Cross-linking process method of enzyme membrane in biosensor and preparation method of enzyme electrode

Abstract

The invention relates to the field of electrochemical biosensors, in particular to a crosslinking process method of an enzyme membrane in a biosensor and a preparation method of an enzyme electrode. A cross-linking process method of an enzyme membrane in a biosensor comprises the following steps: spraying a cross-linking solution to the surface of the enzyme membrane, so that the fogdrop-shaped cross-linking solution permeates from the surface of the enzyme membrane to the inside of the enzyme membrane, and realizing different cross-linking densities of the inside and the outside of the enzyme membrane under the support of a polymer during incubation to obtain an enzyme electrode. According to the cross-linking process method of the enzyme membrane, the cross-linking reagent is atomized and evenly arranged on the enzyme membrane in a spraying mode, the prepared enzyme electrode is high in sensitivity and good in stability, meanwhile, operation is easy, batch production is easy, and the production cost is low.

Description

Technical Field

[0001] This invention relates to the field of electrochemical biosensors, specifically to a cross-linking process for enzyme membranes and a method for preparing enzyme electrodes in a biosensor. Background Technology

[0002] Enzymes are a crucial component of electrochemical biosensors, significantly impacting their stability and reproducibility. However, the chemical and thermal instability of enzymes makes their analyte sensors susceptible to various environmental factors, such as temperature, humidity, and pH. Therefore, developing appropriate enzyme immobilization methods and processes to maintain their optimal catalytic activity is a key technology for preparing high-performance electrochemical biosensors.

[0003] Enzyme immobilization methods can generally be classified into four categories: physical adsorption, covalent bonding, cross-linking, and encapsulation. Among these, cross-linking is the most commonly used immobilization method in the preparation of electrochemical biosensors. Ionic bonds, hydrogen bonds, covalent bonds, or other non-specific interactions formed between groups can generally be represented by the term "cross-linking."

[0004] Cross-linking methods can generally be divided into physical cross-linking and chemical cross-linking. Physical cross-linking usually uses reversible non-covalent bonds or weak covalent bonds, which is one of its drawbacks and may potentially alter the properties of the enzyme. Chemical cross-linking, on the other hand, cross-links enzyme molecules through relatively strong covalent bonds. More specifically, it refers to linking two or more molecules intermolecularly or intramolecularly through covalent bonds. The reagents used to achieve this function are called "cross-linking reagents." The target sites for intermolecular or intramolecular cross-linking are mostly primary amines, carboxyl groups, thiol groups, carbonyl groups, etc.

[0005] In the development of electrochemical biosensors, the combination of materials and processes is synergistic and inseparable. Especially in the realization of enzyme cross-linking processes, most current methods use spot coating, spin coating, etc., but the enzyme membrane electrodes prepared by these methods have poor sensitivity and stability. Summary of the Invention

[0006] To address the above problems, this invention provides a cross-linking process for enzyme membranes, the specific technical solution of which is as follows: A cross-linking process for an enzyme membrane in a biosensor involves spraying a cross-linking solution onto the surface of the enzyme membrane, allowing the droplet-like cross-linking solution to penetrate from the surface of the enzyme membrane into its interior. During incubation, the enzyme membrane achieves different cross-linking densities on its interior and exterior under the support of the polymer, resulting in an enzyme electrode.

[0007] Preferably, the enzyme membrane is obtained by spraying an enzyme membrane solution onto the electrode surface.

[0008] Preferably, the preparation of the enzyme membrane solution includes the following steps: dissolving glucose oxidase and serum albumin in phosphate buffer solution to obtain solution A; diluting the water-soluble polymer to 0.1 mg / L using phosphate buffer solution to obtain solution B; slowly filtering solution A into solution B, mixing well, and incubating for 30-60 minutes to obtain the enzyme membrane solution.

[0009] Furthermore, the diameter of the filter pores is 0.22~0.45μm when filtering solution A.

[0010] Furthermore, the serum albumin includes bovine serum albumin or human serum albumin.

[0011] Furthermore, the polymer includes one or more of polyvinyl alcohol, waterborne polyurethane, cellulose, and polyethylene glycol.

[0012] Preferably, the ambient temperature for preparing the enzyme membrane is 25±5℃ and the ambient humidity is 40~60%.

[0013] Preferably, 10 to 12 layers are sprayed when applying the enzyme film solution.

[0014] Preferably, when preparing the crosslinking solution, a phosphate buffer solution is used to dilute the crosslinking reagent mixture to a concentration of 2.0-2.5% (volume concentration or mass concentration).

[0015] Furthermore, the crosslinking agent includes one or more of EDC, glutaraldehyde, and 1,4-butanediol diglycidyl ether.

[0016] Preferably, after spraying the crosslinking solution, the mixture is incubated at 35-50°C for 2-3 hours.

[0017] A method for preparing an enzyme electrode involves using the cross-linking process for an enzyme membrane in a biosensor, wherein an enzyme membrane solution is sprayed onto the electrode to obtain an enzyme membrane; a cross-linking solution is sprayed onto the surface of the enzyme membrane and incubated to obtain the enzyme electrode.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a crosslinking process for enzyme membranes, which uses a spraying method to atomize the crosslinking reagent and uniformly place it on the enzyme membrane. The prepared enzyme electrode has high sensitivity and good stability, and is simple to operate, easy to mass-produce, and has low production cost.

[0019] Spraying can disperse the modification liquid into uniform and fine droplets, which can be applied to the crosslinking process and then modified onto the surface of the electrode. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the cross-linking of the enzyme membrane; Figure 2This is a schematic diagram of the permeation model of cross-linking agent droplets on the enzyme membrane; Figure 3 This is a schematic diagram of an enzyme electrode; Figure 4 This is a graph showing the rate of change in the sensitivity of the enzyme electrode on day 7 compared to day 1; Figure 5 This is a schematic diagram of a device for preparing enzyme electrodes based on cross-linking. Detailed Implementation

[0021] The present invention will now be further described with reference to the accompanying drawings.

[0022] Optimizing the enzyme cross-linking method and process improves the sensitivity and stability of the enzyme electrode to a certain extent. The membrane setup is simple and efficient to operate, easy to mass-produce, and has low application cost.

[0023] A method for preparing enzyme electrodes based on cross-linking involves spraying a cross-linking solution onto the surface of an enzyme membrane, allowing the droplet-like cross-linking solution to penetrate from the surface of the enzyme membrane into its interior. During incubation, the enzyme membrane achieves different cross-linking densities on the inside and outside under the support of the polymer, resulting in an enzyme electrode. This method greatly protects the activity of the enzyme inside the mesh while fixing the enzymes in both the inner and outer layers through different degrees of covalent bonds.

[0024] The enzyme membrane is obtained by spraying an enzyme membrane solution onto the electrode surface.

[0025] The preparation of the enzyme membrane solution includes the following steps: dissolving glucose oxidase and serum albumin in phosphate buffer solution to obtain solution A; diluting the water-soluble polymer to 0.1 mg / L using phosphate buffer solution to obtain solution B; slowly filtering solution A into solution B, mixing well, and incubating for 30-60 minutes to obtain the enzyme membrane solution.

[0026] When filtering solution A, the diameter of the filter pores is 0.22~0.45μm.

[0027] Serum albumin includes bovine serum albumin or human serum albumin.

[0028] The polymer includes one or more of polyvinyl alcohol, waterborne polyurethane, cellulose, and polyethylene glycol.

[0029] The ambient temperature for enzyme membrane preparation is 25±5℃, and the ambient humidity is 40~60%.

[0030] When applying the enzyme film solution, apply 10 to 12 layers.

[0031] When preparing the crosslinking solution, the crosslinking reagent mixture is diluted to a mass concentration of 2.0-2.5% using a phosphate buffer solution.

[0032] The crosslinking solution is filtered during spraying, with the filter pores having a diameter of 0.22~0.45μm.

[0033] Crosslinking agents include one or more of EDC, glutaraldehyde, and 1,4-butanediol diglycidyl ether.

[0034] After spraying the crosslinking solution, incubate the reaction at 35~50℃ for 2~3 hours.

[0035] like Figure 5 As shown, the apparatus for preparing enzyme electrodes based on cross-linking, and the method for preparing enzyme electrodes based on cross-linking, include a container 1, an injection pump 2, a spray gun 4, and a controller 6. The container 1 is used to store the cross-linking solution or enzyme membrane solution; the injection pump is connected to the container and to the spray gun 4 via a pipe 3. The spray gun 4 is used to spray the electrode 5; the controller 6 is connected to the injection pump 2 and is used to control the injection pump 2. When an automatic spray gun is used, the controller 6 is also connected to the spray gun.

[0036] The cross-linking agent can be one or more of the following water-soluble compounds: 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), glutaraldehyde, 1,4-butanediol diglycidyl ether, etc.

[0037] The polymer can be one or more of the following polymers: polyvinyl alcohol, waterborne polyurethane, cellulose, polyethylene glycol, etc.

[0038] A method for preparing an enzyme electrode involves employing a cross-linking process for an enzyme membrane in a biosensor. The enzyme membrane solution is sprayed onto the electrode to obtain the enzyme membrane; a cross-linking solution is then sprayed onto the surface of the enzyme membrane and incubated to obtain the enzyme electrode.

[0039] The analytes in the biosensors involved in this application may be glucose, lactic acid, etc.

[0040] like Figures 1 to 5 As shown, a method for preparing enzyme electrodes based on cross-linking can be used in the spraying process of proteins or cross-linking reagents. Specifically, it involves spraying the cross-linking reagent evenly onto the surface of a moist enzyme membrane. Figure 1 As shown, this method can maximize the effective contact area between two liquid phases. Figure 3 As shown, by controlling the ambient atmosphere, temperature and humidity, droplet-shaped cross-linking reagents penetrate from the surface of the enzyme membrane into the interior. With the support of the polymer, different cross-linking densities are achieved inside and outside the enzyme membrane, which maximizes the protection of the internal enzyme activity while also improving the stability of the enzyme.

[0041] Preparation of enzyme membrane: The enzyme membrane solution is filtered using a needle filter to remove insoluble impurities. Then, the enzyme membrane solution is pushed into a precision injection pump through an automatic liquid feeding device. The required ambient humidity and temperature are controlled. The enzyme membrane solution is then atomized by spraying and repeatedly applied in multiple layers to the surface of the working electrode.

[0042] Crosslinking method: Use a needle filter to filter the crosslinking agent solution to remove insoluble impurities. Then, use an automatic liquid feeding device to push the crosslinking agent solution into a precision injection pump. Adjust the ambient temperature and humidity, and then use spraying to atomize the crosslinking reagent solution. Repeatedly apply multiple layers to the enzyme membrane surface to obtain the enzyme electrode.

[0043] Example 1: Spray crosslinking Taking an electrochemical biosensor with glucose as the analyte as an example, the preparation is as follows: (1) Preparation of enzyme membrane in glucose sensor: glucose oxidase and serum albumin were dissolved in phosphate buffer solution to obtain solution A. The water-soluble polymer was diluted to 0.1 mg / L with phosphate buffer solution to obtain solution B. Then, solution A was slowly filtered into test tube containing solution B using a syringe filter. After mixing, the mixture was incubated for 45 minutes. The test tube was then connected to a precision injection pump. The ambient temperature was controlled at 25℃ and the ambient humidity was adjusted to 55%. The enzyme solution was then atomized by spraying and repeatedly sprayed 10 layers onto the electrode surface.

[0044] The serum albumin mentioned can be either bovine serum albumin or human serum albumin. The needle filter has a pore size of 0.22 micrometers or 0.45 micrometers.

[0045] (2) Crosslinking method of enzyme membrane in glucose sensor: The crosslinking reagent mixture was diluted to a concentration of 2% using phosphate buffer solution, and then the solution was filtered into a test tube using a syringe filter. The test tube was directly connected to a precision injection pump. The ambient temperature was kept constant and the ambient humidity was adjusted to 70%. The solution was then atomized by spraying, and the nozzle speed was changed to evenly place it on the surface of the enzyme membrane. Finally, the electrode was transferred to 40°C and incubated for 2 hours to obtain the enzyme electrode.

[0046] The needle filter has a pore size of 0.22 micrometers or 0.45 micrometers.

[0047] Comparative Example 1: Dot-coated crosslinking Enzyme electrode preparation method: Using a dispensing device, the enzyme membrane solution prepared in Example 1 was first uniformly applied to the surface of the working electrode. Then, the cross-linking reagent solution prepared in Example 1 was applied to the enzyme surface. Finally, the enzyme electrode was obtained after incubation.

[0048] In the above-described enzyme membrane preparation and crosslinking methods, the parameter settings for the dispensing machine remain unchanged to ensure that the volume of the enzyme membrane solution and the crosslinking reagent solution are consistent. The differential method is used to calculate the amount of enzyme solution and crosslinking reagent solution used, consistent with Example 1. Furthermore, the ambient temperature, humidity, incubation temperature, and duration during the preparation process are consistent with those described in Example 1.

[0049] The enzyme electrodes prepared in Example 1 and Comparative Example 1 were subjected to comparative performance tests, which specifically included: (1) Sensitivity of enzyme electrodes (2) The enzyme electrode was immersed in phosphate buffer solution for 7 days to simulate the implantation state, and its sensitivity was measured daily to evaluate the stability of the enzyme electrode sensitivity.

[0050] The test results are shown in Table 1 below:

[0051] like Figure 4 As shown, preliminary experimental results indicate that when the crosslinking reagent is atomized by spraying and uniformly placed on the enzyme membrane, the sensitivity of the samples in Example 1 is higher than that of the samples in Comparative Example 1, and the sensitivity does not change significantly within 7 days, all being less than ±5%. This also indirectly proves to some extent that the enzyme molecules fixed on the electrode did not fall into the tissue simulation solution.

[0052] The structure of an enzyme electrode is as follows: Figure 3 As shown.

[0053] The technical principles of the present invention have been described above with reference to specific embodiments. These descriptions are merely for explaining the principles of the invention and should not be construed as limiting the scope of protection of the invention in any way. Based on this explanation, those skilled in the art can readily conceive of other specific embodiments of the invention without inventive effort, and these embodiments will all fall within the scope of protection of the claims of the present invention.

Claims

1. A cross-linking process for an enzyme membrane in a biosensor, characterized in that, The cross-linking solution is sprayed onto the surface of the enzyme membrane and incubated. The spraying allows the droplet-shaped cross-linking solution to penetrate from the surface of the enzyme membrane into the interior of the enzyme membrane. During incubation, the enzyme membrane achieves different cross-linking densities on the inside and outside under the support of the polymer.

2. The cross-linking process method for an enzyme membrane in a biosensor according to claim 1, characterized in that, The enzyme membrane is obtained by spraying an enzyme membrane solution onto the electrode surface.

3. The cross-linking process method for an enzyme membrane in a biosensor according to claim 2, characterized in that, The preparation of the enzyme membrane solution includes the following steps: Solution A was prepared by dissolving glucose oxidase and serum albumin in phosphate buffer solution. Solution B was prepared by diluting the water-soluble polymer to 0.1 mg / L using a phosphate buffer solution. Slowly filter solution A into solution B, mix well, and incubate for 30-60 minutes to obtain the enzyme membrane solution.

4. The cross-linking process method for an enzyme membrane in a biosensor according to claim 3, characterized in that, The serum albumin includes bovine serum albumin or human serum albumin.

5. The cross-linking process method for an enzyme membrane in a biosensor according to claim 3, characterized in that, The polymer includes one or more of polyvinyl alcohol, waterborne polyurethane, cellulose, and polyethylene glycol.

6. The cross-linking process method for an enzyme membrane in a biosensor according to claim 3, characterized in that, The ambient temperature for enzyme membrane preparation was 25±5℃, and the ambient humidity was 40~60%.

7. The cross-linking process method for an enzyme membrane in a biosensor according to claim 1, characterized in that, When preparing the crosslinking solution, the crosslinking reagent mixture is diluted to a mass concentration of 2.0-2.5% using a phosphate buffer solution.

8. The cross-linking process method for an enzyme membrane in a biosensor according to claim 7, characterized in that, The crosslinking agent includes one or more of EDC, glutaraldehyde, and 1,4-butanediol diglycidyl ether.

9. The cross-linking process method for an enzyme membrane in a biosensor according to claim 1 or 7, characterized in that, After spraying the crosslinking solution, incubate the reaction at 35~50℃ for 2~3 hours.

10. A method for preparing an enzyme electrode, employing the cross-linking process for the enzyme membrane in a biosensor as described in claim 1, characterized in that, The enzyme membrane solution is sprayed onto the electrode to obtain the enzyme membrane; The cross-linking solution was sprayed onto the surface of the enzyme membrane and incubated to obtain the enzyme electrode.

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