Bio-enzyme electrode, preparation method thereof and wearable electrochemical biosensor

Abstract

The invention discloses a biological enzyme electrode, a preparation method thereof and a wearable electrochemical biosensor, and relates to the technical field of sensors. The bio-enzyme electrode comprises an electrode body and a bio-enzyme layer formed on the surface of the electrode body, and the bio-enzyme layer comprises bio-enzyme, bovine serum albumin, a zwitterionic polymer and a cross-linking agent. According to the technical scheme, the enzyme activity stability of the biological enzyme electrode can be improved, and then the long-term monitoring stability of the wearable electrochemical biosensor is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sensors, in particular to a biological enzyme electrode, a preparation method thereof and a wearable electrochemical biosensor. BACKGROUND

[0002] As a kind of efficient biological catalyst, biological enzyme has the advantages of high selectivity, high catalytic efficiency and mild reaction regulation, and is widely used in food detection, biological pharmacy, biosensor and other fields. Among them, the wearable electrochemical biosensor has great potential in realizing personalized health monitoring, predictive analysis and timely intervention of personalized health care. In the wearable electrochemical biosensor, the biological enzyme layer in the biological enzyme electrode is the most important part for the specific recognition of biological analytes by the sensor, which determines the monitoring performance of the wearable electrochemical biosensor. Therefore, the monitoring performance of the wearable electrochemical biosensor mainly depends on the fixation and enzyme activity maintenance ability of the biological enzyme.

[0003] At present, biological enzyme molecules in the biological enzyme layer are usually cross-linked and fixed on the surface of the electrode body by using a cross-linking agent. However, due to the low affinity between the electrode body and the biological enzyme, unstable cross-linking and other factors, it often leads to the leakage of biological enzyme and the deactivation of biological enzyme in the catalytic reaction process. The enzyme activity stability of the biological enzyme electrode is poor, which further affects the long-term monitoring stability of the wearable electrochemical biosensor.

[0004] The above content is only used to assist in understanding the technical solutions of the present application, and does not represent the acknowledgement of the above content as prior art. SUMMARY

[0005] The main purpose of the present application is to provide a biological enzyme electrode, a preparation method thereof and a wearable electrochemical biosensor, which aims to improve the enzyme activity stability of the biological enzyme electrode, and further improve the long-term monitoring stability of the wearable electrochemical biosensor.

[0006] In order to achieve the purpose of the present application, the present application provides a biological enzyme electrode, which comprises an electrode body and a biological enzyme layer formed on the surface of the electrode body, and the biological enzyme layer comprises biological enzyme, bovine serum albumin, zwitterionic polymer and cross-linking agent.

[0007] In an embodiment, the content of the zwitterionic polymer accounts for 0.1wt%-5wt% of the total amount of the biological enzyme layer; and / or, The zwitterionic polymer comprises at least one of carboxybetaine-based polymer, sulfobetaine-based polymer and phosphobetaine-based polymer.

[0008] In an embodiment, the biological enzyme layer further comprises aqueous polyurethane.

[0009] In an embodiment, the waterborne polyurethane is present in an amount of 0.01wt%-10wt% of the total amount of the bio-enzyme layer.

[0010] In an embodiment, the bio-enzyme layer further comprises a hydrophilic polymer.

[0011] In an embodiment, the hydrophilic polymer is present in an amount of 0.2wt%-2wt% of the total amount of the bio-enzyme layer; and / or, The hydrophilic polymer comprises at least one of polyvinylpyrrolidone and cellulose acetate.

[0012] In an embodiment, the bio-enzyme layer has a thickness of 1-20 microns.

[0013] The present application also provides a preparation method of a bio-enzyme electrode, comprising the following steps: providing an electrode body, a bio-enzyme, a buffer, bovine serum albumin, a zwitterionic polymer and a cross-linking agent; dissolving the bio-enzyme into the buffer to obtain a bio-enzyme solution; adding bovine serum albumin, a zwitterionic polymer and a cross-linking agent into the bio-enzyme solution in sequence, and stirring and dissolving to obtain a bio-enzyme mixture solution; coating the bio-enzyme mixture solution on the surface of the electrode body, and drying to obtain a bio-enzyme electrode.

[0014] In an embodiment, the concentration of the bio-enzyme in the bio-enzyme solution is 30-50mg / mL; and / or, The concentration of the bovine serum albumin in the bio-enzyme mixture solution is 8-12mg / mL; and / or, The amount of the zwitterionic polymer added is 0.1wt%-5wt% of the total solid content in the bio-enzyme mixture solution; and / or, The concentration of the cross-linking agent is 1wt%-1.5wt%.

[0015] In an embodiment, the step of adding bovine serum albumin, a zwitterionic polymer and a cross-linking agent into the bio-enzyme solution in sequence, and stirring and dissolving to obtain a bio-enzyme mixture solution, comprises: adding bovine serum albumin, a zwitterionic polymer, a hydrophilic polymer and / or waterborne polyurethane, and a cross-linking agent into the bio-enzyme solution in sequence, and stirring and dissolving to obtain a bio-enzyme mixture solution.

[0016] The present application also provides a wearable electrochemical biosensor, comprising a working electrode, wherein the working electrode is the bio-enzyme electrode as described above.

[0017] The bio-enzyme electrode provided by the application comprises an electrode body and a bio-enzyme layer formed on the surface of the electrode body, and the bio-enzyme layer comprises bio-enzyme, bovine serum albumin, a zwitterionic polymer and a cross-linking agent. The bio-enzyme, the bovine serum albumin and the zwitterionic polymer are cross-linked by the cross-linking agent to form the bio-enzyme layer. The bovine serum albumin has high compatibility with the bio-enzyme, and forms a protective layer by hydrogen bonding between the molecules of the bio-enzyme, thereby protecting the activity of the bio-enzyme. Meanwhile, the bovine serum albumin can effectively prevent the bio-enzyme from being excessively cross-linked and affecting the activity of the bio-enzyme. The zwitterionic polymer has zero total charge in a wide pH range, has high hydrophilicity and can retain water, thereby helping to prevent the bio-enzyme from being denatured. Meanwhile, the zwitterionic group of the zwitterionic polymer provides a charge center which can have strong charge-charge interaction with the ionic group in the bio-enzyme, thereby stably fixing the bio-enzyme in the enzyme layer and reducing the leaching of the bio-enzyme from the bio-enzyme layer. Therefore, the enzyme activity stability of the bio-enzyme electrode can be strengthened by the synergy of the cross-linking and electrostatic effects, and the long-term monitoring stability of the wearable electrochemical biosensor can be improved. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other related drawings can be obtained by those skilled in the art without any creative effort.

[0019] Figure 1 It is a structural schematic diagram of an embodiment of the bio-enzyme electrode of the present application. Figure 2 It is a schematic diagram of the response current change of the electrochemical biosensor of embodiment 1 of the present application under different glucose concentrations. Figure 3 It is a schematic diagram of the change of the enzyme activity of the electrochemical biosensor of embodiment 1 of the present application with running time.

[0020] LIST OF DRAWINGS 100, bio-enzyme electrode; 1, electrode body; 11, substrate; 12, conductive layer; 2, bio-enzyme layer.

[0021] The implementation, functional features and advantages of the present application will be further described with reference to the embodiments and the drawings. DETAILED DESCRIPTION

[0022] It should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0023] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the reagents or instruments are not specified by the manufacturers, they are all the conventional products that can be purchased in the market. In addition, the meaning of "and / or" appearing in the whole text includes three parallel solutions. Taking "A and / or B" as an example, it includes the solutions of A, or B, or A and B. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the fact that the person skilled in the art can realize it. When the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist and is not within the protection scope of the present application. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without creative labor are within the protection scope of the present application.

[0024] As a kind of efficient biological catalyst, biological enzyme has the advantages of high selectivity, high efficient catalytic nature, reaction regulation mildness and is widely used in food detection, biological pharmacy, biological sensor and other fields. Among them, wearable electrochemical biosensor has great potential in realizing personalized health monitoring, prediction analysis and timely intervention of personalized health care. In wearable electrochemical biosensor, the biological enzyme layer in biological enzyme electrode is the most important part for the specific recognition of biological analyte of sensor, which determines the monitoring performance of wearable electrochemical biosensor, so the monitoring performance of wearable electrochemical biosensor mainly depends on the fixation and enzyme activity maintenance ability of biological enzyme.

[0025] At present, biological enzyme molecules in biological enzyme layer are usually cross-linked and fixed on the surface of electrode body by cross-linking agent. However, due to the low affinity between electrode body and biological enzyme, unstable cross-linking and other factors, biological enzyme leakage and biological enzyme deactivation in the process of catalytic reaction often occur, the enzyme activity stability of biological enzyme electrode is poor, and then the long-term monitoring stability of wearable electrochemical biosensor is affected.

[0026] In order to solve the above technical problems, the present application provides a biological enzyme electrode, which aims to improve the enzyme activity stability of biological enzyme electrode, and then improve the long-term monitoring stability of wearable electrochemical biosensor.

[0027] The present application crosslinks the biological enzyme, the bovine serum albumin and the zwitterionic polymer by the crosslinking agent to form the biological enzyme layer, wherein the molecular structure of the bovine serum albumin has higher compatibility with the biological enzyme, and a protective layer is formed by the hydrogen bond interaction between the biological enzyme molecules, so as to protect the activity of the biological enzyme, and the bovine serum albumin can also effectively avoid the excessive crosslinking of the biological enzyme and affect the activity of the biological enzyme. The zwitterionic polymer has zero total charge in a wide pH range, has high hydrophilicity and can retain water, which helps to prevent the biological enzyme from denaturation; and the zwitterionic group of the zwitterionic polymer provides a charge center which can have strong charge-charge interaction with the ionic group in the biological enzyme, so that the biological enzyme can be stably fixed in the enzyme layer, and the leaching of the biological enzyme from the biological enzyme layer is reduced. Therefore, the activity stability of the biological enzyme electrode can be strengthened by the synergy of crosslinking and electrostatic interaction, and the long-term monitoring stability of the wearable electrochemical biosensor can be improved.

[0028] Referring to Figure 1 As shown in the figure, in an embodiment of the present application, the biological enzyme electrode 100 comprises an electrode body 1 and a biological enzyme layer 2 formed on the surface of the electrode body 1, and the biological enzyme layer 2 comprises biological enzyme, bovine serum albumin, zwitterionic polymer and crosslinking agent.

[0029] The electrode body 1 comprises a substrate 11 and a conductive layer 12 formed on the surface of the substrate 11, and the material of the substrate 11 comprises at least one of polyimide, polyethylene terephthalate and polydimethylsiloxane, and the material of the conductive layer 12 comprises at least one of platinum, gold, silver, copper, chromium and carbon. In the preparation of the conductive layer 12, the electrode design pattern is first made, and then the required conductive circuit and electrode are formed on the surface of the substrate 11 by sputtering, evaporation, electroplating or screen printing, etc., which is the conductive layer 12.

[0030] The biological enzyme layer 2 is formed on the surface of the electrode body 1, and can be formed by reasonable coating methods such as dip coating, spot coating and spraying. The biological enzyme layer 2 is the key recognition element of the electrochemical biosensor, and the activity and enzyme activity retention ability of the enzyme directly affect the sensitivity of the electrochemical biosensor. The biological enzyme layer 2 is fixed with biological enzyme and bovine serum albumin, the material of the biological enzyme layer 2 is zwitterionic polymer, and the crosslinking agent is also contained, that is, the biological enzyme layer 2 is formed after the crosslinking treatment of biological enzyme, bovine serum albumin and zwitterionic polymer.

[0031] The bioenzyme, as a core functional component of the bioenzyme layer 2, is the core of the bioenzyme electrode 100 for recognition and catalysis. The bioenzyme has structural specificity, for example, glucose oxidase only recognizes glucose molecules, can accurately bind to the target substrate in a complex sample (such as blood, urine), avoids interference of non-target substances, and ensures the detection specificity of the bioenzyme electrode 100. The bioenzyme includes, but is not limited to, at least one of glucose oxidase, glucose dehydrogenase, lactic acid oxidase, and lactic acid dehydrogenase.

[0032] The bovine serum albumin, as an auxiliary stable component of the bioenzyme layer 2, is a water-soluble globular protein, and has high compatibility with the molecular structure of the bioenzyme. The bovine serum albumin can form a wrapping layer through hydrogen bonds and hydrophobic interactions between enzyme molecules, thereby protecting the activity of the bioenzyme. Meanwhile, the bovine serum albumin can also effectively avoid excessive cross-linking of the bioenzyme to affect the activity thereof.

[0033] The amphoteric ion polymer has equal amounts of positive and negative charge groups in the molecular structure, and is overall electroneutral. The positive and negative charge groups in the amphoteric ion polymer can form strong hydrogen bonds with water molecules to form a hydration layer on the surface, thereby improving biocompatibility and reducing biological pollution. Moreover, one charge center provided by the amphoteric ion group of the amphoteric ion polymer can have strong charge-charge interaction with the ion group in the bioenzyme, so that the bioenzyme can be relatively stably fixed in the enzyme layer, thereby reducing leaching of the bioenzyme from the bioenzyme layer 2. Meanwhile, the hydration layer on the surface of the amphoteric ion polymer can also accelerate the diffusion rate of the target substrate or product in the bioenzyme layer 2, thereby shortening the response time of the bioenzyme electrode 100. In addition, the amphoteric ion polymer has good chemical stability, can form a stable cross-linking network with the bioenzyme and the bovine serum albumin, thereby improving the stability of the bioenzyme layer 2. Moreover, the flexible chain structure of the amphoteric ion polymer can enhance the flexibility of the bioenzyme layer 2, reduce cracking of the bioenzyme layer 2, and thus improve the mechanical stability of the bioenzyme electrode 100.

[0034] The cross-linking agent is used to realize the structural stability of the bioenzyme layer 2 through chemical covalent bonds, thereby preventing loss of the bioenzyme. The cross-linking agent includes, but is not limited to, glutaraldehyde.

[0035] The present application uses the synergy of cross-linking and electrostatic effects to strengthen the enzyme activity stability of the bioenzyme electrode 100, and thus improves the long-term monitoring stability of the wearable electrochemical biosensor.

[0036] In an optional embodiment of the present application, the content of the amphoteric ion polymer accounts for 0.1wt%-5wt% of the total amount of the bioenzyme layer 2, such as 0.1wt%, 1wt%, 2wt%, 3wt%, 4wt%, 5wt% and any interval value between any two endpoint values.

[0037] The content of the zwitterionic polymer is limited in the above range, which can not only effectively construct a hydration layer, ensure the enzyme activity basis, provide sufficient charge sites, effectively fix the biological enzyme through the electrostatic effect, thereby improving the enzyme activity stability of the biological enzyme electrode 100, but also effectively avoid excessive use of the zwitterionic polymer to cause the enzyme layer to be excessively expanded, thereby ensuring the density and stability of the biological enzyme layer 2.

[0038] In an optional embodiment of the present application, the zwitterionic polymer includes at least one of a carboxybetaine polymer, a sulfobetaine polymer, and a phosphobetaine polymer.

[0039] The positive charge center of the carboxybetaine polymer is a quaternary ammonium salt, the negative charge center is a carboxyl group, and the total charge is an electric center. The negative charge intensity of the carboxyl group is weak, and a mild electrostatic force is formed with the positive charge group on the surface of the biological enzyme, so that the biological enzyme can be fixed mildly, and is especially suitable for enzymes sensitive to charges, such as lactate dehydrogenase.

[0040] The positive charge center of the sulfobetaine polymer is a quaternary ammonium salt, and the negative charge center is a sulfonic acid group. The hydrophilicity of the sulfonic acid group is much stronger than that of the carboxyl group, and a more compact hydration layer can be formed with water molecules, so that the biological enzyme layer 2 has strong resistance to biological pollution, and is suitable for detection of complex samples in vivo. The compact hydration layer formed has strong water locking ability, which can effectively improve the long-term stability of the biological enzyme layer 2.

[0041] The positive charge center of the phosphobetaine polymer is a quaternary ammonium salt, and the negative charge center is a phosphoric acid group. The molecular structure of the phosphobetaine is highly similar to that of the biological membrane component, has good biocompatibility, and the phosphoric acid group can form a hydrogen bond and an electrostatic double interaction with the hydroxyl group and the amino group on the surface of the biological enzyme molecule, so that the enzyme can be fixed more firmly and the leaching rate of the enzyme can be reduced more effectively.

[0042] When the zwitterionic polymer is selected, one of the above types can be selected, or a mixture of at least two of them can be selected, which is not limited herein.

[0043] In an optional embodiment of the present application, the biological enzyme layer 2 further includes a water-based polyurethane.

[0044] The water-based polyurethane contains hydrophilic groups (such as carboxyl, hydroxyl, and urethane groups) and hydrophobic segments (such as polyester and polyether segments) in the molecular chain, and can form a uniform and dense enzyme layer as a film-forming agent. Moreover, the molecular chain structure of the water-based polyurethane has soft segments and hard segments, which can improve the flexibility and ductility of the enzyme layer 2, effectively prevent the enzyme layer 2 from cracking, and thus improve the storage stability of the enzyme electrode 100. In addition, the self-crosslinking network of the water-based polyurethane can interpenetrate with the crosslinking network formed by the crosslinking agent, enhance the structural strength and stability of the enzyme layer 2, avoid excessive crosslinking, and balance the structural stability and enzyme activity maintenance capability.

[0045] Optionally, the content of the water-based polyurethane accounts for 0.01wt%-10wt% of the total amount of the enzyme layer 2, such as 0.01wt%, 0.1wt%, 1wt%, 3wt%, 5wt%, 8wt%, 10wt%, and any interval value between any two endpoint values.

[0046] In the embodiments of the present application, the content of the water-based polyurethane is limited within the above range, which can fully play the role of the film-forming agent, effectively prevent the enzyme layer 2 from cracking, improve the storage stability of the enzyme layer 2, and effectively avoid enzyme activity inhibition and monitoring sensitivity reduction caused by excessive content.

[0047] In optional embodiments of the present application, the enzyme layer 2 further includes a hydrophilic polymer.

[0048] The hydrophilic polymer has a large number of strong hydrophilic groups in the molecular structure, which can form strong interaction with water molecules through hydrogen bonds, electrostatic interaction, etc., shorten the water absorption time of the enzyme layer 2, and quickly reach a stable state, so as to not have a negative impact on the start of the electrochemical biosensor. The combination of the hydrophilic polymer and the zwitterionic polymer forms a double hydrophilic system, which can effectively improve the water absorption of the plastic and quickly reach a stable state. Moreover, the water-based polymer can form hydrogen bonds with the enzyme molecules, improve the activity stability of the enzyme, and has excellent compatibility with biological proteins such as enzymes and bovine serum albumin, so as to form a more uniform enzyme layer 2.

[0049] Optionally, the content of the hydrophilic polymer accounts for 0.2wt%-2wt% of the total amount of the enzyme layer 2, such as 0.2wt%, 1wt%, 1.5wt%, 2wt%, and any interval value between any two endpoint values.

[0050] In the embodiments of the present application, the amount of the hydrophilic polymer is limited within the above range, which can realize the functions of fast water absorption and quick reaching of a stable state, avoid the start lag of the electrochemical biosensor, and avoid excessive swelling caused by excessive content, so as to ensure the diffusion efficiency of the target substance and the structural stability of the enzyme layer 2.

[0051] Optionally, the hydrophilic polymer includes at least one of polyvinylpyrrolidone and cellulose acetate.

[0052] In an optional embodiment of the present application, the thickness of the biological enzyme layer is 1 μm-20 μm, such as 1 μm, 5 μm, 10 μm, 15 μm, 20 μm and interval values between any two endpoint values.

[0053] The present application limits the thickness of the biological enzyme layer 2 to the above range, which can not only ensure sufficient enzyme loading and meet the sensitivity requirement of trace detection, but also reduce the diffusion resistance of the target substrate and ensure the rapid response of the electrochemical biosensor.

[0054] The present application also provides a preparation method of a biological enzyme electrode, including the following steps: (1) providing an electrode body, biological enzymes, a buffer solution, bovine serum albumin, a zwitterionic polymer and a crosslinking agent.

[0055] The specific structure and material composition of the electrode body in step (1) can be referred to the above embodiments, which will not be repeated here. The biological enzymes include but are not limited to at least one of glucose oxidase, glucose dehydrogenase, lactic acid oxidase and lactic acid dehydrogenase. The buffer solution includes but is not limited to a phosphate buffer solution. The zwitterionic polymer includes at least one of carboxybetaine-based polymer, sulfobetaine-based polymer and phosphobetaine-based polymer. The crosslinking agent includes but is not limited to glutaraldehyde.

[0056] (2) dissolving the biological enzymes into the buffer solution to obtain a biological enzyme solution.

[0057] The specific operation of step (2) is as follows: the biological enzymes are added into the buffer solution, and vortexed by a vortex oscillator until the biological enzymes are completely dissolved to obtain a biological enzyme solution. In the prepared biological enzyme solution, the concentration of the biological enzymes is 30 mg / mL-50 mg / mL, such as 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL and interval values between any two endpoint values.

[0058] (3) sequentially adding the bovine serum albumin, the zwitterionic polymer and the crosslinking agent into the biological enzyme solution, and stirring and dissolving to obtain a biological enzyme mixture.

[0059] The specific operation of step (3) is as follows: first, add bovine serum albumin to the biological enzyme solution, shake to dissolve completely, then add the zwitterionic polymer, stir to disperse uniformly, finally add the crosslinking agent, stir uniformly, and stand at room temperature for 20-40 min to obtain the biological enzyme mixture. The concentration of bovine serum albumin is 8-12 mg / mL, such as 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL and interval values between any two endpoint values. The addition amount of the zwitterionic polymer is 0.1-5 wt% of the total solid content in the biological enzyme mixture. The crosslinking agent can be selected from glutaraldehyde, and the concentration of glutaraldehyde is 1-1.5 wt%, such as 1 wt%, 1.25 wt%, 1.5 wt% and interval values between any two endpoint values.

[0060] (4) Coating the biological enzyme mixture on the surface of the electrode body, and drying to obtain a biological enzyme electrode.

[0061] The specific operation of step (4) is as follows: at room temperature, the biological enzyme mixture is coated on the surface of the electrode body, and the coating method can be dip coating, dot coating or reasonable method, which can be determined according to the shape and size of the electrode. After drying, a biological enzyme layer is formed, and the biological enzyme layer and the electrode body together constitute a biological enzyme electrode. The coating thickness of the biological enzyme layer can be selected to be 1-20 μm.

[0062] In an optional embodiment of the application, step (3) comprises: The biological enzyme mixture is obtained by sequentially adding bovine serum albumin, zwitterionic polymer, hydrophilic polymer and / or aqueous polyurethane, and crosslinking agent to the biological enzyme solution, and stirring to dissolve.

[0063] Specifically, first, add bovine serum albumin to the biological enzyme solution, shake to dissolve completely, then sequentially add the zwitterionic polymer, hydrophilic polymer and / or aqueous polyurethane, stir to disperse uniformly, finally add the crosslinking agent, stir uniformly, and stand at room temperature for 20-40 min to obtain the biological enzyme mixture. The specific types and amounts of the hydrophilic polymer and / or aqueous polyurethane can refer to the above embodiments, which will not be repeated here.

[0064] In the embodiment of the present application, the hydrophilic polymer is added to the raw material of the biological enzyme layer, which can effectively shorten the water absorption time of the biological enzyme layer, quickly reach a stable state, avoid the start-up lag of the electrochemical biosensor, and improve the activity stability of the biological enzyme and facilitate the formation of a more uniform biological enzyme layer. The addition of waterborne polyurethane to the raw material of the biological enzyme layer can not only improve the flexibility and ductility of the biological enzyme layer, effectively prevent the biological enzyme layer from cracking, thereby improving the storage stability of the biological enzyme electrode, but also enhance the structural strength and stability of the biological enzyme layer, avoid excessive cross-linking, and balance the structural stability and enzyme activity maintenance capability.

[0065] The present application also provides a wearable electrochemical biosensor, which comprises a working electrode, and the working electrode is the biological enzyme electrode as described above.

[0066] The wearable electrochemical biosensor can be a three-electrode system or a two-electrode system, and the different systems can be selected according to the electrode design and electrode area size. The three-electrode system comprises a working electrode, a reference electrode and a counter electrode, and the two-electrode system comprises a working electrode and a counter electrode. The working electrode in the two-electrode system and the working electrode in the three-electrode system are both the biological enzyme electrode of the present application, and the specific structure, composition and preparation method of the biological enzyme electrode can be referred to the above embodiments, which will not be described here.

[0067] Hereinafter, the embodiments of the present application will be described. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application. If the specific technology or condition is not specified in the embodiments, the technology or condition described in the literature in the art or according to the product instruction is used. If the reagent or instrument is not specified by the manufacturer, it is a conventional product that can be obtained by market purchase.

[0068] Example 1 The present embodiment provides a biological enzyme electrode, and the specific preparation comprises the following steps: (1) 80 mg of glucose oxidase is added to 2 mL of phosphate buffer, and the glucose oxidase is completely dissolved by vortexing to obtain a glucose oxidase solution.

[0069] (2) 20 mg of bovine serum albumin is added to 2 mL of the glucose oxidase solution, and the bovine serum albumin is completely dissolved by vortexing. Then, 3% wt of sulfobetaine, 2% wt of PVP hydrophilic polymer and 5 wt% of waterborne polyurethane are sequentially added, and after stirring and dissolving, 1.25 wt% of glutaraldehyde is added, stirred uniformly and placed at room temperature for 30 min to obtain a glucose oxidase mixture.

[0070] (3) At room temperature, the glucose oxidase mixture is coated on the surface of the electrode body (the electrode material is platinum) with a coating thickness of 15 μm. After drying, the bio-enzyme electrode is obtained.

[0071] An electrochemical biosensor was assembled using a bioenzyme electrode as the working electrode, and it was tested against different concentrations of glucose. The response currents corresponding to different glucose concentrations were obtained, and the test results are as follows: Figure 2 As shown, by Figure 2 As can be seen, the electrochemical biosensor of Example 1 exhibits a good linear relationship with different concentrations of glucose, and the linear correlation coefficient is close to 1, indicating high sensitivity. It can be used as a wearable electrochemical biosensor.

[0072] The electrochemical biosensor of Example 1 was run continuously for 20 days, and tests were conducted every few days to obtain changes in enzyme activity during long-term operation. Figure 3 As shown, from Figure 3 As can be seen, after 20 days of continuous operation, the enzyme activity still maintained more than 95% of the initial enzyme activity, indicating that the electrochemical biosensor of Example 1 has excellent long-term operational stability. Meanwhile, the storage stability of the electrochemical biosensor of Example 1 was also investigated. It was found that after 120 days of storage at room temperature and pressure, the enzyme activity of the electrochemical biosensor of Example 1 still maintained more than 94% of the initial enzyme activity, indicating that the electrochemical biosensor of Example 1 has excellent storage stability.

[0073] Example 2 The difference from Example 1 is that the amount of zwitterionic polymer used is 0.1 wt%, while everything else is the same as in Example 1.

[0074] Example 3 The difference from Example 1 is that the amount of zwitterionic polymer used is 1 wt%, while everything else is the same as in Example 1.

[0075] Example 4 The difference from Example 1 is that the amount of zwitterionic polymer used is 5 wt%, while everything else is the same as in Example 1.

[0076] Example 5 The difference from Example 1 is that the amount of waterborne polyurethane used is 0.01 wt%, while all other aspects are the same as in Example 1.

[0077] Example 6 The difference from Example 1 is that the amount of waterborne polyurethane used is 1 wt%, while everything else is the same as in Example 1.

[0078] Example 7 The difference from Example 1 is that the amount of the aqueous polyurethane is 10 wt%, and the others are the same as Example 1.

[0079] Example 8 The difference from Example 1 is that the amount of the hydrophilic polymer is 0.2 wt%, and the others are the same as Example 1.

[0080] Example 9 The difference from Example 1 is that the amount of the hydrophilic polymer is 1 wt%, and the others are the same as Example 1.

[0081] Example 10 The difference from Example 1 is that the coating thickness of the biological enzyme layer on the surface of the electrode body is 1 μm, and the others are the same as Example 1.

[0082] Example 11 The difference from Example 1 is that the coating thickness of the biological enzyme layer on the surface of the electrode body is 5 μm, and the others are the same as Example 1.

[0083] Example 12 The difference from Example 1 is that the coating thickness of the biological enzyme layer on the surface of the electrode body is 20 μm, and the others are the same as Example 1.

[0084] Comparative Example 1 The difference from Example 1 is that the glucose oxidase mixed solution only contains glucose oxidase, bovine serum albumin, and a crosslinking agent, and the other operations are the same as Example 1.

[0085] Comparative Example 2 The difference from Example 1 is that the glucose oxidase mixed solution only contains glucose oxidase, bovine serum albumin, PVP hydrophilic polymer, and a crosslinking agent, and the other operations are the same as Example 1.

[0086] The electrochemical biosensors of each example and comparative example are tested for sensitivity, enzyme activity retention rate after 20 days of operation, and enzyme activity retention rate after 120 days of storage, and the test results are recorded in Table 1.

[0087] Table 1 Performance parameters of the electrochemical biosensors of each example and comparative example

[0088] As can be seen from the performance data in Table 1, compared with Comparative Example 1-2, the electrochemical biosensors obtained in Inventive Examples 1 to 12 all have higher sensitivity, long-term operation stability and storage stability by reasonably adjusting the amount of zwitterionic polymer, the amount of waterborne polyurethane, the amount of hydrophilic polymer and the thickness of the biological enzyme layer in the preparation of the biological enzyme layer.

[0089] It should be noted that in this document, the terms "comprising", "containing" or any other variant thereof are intended to cover non-exclusive inclusions, so that a process, method, article or system that includes a series of elements not only includes those elements, but also includes other elements not explicitly listed, or inherent to such a process, method, article or system. Without more limitations, the element defined by the statement "comprising a" does not exclude the presence of other identical elements in the process, method, article or system that includes the element.

[0090] The above is only the preferred embodiment of the present application, and does not limit the patent scope of the present application, and any equivalent article or equivalent process transformation, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A bio-enzyme electrode, characterized in that, The bio-enzyme electrode comprises an electrode body and a bio-enzyme layer formed on the surface of the electrode body, and the bio-enzyme layer comprises bio-enzyme, bovine serum albumin, a zwitterionic polymer and a cross-linking agent.

2. The bioenzyme electrode as described in claim 1, characterized in that, The content of the zwitterionic polymer accounts for 0.1wt%-5wt% of the total amount of the bio-enzyme layer; and / or, The zwitterionic polymer comprises at least one of a carboxybetaine polymer, a sulfobetaine polymer and a phosphobetaine polymer.

3. The bioenzyme electrode as described in claim 1, characterized in that, The bio-enzyme layer further comprises an aqueous polyurethane.

4. The bioenzyme electrode as described in claim 3, characterized in that, The content of the aqueous polyurethane accounts for 0.01wt%-10wt% of the total amount of the bio-enzyme layer.

5. The bioenzyme electrode as described in claim 1, characterized in that, The bio-enzyme layer further comprises a hydrophilic polymer.

6. The bioenzyme electrode as described in claim 5, characterized in that, The content of the hydrophilic polymer accounts for 0.2wt%-2wt% of the total amount of the bio-enzyme layer; and / or, The hydrophilic polymer comprises at least one of polyvinylpyrrolidone and cellulose acetate.

7. The bio-enzyme electrode according to any one of claims 1 to 6, wherein The thickness of the bio-enzyme layer is 1μm-20μm.

8. A method for preparing a bio-enzyme electrode, characterized by, The method comprises the following steps: providing an electrode body, bio-enzyme, buffer, bovine serum albumin, zwitterionic polymer and cross-linking agent; dissolving the bio-enzyme into the buffer to obtain a bio-enzyme solution; adding the bovine serum albumin, zwitterionic polymer and cross-linking agent into the bio-enzyme solution in sequence, and stirring and dissolving to obtain a bio-enzyme mixture; coating the bio-enzyme mixture on the surface of the electrode body, and drying to obtain a bio-enzyme electrode.

9. The method of claim 8, wherein the enzyme electrode is prepared by the steps of: (a) preparing a solution of the enzyme and the polymer; (b) adding the solution to the electrode; and (c) drying the electrode. The concentration of the bio-enzyme in the bio-enzyme solution is 30mg / mL-50mg / mL; and / or, The concentration of the bovine serum albumin in the bio-enzyme mixture solution is 8mg / mL-12mg / mL; and / or, The addition amount of the zwitterionic polymer accounts for 0.1wt%-5wt% of the total solid content in the bio-enzyme mixture; and / or, The concentration of the cross-linking agent is 1wt%-1.5wt%.

10. The method for preparing the bioenzyme electrode as described in claim 8 or 9, characterized in that, The step of adding the bovine serum albumin, zwitterionic polymer and cross-linking agent into the bio-enzyme solution in sequence, and stirring and dissolving to obtain a bio-enzyme mixture comprises: The step of adding the bovine serum albumin, zwitterionic polymer, hydrophilic polymer and / or aqueous polyurethane, and cross-linking agent into the bio-enzyme solution in sequence, and stirring and dissolving to obtain a bio-enzyme mixture.

11. A wearable electrochemical biosensor, characterized in that, The working electrode is the bio-enzyme electrode according to any one of claims 1 to 7, or the bio-enzyme electrode prepared by the preparation method of the bio-enzyme electrode according to any one of claims 8 to 10.

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