example 2, N,N'-methylenediacrylamide was replaced with 15 mg of acrylamide. The remaining steps remained unchanged.

[0077] Following the detection method described in Example 1 above, the R-squared value of the logarithmic relationship between grayscale value and glucose concentration was measured. 2 =0.9774, the linear relationship is not good.

[0078] Comparative Example 4

[0079] The difference between Comparative Example 4 and Example 2 is that in Example 2, acrylated GOX was replaced with an equal weight of GOX, and acrylated HRP was replaced with HRP, meaning that neither GOX nor HRP was grafted with acrylate groups. The remaining steps remained unchanged.

[0080] Following the detection method described in Example 1 above, the R-squared value of the logarithmic relationship between grayscale value and glucose concentration was measured. 2 =0.9349, the linear relationship is not good.

[0081] Example 7

[0082] 20 mg of acrylated GOX from Preparation Example 1 and 1 mg of HRP from Preparation Example 1 were dissolved in 10 ml of PBS buffer (20 mM, pH = 7.0) and stirred until homogeneous. Then, 1 ml of DMSO solution containing 10 mg of N,N'-methylenediacrylamide was added and stirred until homogeneous. The reaction system was purged with nitrogen for 30 min. Immediately after adding 0.5 ml of APS aqueous solution (12 mg / ml), 0.5 ml of TEMED aqueous solution (30 μL / ml) was added, and the polymerization reaction was carried out at room temperature for 3 h. The reaction product was dialyzed for 12 h using a dialysis bag with a molecular weight cutoff of 3500, with the water changed every 2 h. After dialysis, the product was freeze-dried under vacuum to obtain the composite enzyme material.

[0083] Following the detection method described in Example 1 above, the R-squared value of the logarithmic relationship between grayscale value and glucose concentration was measured. 2 =0.9925, indicating a good linear relationship. The fitted equation is y = -0.4283x + 4.4511.

[0084] Example 8

[0085] 1 mg of acrylated GOX and 1 mg of HRP from Preparation Example 1 were dissolved in 10 ml of PBS buffer (20 mM, pH 7.0) and stirred until homogeneous. Then, 1 ml of DMSO solution containing 0.1 mg of N,N'-methylenediacrylamide was added and stirred until homogeneous. The reaction system was purged with nitrogen for 30 min. Immediately after adding 0.5 ml of APS aqueous solution (12 mg / ml), 0.5 ml of TEMED aqueous solution (30 μL / ml) was added, and the polymerization reaction was carried out at room temperature for 2 h. The reaction product was dialyzed for 12 h using a dialysis bag with a molecular weight cutoff of 3500, with the water changed every 2 h. After dialysis, the product was freeze-dried under vacuum to obtain the composite enzyme material.

[0086] Following the detection method described in Example 1 above, the R-squared value of the logarithmic relationship between grayscale value and glucose concentration was measured. 2 =0.9908, indicating a good linear relationship. The fitted equation is y = -0.4037x + 4.5271.

[0087] Example 9

[0088] 1 mg of acrylated GOX from Preparation Example 1, 1 mg of HRP from Preparation Example 1, and 1 mg of hydroxyethyl acrylate were dissolved in 10 ml of PBS buffer (20 mM, pH = 7.0) and stirred until homogeneous. Then, 1 ml of DMSO solution containing 0.1 mg of N,N'-methylenediacrylamide was added and stirred until homogeneous. The reaction system was purged with nitrogen for 30 min. Immediately after adding 0.5 ml of APS aqueous solution (12 mg / ml), 0.5 ml of TEMED aqueous solution (30 μL / ml) was added, and the polymerization reaction was carried out at room temperature for 2 h. The reaction product was dialyzed for 12 h using a dialysis bag with a molecular weight cutoff of 3500, with the water changed every 2 h. After dialysis, the product was freeze-dried under vacuum to obtain the composite enzyme material.

[0089] Following the detection method described in Example 1 above, the R-squared value of the logarithmic relationship between grayscale value and glucose concentration was measured. 2 =0.9923, indicating a good linear relationship. The fitted equation is y = -0.4085x + 4.3028.

[0090] Glucose aqueous solutions with concentrations of 0.005 mmol / L, 0.03 mmol / L, 0.20 mmol / L, 0.60 mmol / L, and 1.20 mmol / L were prepared and tested using the assay enzyme material. Each glucose aqueous solution sample was tested three times, and the average value was taken. The results are shown in Table 1 below.

[0091] Table 1. Glucose concentration test results (mmol / L)

[0092] sample 0.005 0.03 0.20 0.60 1.20 Example 2 0.004±0.0006 0.029±0.005 0.21±0.03 0.58±0.10 1.22±0.18 Example 3 0.005±0.0003 0.032±0.002 0.18±0.02 0.60±0.07 1.19±0.11 Example 4 0.005±0.0002 0.030±0.002 0.19±0.01 0.60±0.03 1.20±0.07 Example 6 0.005±0.0002 0.029±0.002 0.20±0.02 0.61±0.04 1.22±0.10 Comparative Example 1 0.004±0.0016 0.027±0.010 0.25±0.09 0.64±0.18 1.20±0.47 Comparative Example 2 0.005±0.0011 0.026±0.011 0.28±0.10 0.61±0.15 1.23±0.40 Comparative Example 4 0.003±0.0020 0.032±0.015 0.21±0.14 0.57±0.22 1.22±0.53

[0093] As can be seen from the test results in Table 1, the composite enzyme material of the present invention has relatively accurate detection results for a wide range of glucose concentrations, and can achieve high accuracy even at low glucose concentrations.

[0094] Saliva samples were collected from two volunteers: Volunteer 1 had normal blood sugar, while Volunteer 2 had hyperglycemia. Both samples were collected in the morning on an empty stomach. The samples were compared using the composite enzyme materials from Examples 2, 3, and 4, and Comparative Example 2, as well as a commercial glucose assay kit (O-toluidine method). Each saliva sample was tested three times for each assay, and the results were averaged. The results are shown in Table 2 below.

[0095] Table 2. Results of glucose detection in saliva (mM)

[0096]

[0097] Based on the test results in Table 2 above, it is shown that the composite enzyme material of the present invention, when used for the detection of glucose concentration in saliva, yields results that are similar to or more accurate than those of existing commercial glucose detection kits.

[0098] As described above, the basic principles, main features, and advantages of the present invention have been shown and described. Those skilled in the art should understand that the present invention is not limited to the above embodiments, which are merely preferred embodiments and should not be construed as limiting the scope of the invention. All equivalent changes and modifications made in accordance with the scope of the patent and the description should still fall within the scope of the present invention. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A composite enzyme material, characterized in that, The composite enzyme material is a chemically cross-linked GOX / HRP composition.

2. A method for preparing a composite enzyme material, characterized in that, The composite enzyme material is a chemically cross-linked GOX / HRP composition, obtained by polymerization, dialysis and drying of reaction raw materials containing the following materials (i)-(iii); Material (i), GOX with chemically bonded carbon-carbon double bonds; Material (ii), HRP with chemically bonded carbon-carbon double bonds; And, materials (iii), water-soluble multifunctional polymerizable monomers.

3. The method for preparing the composite enzyme material according to claim 2, characterized in that, The chemically bonded carbon-carbon double bond GOX is acrylated GOX or acrylamide GOX.

4. The method for preparing the composite enzyme material according to claim 2, characterized in that, The chemically bonded carbon-carbon double bond HRP is acrylated HRP or acrylamided HRP.

5. The method for preparing the composite enzyme material according to claim 2, characterized in that, The water-soluble multifunctional polymerizable monomer is selected from one or a combination of two or more of the following: acrylamide monomers and polyethylene glycol polyacrylates.

6. The method for preparing the composite enzyme material according to claim 2, characterized in that, The weight ratio of the chemically bonded carbon-carbon double bond GOX, the chemically bonded carbon-carbon double bond HRP, and the water-soluble multifunctional polymerizable monomer is 20:1:10-1:1:0.

1.

7. The method for preparing the composite enzyme material according to claim 2, characterized in that, The reaction raw materials also include material (iv) a water-soluble monofunctional polymerizable monomer.

8. The method for preparing the composite enzyme material according to claim 7, characterized in that, The water-soluble monofunctional polymerizable monomer is selected from one or a combination of two or more of amino or quaternary ammonium salt monomers, acrylamide monomers, hydroxyalkyl acrylates, (meth)acrylic acid and its salts, vinyl benzoic acid and its salts, and polyethylene glycol monoacrylates. Preferably, the water-soluble monofunctional polymerizable monomer accounts for 20-80% of the weight of the reactants.

9. The method for preparing the composite enzyme material according to claim 2, characterized in that, The reaction medium for the polymerization reaction is selected from a buffer solution, and the reaction temperature for the polymerization reaction is 5-35℃.

10. The application of a composite enzyme material obtained by the preparation method of the composite enzyme material according to claim 1 or any one of claims 2-9, characterized in that, As a material for detecting glucose in body fluids.

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