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, a bio-enzyme recognition layer arranged on the surface of the electrode body and an anti-interference layer arranged on the surface of the bio-enzyme recognition layer, the anti-interference layer adopts a molecularly imprinted polymer film, and adsorption sites matched with a target interferent structure are formed in the molecularly imprinted polymer film. The adsorption site is configured to adsorb a target interferent. According to the technical scheme, the anti-interference performance can be improved, and therefore the accuracy of the detection result of the target to-be-detected object 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] The wearable electrochemical biosensor has great application potential in realizing personalized health monitoring, predictive analysis and timely intervention of personalized healthcare. The working electrode of the wearable electrochemical biosensor usually adopts a biological enzyme electrode. When detecting a complex fluid (such as blood, urine, tissue fluid, sweat, etc.) of the human body, the target detection substance in the complex fluid can produce an identifiable electrical signal through oxidation or reduction reaction at the interface of the biological enzyme recognition layer of the biological enzyme electrode, so that the concentration of the target detection substance can be calculated. However, the complex fluid of the human body may contain interfering substances, and the interfering substances can also produce interference signals through electrochemical reaction at the interface of the biological enzyme recognition layer of the biological enzyme electrode, thereby affecting the detection result of the target detection substance.

[0003] At present, an anti-interference layer is usually formed on the surface of the biological enzyme recognition layer of the biological enzyme electrode by using an anti-interference material, so as to prevent the interfering substances from transmitting to the interface of the biological enzyme recognition layer and reduce the interference signals. However, the anti-interference performance of the anti-interference layer formed by this method is relatively poor in the detection process, especially when the content of the target detection substance in the complex fluid is extremely low or the types of the interfering substances are relatively large, thereby affecting the detection result of the target detection substance.

[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, aiming to improve the anti-interference performance and thus improve the accuracy of the detection result of the target detection substance.

[0006] To achieve the purpose of the present application, the present application provides a biological enzyme electrode, which comprises an electrode body, a biological enzyme recognition layer arranged on the surface of the electrode body and an anti-interference layer arranged on the surface of the biological enzyme recognition layer, wherein the anti-interference layer is a molecularly imprinted polymer film, the inside of the molecularly imprinted polymer film is formed with adsorption sites matching the structure of target interfering substances, and the adsorption sites are configured to adsorb the target interfering substances.

[0007] In an embodiment, the molecularly imprinted polymer film comprises at least one of a sulfobetaine polymer monomer, a carboxybetaine polymer monomer, a phosphocholine polymer monomer and a methacrylic acid polymer monomer, and a crosslinking agent; or, The molecularly imprinted polymer film comprises a conductive polymer.

[0008] In an embodiment, the molecularly imprinted polymer film further comprises chitosan.

[0009] In an embodiment, the content of the chitosan accounts for 0.1wt%-5wt% of the total amount of the molecularly imprinted polymer film.

[0010] In an embodiment, the molecularly imprinted polymer film further comprises cellulose acetate.

[0011] In an embodiment, the content of the cellulose acetate accounts for 0.05wt%-3wt% of the total amount of the molecularly imprinted polymer film.

[0012] In an embodiment, the thickness of the anti-interference layer is 10-30μm.

[0013] In an embodiment, a hydrophilic layer is further arranged between the electrode body and the biological enzyme recognition layer.

[0014] The application further provides a preparation method of a biological enzyme electrode, comprising the following steps: providing an electrode body, a biological enzyme solution, a high polymer material, a target interference substance and a solvent; forming a biological enzyme recognition layer on the surface of the electrode body by using the biological enzyme solution; mixing the high polymer material, the target interference substance and the solvent, and obtaining a polymer mixed solution after uniform stirring; forming a polymer film layer on the surface of the biological enzyme recognition layer by using the polymer mixed solution; eluting and removing the target interference substance in the polymer film layer by using an eluent to form an anti-interference layer, thereby obtaining a biological enzyme electrode.

[0015] In an embodiment, the high polymer material comprises at least one of a sulfobetaine polymer monomer, a carboxybetaine polymer monomer, a phosphocholine polymer monomer and a methacrylic acid polymer monomer, and a crosslinking agent and an initiator, and the step of forming a polymer film layer on the surface of the biological enzyme recognition layer by using the polymer mixed solution comprises: coating the polymer mixed solution on the surface of the biological enzyme recognition layer, and forming a polymer film layer after solidification; or, the high polymer material comprises a conductive polymer monomer, and the step of forming a polymer film layer on the surface of the biological enzyme recognition layer by using the polymer mixed solution comprises: depositing the polymer mixed solution on the surface of the biological enzyme recognition layer by using an electrodeposition method to form a polymer film layer.

[0016] In an embodiment, the target interferent is used in an amount of 0.1wt%-10wt% of the total amount of the polymer material.

[0017] In an embodiment, the step of mixing the polymer material, the target interferent, and the solvent to obtain a polymer mixed solution after stirring uniformly comprises: The polymer material, the target interferent, chitosan and / or cellulose acetate, and the solvent are mixed to obtain a polymer mixed solution after stirring uniformly.

[0018] In an embodiment, the step of preparing a bio-enzyme recognition layer on the surface of the electrode body using the bio-enzyme solution comprises: A hydrophilic layer is prepared on the surface of the electrode body using a hydrophilic material. A bio-enzyme recognition layer is prepared on the surface of the hydrophilic layer using the bio-enzyme solution.

[0019] The application also provides a wearable electrochemical biosensor, which comprises a working electrode, and the working electrode is a bio-enzyme electrode as described above or is prepared by the bio-enzyme electrode preparation method.

[0020] The bio-enzyme electrode provided by the application comprises an electrode body, a bio-enzyme recognition layer arranged on the surface of the electrode body, and an anti-interference layer arranged on the surface of the bio-enzyme recognition layer, wherein the anti-interference layer is a molecularly imprinted polymer film, and the molecularly imprinted polymer film has adsorption sites matching the structure of the target interferent formed inside, and the adsorption sites are configured to adsorb the target interferent. When the bio-enzyme electrode of the application is used to detect the target analyte in the complex human fluid, because the anti-interference layer is a molecularly imprinted polymer film, the adsorption sites in the anti-interference layer match the structure of the target interferent in the complex human fluid, and the adsorption sites can re-adsorb the target interferent in the complex human fluid through hydrogen bonding, electrostatic interaction, etc., so as to effectively avoid the target interferent in the complex human fluid from transmitting to the interface of the bio-enzyme recognition layer to generate an interference signal, and the molecularly imprinted polymer film used as the anti-interference layer can effectively adsorb multiple interferents or trace interferents in the complex human fluid. Therefore, the bio-enzyme electrode of the application has good anti-interference performance, and the accuracy of the detection result of the target analyte is relatively high. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the 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 application, and for those skilled in the art, other related drawings can also be obtained without creative labor.

[0022] Figure 1 Structure diagram of one embodiment of the biological enzyme electrode provided by the present application; Figure 2 Anti-interference principle diagram of the molecularly imprinted polymer film used in the anti-interference layer of the biological enzyme electrode provided by the present application; Figure 3 Anti-interference performance verification curve diagram of the electrochemical biosensor of embodiment 1 of the present application when used for detecting hydrogen peroxide; Figure 4 Anti-interference performance verification curve diagram of the electrochemical biosensor of embodiment 1 of the present application when used for detecting glucose.

[0023] Reference Signs List: 1, electrode body; 2, hydrophilic layer; 3, biological enzyme recognition layer; 4, anti-interference layer; 41, adsorption site.

[0024] The implementation, functional features and advantages of the present application will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0025] It should be understood that the specific embodiments described herein are merely intended to explain the present application, and are not intended to limit the present application.

[0026] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturers are used. If the reagents or instruments used are not specified by the manufacturers, they are all conventional products that can be purchased in the market. In addition, the meaning of "and / or" appearing in the full text includes three parallel solutions. For example, "A and / or B" includes A solution, or B solution, or A and B solutions. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the fact that the ordinary 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 scope of protection claimed by the present application. Based on the embodiments in the present application, all other embodiments obtained by the ordinary skilled in the art without creative labor are within the scope of protection of the present application.

[0027] Wearable electrochemical biosensors have great application potential in realizing personalized health monitoring, predictive analysis and timely intervention of personalized healthcare. The working electrode of the wearable electrochemical biosensor usually adopts a biological enzyme electrode. When detecting complex fluids of the human body (such as blood, urine, tissue fluid, sweat, etc.), the target detection substance in the complex fluid can produce an identifiable electrical signal through oxidation or reduction reaction at the biological enzyme recognition layer interface of the biological enzyme electrode, so that the concentration of the target detection substance can be calculated. However, the complex fluid of the human body may contain interfering substances, and the interfering substances will also produce interference signals through electrochemical reaction at the biological enzyme recognition layer interface of the biological enzyme electrode, thereby affecting the detection result of the target detection substance.

[0028] At present, an anti-interference layer is usually formed on the surface of the biological enzyme recognition layer of the biological enzyme electrode by using an anti-interference material, so as to prevent the transmission of the interfering substances to the biological enzyme recognition layer interface and reduce the interference signals. However, the anti-interference layer formed by this method has relatively poor anti-interference performance in the detection process, especially when the content of the target detection substance in the complex fluid is extremely low or the types of the interfering substances are relatively large, thereby affecting the detection result of the target detection substance.

[0029] In order to solve the above problems, the present application provides a biological enzyme electrode, which aims to improve the anti-interference performance and thereby improve the accuracy of the detection result of the target detection substance.

[0030] In the biological enzyme electrode provided by the present application, the anti-interference layer adopts a molecularly imprinted polymer film, the adsorption sites in the molecularly imprinted polymer film are matched with the structure of the target interfering substance in the complex fluid of the human body, and when the biological enzyme electrode of the present application is used to detect the target detection substance in the complex fluid of the human body, the adsorption sites will re-adsorb the target interfering substance in the complex fluid of the human body through hydrogen bonding, electrostatic interaction and other ways, so as to effectively avoid the transmission of the target interfering substance in the complex fluid of the human body to the biological enzyme recognition layer interface to produce interference signals, and the molecularly imprinted polymer film as the anti-interference layer can effectively adsorb multiple interfering substances or trace interfering substances in the complex fluid of the human body. Therefore, the biological enzyme electrode of the present application has good anti-interference performance, and the accuracy of the detection result of the target detection substance is relatively high.

[0031] Please refer to Figure 1 and Figure 2 In an embodiment of the present application, the biological enzyme electrode includes an electrode body 1, a biological enzyme recognition layer 3 arranged on the surface of the electrode body 1, and an anti-interference layer 4 arranged on the surface of the biological enzyme recognition layer 3, wherein the anti-interference layer 4 adopts a molecularly imprinted polymer film, the inside of the molecularly imprinted polymer film is formed with adsorption sites 41 matched with the structure of the target interfering substance, and the adsorption sites 41 are configured to adsorb the target interfering substance.

[0032] The electrode body 1 comprises a substrate and a conductive layer formed on the surface of the substrate, the material of the substrate comprises at least one of polyimide, polyethylene terephthalate, polydimethylsiloxane, and the material of the conductive layer comprises at least one of platinum, gold, silver, copper, chromium and carbon. In the preparation of the conductive layer, the electrode design pattern is first formed, and then the required conductive circuit and electrode are formed on the surface of the substrate by sputtering, evaporation, electroplating or screen printing, etc., that is, the conductive layer.

[0033] The biological enzyme recognition layer 3 is formed on the surface of the electrode body 1, and can be formed on one side surface or both side surfaces of the electrode body 1 by reasonable coating methods such as dip coating, spot coating and spraying. The biological enzyme recognition layer 3 is a key recognition element of the electrochemical biosensor, and the activity and enzyme activity retention of the enzyme directly affect the sensitivity of the electrochemical biosensor. The specific composition of the biological enzyme recognition layer 3 is not limited here, as long as the target detection substance in the complex fluid can produce an identifiable electrical signal by oxidation or reduction reaction at the interface of the biological enzyme recognition layer 3.

[0034] Referring again to Figure 2 The anti-interference layer 4 is formed on the surface of the biological enzyme recognition layer 3, and the anti-interference layer 4 is a molecularly imprinted polymer film. The molecularly imprinted polymer film is a functional film material with molecular recognition sites, which realizes high-efficiency recognition and adsorption of specific molecules by simulating the specific recognition principle of antigen-antibody in the body and constructing imprinted sites complementary to the spatial and chemical shape of the template molecules in the polymer film. In the present application, the template molecules are target interference molecules in the complex fluid of the human body, and the imprinted sites 41 in the molecularly imprinted polymer film formed by the template molecules are adsorption sites matching the structure of the target interference molecules, which can adsorb the target interference molecules in the complex fluid of the human body again through hydrogen bonds, electrostatic interactions and the like.

[0035] It should be noted that before detecting the target to-be-detected substance in the complex fluid of the human body, the types of the target interference molecules in the complex fluid of the human body are determined, and then the molecularly imprinted polymer film, i.e. the anti-interference layer 4, is prepared based on the target interference molecules in the complex fluid of the human body.

[0036] Optionally, the target interference molecules in the complex fluid of the human body comprise at least one of uric acid (UA), ascorbic acid (AA) and acetaminophen (AP).

[0037] When the bio-enzyme electrode of the application is used to detect the target analyte in the complex human fluid, the anti-interference layer 4 in the bio-enzyme electrode adopts the molecularly imprinted polymer film, the adsorption sites 41 in the molecularly imprinted polymer film are matched with the structure of the target interference in the complex human fluid, and the adsorption sites 41 can re-adsorb the target interference in the complex human fluid through hydrogen bonds, electrostatic interactions and the like, so as to effectively avoid the target interference in the complex human fluid from transmitting to the interface of the bio-enzyme recognition layer 3 to generate an interference signal, and the molecularly imprinted polymer film as the anti-interference layer 4 can effectively adsorb various interference substances or trace interference substances in the complex human fluid, so that the bio-enzyme electrode of the application has good anti-interference performance, and the accuracy of the detection result of the target analyte is relatively high.

[0038] In some embodiments of the application, the molecularly imprinted polymer film comprises at least one of a sulfobetaine polymer monomer, a carboxybetaine polymer monomer, a phosphocholine polymer monomer, a methacrylic acid polymer monomer, and a crosslinking agent.

[0039] In the embodiments of the application, the zwitterionic polymer monomer and the methacrylic acid polymer monomer are used as the polymer monomer, and the crosslinking agent is used to construct a film structure with specific recognition ability. The polymer monomer is the key to realizing specific adsorption of interference substances and compatibility with the biological fluid environment of the molecularly imprinted polymer film.

[0040] The zwitterionic polymer monomer has a structure that can form a hydration layer on the surface of the molecularly imprinted polymer film through solvation, so as to ensure that the target interference in the complex human fluid can diffuse to the adsorption sites 41 to be adsorbed, and at the same time, ensure that the target analyte in the complex human fluid can pass through the anti-interference layer 4 to reach the interface of the bio-enzyme recognition layer 3 to be recognized.

[0041] The sulfobetaine polymer monomer molecule contains quaternary ammonium cations and sulfonate anions, and the sulfonate can form electrostatic interaction with the anions in the target interference, thereby enhancing the adsorption of polar interference substances. The carboxybetaine polymer monomer molecule contains quaternary ammonium cations and carboxyl anions, and the carboxyl groups can be partially protonated in an acidic environment to form hydrogen bonds with interference substances containing amino groups; in a physiological pH environment, the carboxyl groups are anions, and the carboxyl anions produce electrostatic interaction with positively charged interference substances, thereby enhancing the adsorption of various target interference substances. The phosphocholine polymer monomer molecule contains phosphate anions and quaternary ammonium cations, and the phosphocholine group has a very high binding energy with water molecules, and the hydration layer formed can effectively repel non-target biological molecules such as red blood cells and platelets, thereby having good anti-fouling performance. In addition, the structure is similar to a biological membrane, and can enhance the adsorption of interference substances similar to biological molecules.

[0042] The molecular structure of the methacrylic polymer monomer contains a carboxyl group or an ester group, wherein the carboxyl group can form a strong hydrogen bond with the interferent, thereby enhancing the adsorption of neutral / weakly polar interferents; and the carboxyl group has high reactivity with the crosslinking agent, so that the arrangement of the monomers can be fixed by a covalent bond, thereby improving the stability of the adsorption site 41.

[0043] The crosslinking agent serves as the skeleton of the molecularly imprinted polymer film. During the polymerization process, the crosslinking agent bridges the monomers by a covalent bond to form a three-dimensional crosslinked network. After the target interferent molecules are eluted, the crosslinked network serves to fix the adsorption site 41.

[0044] In the embodiments of the present application, the polymer monomers of the molecularly imprinted polymer film are selected from at least two kinds of mixed zwitterionic polymer monomers and methacrylic monomers. In this way, the adsorption requirements for target interferents with different polarities and different functional groups can be covered, and the anti-interference performance is better.

[0045] In optional embodiments of the present application, the crosslinking agent includes, but is not limited to, at least one of ethylene glycol dimethacrylate and (3-(trimethoxysilyl)propane methacrylate).

[0046] In some embodiments of the present application, the molecularly imprinted polymer film includes a conductive polymer.

[0047] In the embodiments of the present application, the molecularly imprinted polymer film introduces a conductive polymer, so that the molecularly imprinted polymer film has an electronic transmission capability, which meets the signal detection requirements of an electrochemical biosensor. In addition, the conductive polymer can spontaneously aggregate to form a continuous and stable polymer film structure through strong intermolecular interactions (such as π-π conjugation, electrostatic interaction, hydrogen bonding, etc.). The driving force for film formation is derived from the molecular structure characteristics of the conductive polymer itself, and no additional chemical crosslinking agent is required, thereby simplifying the preparation process and achieving the dual functions of anti-interference adsorption and conductive signal transmission.

[0048] In some embodiments of the present application, the molecularly imprinted polymer film further includes chitosan. Chitosan contains a large number of hydroxyl groups and amino groups, and can form a three-dimensional porous network structure, thereby improving the transmission efficiency of the target analyte in a complex human fluid, reducing the diffusion resistance, and improving the detection sensitivity. In addition, chitosan can be combined with the biological enzyme recognition layer 3 through covalent action, effectively preventing the anti-interference layer 4 from falling off, and improving the structural stability of the biological enzyme electrode. Furthermore, the amino group can be protonated under acidic conditions, thereby adsorbing and fixing negatively charged interferents, and further improving the anti-interference performance.

[0049] In optional embodiments of the present application, the content of chitosan accounts for 0.1wt%-5wt% of the total amount of the molecularly imprinted polymer film, such as 0.1wt%, 1wt%, 3wt%, 5wt%, and interval values between any two endpoint values.

[0050] The content of chitosan is limited in the above range, which can fully play the role of improving the detection sensitivity, structural stability and anti-interference performance, and at the same time, avoid unnecessary signal attenuation caused by too high content. And limited in the above content, the formed molecularly imprinted polymer film can ensure good structural integrity, ensure better combination between the anti-interference layer 4 and the biological enzyme recognition layer 3, thereby guaranteeing the stability and reliability of the biological enzyme electrode.

[0051] In some embodiments of the present application, the molecularly imprinted polymer film further comprises cellulose acetate. The cellulose acetate has good film-forming property, mechanical strength and chemical stability, which can improve the stability of the molecularly imprinted polymer film. In addition, the acetyl group of the cellulose acetate can form a hydrogen bond with the interferent, further reducing the migration rate of the interferent and reducing its influence on the target signal, thereby further improving the interference signal.

[0052] In an optional embodiment of the present application, the content of cellulose acetate accounts for 0.05wt%-3wt% of the total amount of the molecularly imprinted polymer film, such as 0.05wt%, 0.1wt%, 1wt%, 3wt%, 5wt% and any interval value between any two endpoint values.

[0053] The content of cellulose acetate is limited in the above range, which can effectively improve the problem and anti-pollution of the anti-interference layer 4, and at the same time, avoid the influence of the detection sensitivity caused by the high content of the target to be measured.

[0054] In an optional embodiment of the present application, the molecularly imprinted polymer film simultaneously comprises chitosan and cellulose acetate. The hydrophobic skeleton of the cellulose acetate can form a "hydrophilic-hydrophobic alternating structure" with the hydrophilic group of the chitosan, which provides a stable microenvironment for the biological enzyme recognition layer 3, reduces the influence of temperature and pH changes in the detection environment on its activity (such as preventing enzyme denaturation), and further maintains the signal stability. The chitosan provides biological active sites and ion channels, and the cellulose acetate provides mechanical support and physical barrier. The biological enzyme electrode formed by the molecularly imprinted polymer film comprising both has high detection sensitivity, strong anti-interference and mechanical stability.

[0055] In an optional embodiment of the present application, the thickness of the anti-interference layer 4 is 10μm-30μm, such as 10μm, 20μm, 30μm and any interval value between any two endpoint values.

[0056] The thickness of the anti-interference layer 4 is limited in the above range, which can fully play the role of efficient anti-interference, and at the same time, avoid the influence on the detection sensitivity caused by too thick.

[0057] Again refer to Figure 1In an alternative embodiment of the present application, a hydrophilic layer 2 is arranged between the electrode body 1 and the biological enzyme recognition layer 3. The hydrophilic layer 2 provides a moist environment for the biological enzyme recognition layer 3 by virtue of its high water absorption capacity and strong water retention capacity, thereby shortening the preheating start-up time of the biological enzyme electrode and ensuring the biological enzyme activity and detection stability.

[0058] The present application also provides a preparation method of the biological enzyme electrode.

[0059] In an embodiment of the present application, the preparation method of the biological enzyme electrode comprises the following steps: (1) providing an electrode body, a biological enzyme solution, a high polymer material, a target interferent and a solvent.

[0060] The specific structure and material composition of the electrode body in step (1) can refer to the above-mentioned embodiments, which will not be repeated here. The biological enzyme in the biological enzyme solution includes but is not limited to at least one of glucose oxidase, glucose dehydrogenase, lactic acid oxidase and lactic acid dehydrogenase, and the specific composition of the biological enzyme solution is not limited here as long as it can form a biological enzyme recognition layer. The specific composition of the high polymer material is also not limited as long as it can form a polymer film. The target interferent includes but is not limited to at least one of uric acid (UA), ascorbic acid (AA) and acetaminophen (AP). The solvent can be selected as an organic solvent, and the specific selection can be determined according to the type of the high polymer material. The organic solvent includes but is not limited to at least one of chloroform, toluene and acetonitrile.

[0061] (2) preparing and forming a biological enzyme recognition layer on the surface of the electrode body by using the biological enzyme solution.

[0062] In step (2), the biological enzyme solution can be coated on at least one side surface of the electrode body by using a coating method to form the biological enzyme recognition layer, wherein the coating method includes but is not limited to dipping or dot coating.

[0063] (3) mixing the high polymer material, the target interferent and the solvent, and stirring uniformly to obtain a polymer mixed solution.

[0064] The sequence of steps (2) and (3) can be exchanged, or steps (2) and (3) can be performed simultaneously.

[0065] (4) preparing and forming a polymer film layer on the surface of the biological enzyme recognition layer by using the polymer mixed solution. In step (4), the polymer film layer can be prepared by using a coating or deposition method, and the specific method is determined according to the type of the high polymer material.

[0066] (5) removing the target interferent in the polymer film layer by using an eluent to form an anti-interference layer, thereby obtaining the biological enzyme electrode.

[0067] The eluent in step (5) can be a polar organic solvent, such as methanol. After removing the target interferent in the polymer film layer, an anti-interference layer is formed, which is a molecularly imprinted polymer film.

[0068] The bio-enzyme electrode can be prepared by the above steps, and the operation is simple. In the bio-enzyme electrode prepared by the present application, the anti-interference layer adopts a molecularly imprinted polymer film. The adsorption sites in the molecularly imprinted polymer film are structurally matched with the target interferent in the complex human fluid. When the bio-enzyme electrode prepared by the present application is used to detect the target analyte in the complex human fluid, the adsorption sites will re-adsorb the target interferent in the complex human fluid through hydrogen bonding, electrostatic interaction and other ways, so as to effectively avoid the target interferent in the complex human fluid from transmitting to the interface of the bio-enzyme recognition layer to generate an interference signal. In addition, the molecularly imprinted polymer film as the anti-interference layer can effectively adsorb various types of interferents or trace interferents in the complex human fluid. Therefore, the bio-enzyme electrode prepared by the present application has good anti-interference performance, and the accuracy of the detection result of the target analyte is relatively high.

[0069] In optional embodiments of the present application, the amount of the target interferent accounts for 0.1wt%-10wt% of the total amount of the high molecular polymer material, such as 0.1wt%, 1wt%, 3wt%, 5wt%, 8wt%, 10wt% and any interval value between any two endpoint values.

[0070] The amount of the target interferent is limited in the above range, which not only ensures that the adsorption site density in the formed molecularly imprinted polymer film is moderate, and the anti-interference capacity is improved, but also ensures the structural integrity of the formed molecularly imprinted polymer film, and prolongs the service life of the anti-interference layer.

[0071] In some embodiments of the present application, the high molecular polymer material includes at least one of a sulfobetaine polymer monomer, a carboxybetaine polymer monomer, a phosphocholine polymer monomer, a methacrylic acid polymer monomer, a crosslinking agent and an initiator, and step (4) includes: The polymer mixed solution is coated on the surface of the bio-enzyme recognition layer, and after solidification, a polymer film layer is formed.

[0072] In the embodiments of the present application, the concentration of the polymer monomer in the polymer mixed solution 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. The crosslinking agent includes but is not limited to at least one of dimethyl acrylate, (3-(trimethoxysilyl) propane methacrylate). The amount of the crosslinking agent is 1wt%-3wt% of the total amount of the polymer monomer, such as 1wt%, 2wt%, 3wt% and interval values between any two endpoint values. The initiator includes a photoinitiator and a thermal initiator, the photoinitiator includes but is not limited to at least one of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959), 2,2-dimethoxy-2-phenylacetophenone (DMPA, I1173), 2,4,6-trimethylbenzoyl-diphenyl phosphine oxide (TPO), bis(2,4,6-trimethylbenzoyl)-phenyl phosphine oxide (819); the thermal initiator includes but is not limited to at least one of azobisisobutyronitrile, 2,2'-azobis isobutyronitrile, azobis cyanovaleric acid. The specific type of initiator can be determined according to the type of polymer monomer. The amount of the initiator is 0.5wt%-2wt% of the total amount of the polymer monomer, such as 1wt%, 2wt%, 3wt% and interval values between any two endpoint values.

[0073] When the photoinitiator is used, the curing is performed by ultraviolet curing, and step (4) includes: coating the polymer mixed solution on the surface of the biological enzyme recognition layer, and forming a polymer film layer after ultraviolet curing.

[0074] When the thermal initiator is used, the curing is performed by thermal curing, and step (4) includes: coating the polymer mixed solution on the surface of the biological enzyme recognition layer, and forming a polymer film layer after heating curing.

[0075] In some other embodiments of the present application, the high molecular polymer material includes a conductive polymer monomer, and step (4) includes: The polymer mixed solution is deposited on the surface of the biological enzyme recognition layer by an electrodeposition method to form a polymer film layer.

[0076] In the embodiments of the present application, the conductive polymer monomer can be selected as o-aminophenol, and the specific operation of forming the polymer film layer by the electrodeposition method is: a certain speed is cycled for a certain number of times in a certain potential range to form a polymer film layer, wherein the potential range, the cycling speed and the cycling number are determined according to the thickness of the polymer film layer, and are not limited in particular.

[0077] The conductive polymer monomer is used to form a polymer film layer by an electrodeposition method in the embodiment of the present application, without an additional chemical cross-linking agent, so that the preparation process is simplified, and the anti-interference layer prepared after elution by an eluent has the dual functions of anti-interference adsorption and conductive signal transmission.

[0078] In some embodiments of the present application, step (4) comprises: The high-molecular polymer material, the target interference, chitosan and / or cellulose acetate, and the solvent are mixed and stirred uniformly to obtain a polymer mixed solution.

[0079] In the embodiment of the present application, chitosan and / or cellulose acetate are added in the preparation of the polymer mixed solution, so that the detection sensitivity and structural stability of the bioelectrode are improved to a certain extent, and the anti-interference performance is further improved. The amount of chitosan and cellulose acetate can refer to the above embodiments, which will not be repeated here.

[0080] In some embodiments of the present application, step (2) comprises: A hydrophilic layer is prepared on the surface of the electrode body by using a hydrophilic material; A bioenzyme recognition layer is prepared on the surface of the hydrophilic layer by using a bioenzyme solution.

[0081] In the embodiment of the present application, the hydrophilic layer is prepared between the electrode body and the bioenzyme recognition layer, which can continuously provide a humid environment for the bioenzyme recognition layer, shorten the preheating start-up time of the bioenzyme electrode, and ensure the bioenzyme activity and detection stability. The specific type of the hydrophilic material is not limited, as long as the prepared hydrophilic layer has high water absorption capacity and strong water retention capacity.

[0082] The present application also provides a wearable electrochemical biosensor, which comprises a working electrode, and the working electrode is the bioenzyme electrode as described above.

[0083] 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 the 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 bioenzyme electrode of the present application, and the specific structure, composition and preparation method of the bioenzyme electrode can refer to the above embodiments, which will not be repeated here.

[0084] Embodiments The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0085] Example 1 This embodiment provides a bio-enzyme electrode, the preparation method of which includes the following steps: (1) Preparation of polymer mixed solution: N-(3-sulfopropyl)-N-(methacryloyloxyethyl)-N,N-dimethylammonium betaine (SBMA) monomer, ethylene glycol dimethacrylate, photoinitiator I2959, interfering agent AP, and chloroform were mixed and stirred evenly to obtain a polymer mixed solution. The concentration of SBMA monomer was 40 mg / mL, the amount of crosslinking agent was 2 wt% of the total polymer monomers, the amount of photoinitiator I2959 was 1 wt% of the total polymer monomers, and the amount of interfering agent AP was 1 wt% of the total polymer monomers.

[0086] (2) The prepared polymer mixture solution was coated on the surface of the glucose oxidase recognition layer and cured with ultraviolet light to form a polymer film. The ultraviolet curing power was 100W and the time was 15min. Then, methanol was used to elute the AP in the polymer film to form a molecularly imprinted polymer film, i.e., the anti-interference layer. The thickness of the anti-interference layer was 25 μm. Thus, the bio-enzyme electrode was obtained.

[0087] The bioenzyme electrode prepared in this embodiment was used as the working electrode of the wearable electrochemical biosensor. Hydrogen peroxide detection was performed to verify its anti-interference capability. The response current of the wearable electrochemical biosensor after the addition of the interfering agent was obtained by detection, as shown in the figure. Figure 3 As shown.

[0088] from Figure 3 It can be seen that the current signal of the electrochemical biosensor increases after adding a certain amount of H2O2. When H2O2 is added in three stages, the response current shows a stepwise linear increase with each addition of a certain concentration of H2O2. Thus, the response current of the electrochemical biosensor is basically linearly related to the concentration of H2O2, indicating that the electrochemical biosensor has excellent detection performance for the target H2O2. When different interfering substances (uric acid, ascorbic acid, and acetaminophen) are added, the change in current of the electrochemical biosensor is extremely small (<3%), indicating that the electrochemical biosensor has excellent anti-interference ability and the interference signal is negligible.

[0089] Further, a glucose oxidase recognition layer is prepared on the surface of the platinum electrode body.

[0090] The bio-enzyme electrode prepared in this example is used as the working electrode of a wearable electrochemical biosensor, and the detection of glucose is carried out to verify the anti-interference ability. The response current-time curve of the wearable electrochemical biosensor after the addition of interference is obtained by detection, as shown in FIG. 2. Figure 4

[0091] From Figure 4 It can be seen that the sensor current signal increases after the addition of a certain amount of glucose, indicating that the electrochemical biosensor has excellent detection performance for the target glucose; when different interference substances (uric acid, ascorbic acid, acetaminophen, sodium chloride NaCl and potassium chloride KCl) are added, the current change of the electrochemical biosensor is very small (<2%), indicating that the electrochemical biosensor has excellent anti-interference ability, and the interference signal can be ignored.

[0092] Example 2 The difference from Example 1 is that the amount of interference AP is 0.1wt% of the total amount of polymer monomers, and the others are the same as Example 1.

[0093] Example 3 The difference from Example 1 is that the amount of interference AP is 5wt% of the total amount of polymer monomers, and the others are the same as Example 1.

[0094] Example 4 The difference from Example 1 is that the amount of interference AP is 10wt% of the total amount of polymer monomers, and the others are the same as Example 1.

[0095] Example 5 The difference from Example 1 is that the thickness of the anti-interference layer is 10 μm, and the others are the same as Example 1.

[0096] Example 6 The difference from Example 1 is that the thickness of the anti-interference layer is 30 μm, and the others are the same as Example 1.

[0097] Example 7 The difference from Example 1 is that chitosan is added in step (2), and the amount of chitosan is 0.1wt%, and the others are the same as Example 1.

[0098] Example 8 The difference from Example 7 is that the amount of chitosan is 2wt%, and the others are the same as Example 1.

[0099] Example 9 ​The difference from Example 7 is that the amount of chitosan is 5 wt%, and the other conditions are the same as those in Example 1.

[0100] Example 10 The difference from Example 1 is that cellulose acetate is added in step (2), and the amount of cellulose acetate is 0.05 wt%, and the other conditions are the same as those in Example 1.

[0101] Example 11 The difference from Example 10 is that the amount of cellulose acetate is 3 wt%, and the other conditions are the same as those in Example 1.

[0102] Example 12 The difference from Example 1 is that chitosan and cellulose acetate are added in step (2), and the amount of chitosan is 0.2 wt% and the amount of cellulose acetate is 0.1 wt%, and the other conditions are the same as those in Example 1.

[0103] Example 13 The difference from Example 1 is that chitosan and cellulose acetate are added in step (2), and the amount of chitosan is 5 wt% and the amount of cellulose acetate is 3 wt%, and the other conditions are the same as those in Example 1.

[0104] Example 14 The difference from Example 1 is that the monomer of the polymer is methacrylic acid, the crosslinking agent is (3-(trimethoxysilyl)propane methacrylate), and the initiator is azobisisobutyronitrile (AIBN), and after the preparation of the polymer mixed solution, the polymer mixed solution is purged with argon for 5 minutes to remove oxygen, and a thermal initiation polymerization reaction is used, and after curing, a polymer film is formed, wherein the temperature of the thermal initiation is 55℃, and the time is 30 minutes, and the other conditions are the same as those in Example 1.

[0105] Example 15 The difference from Example 1 is that the polymer mixed solution includes 2.0×10 -3 mol / L o-aminophenol, 1.0×10 -2 mol / L AP and 0.1 mol / L HClO4, and an electrodeposition method is used to form a polymer film layer, specifically: 30 cycles are performed between 0V and 1.3V at a speed of 0.1V / s by cyclic voltammetry, and a polymer film layer is deposited, and the thickness of the polymer film layer is 10 μm, and the other conditions are the same as those in Example 1.

[0106] Comparative Example 1 The bioenzyme electrode only includes an electrode body and a glucose oxidase recognition layer, and does not include an anti-interference layer.

[0107] The sensitivity, stability and anti-interference ability of the electrochemical biosensor of each embodiment and the comparative example were tested, and the test results were recorded in Table 1. Among them, the stability refers to the percentage of the sensitivity of the electrochemical biosensor after being placed in the phosphate buffer for 7 days relative to the initial sensitivity; the anti-interference ability refers to the maximum concentration of AP that the electrode can resist.

[0108] Table 1 Performance parameters of the electrochemical biosensor of each embodiment and the comparative example

[0109] As can be seen from the performance data in Table 1, compared with Comparative Example 1, the electrochemical biosensors of Embodiments 1 to 15 all have high sensitivity, stability and anti-interference ability by reasonably adjusting the amount of interference AP in the preparation raw material of the anti-interference layer, the thickness of the interference layer, the addition amount of chitosan and / or cellulose acetate, and the film forming method of different polymer monomers.

[0110] It should be noted that in this document, the terms "comprising", "including", or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or system including a series of elements includes not only those elements, but also 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 including the element.

[0111] 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 electrode body, the biological enzyme recognition layer arranged on the surface of the electrode body, and the anti-interference layer arranged on the surface of the biological enzyme recognition layer are provided, wherein the anti-interference layer is a molecularly imprinted polymer film, the inside of the molecularly imprinted polymer film is formed with adsorption sites matching the structure of target interference substances, and the adsorption sites are configured to adsorb the target interference substances.

2. The bioenzyme electrode as described in claim 1, characterized in that, The molecularly imprinted polymer film comprises at least one of a sulfobetaine polymer monomer, a carboxybetaine polymer monomer, a phosphocholine polymer monomer, and a methacrylic acid polymer monomer, and a crosslinking agent; or The molecularly imprinted polymer film comprises a conductive polymer.

3. The bioenzyme electrode as described in claim 2, characterized in that, The molecularly imprinted polymer film further comprises chitosan.

4. The bioenzyme electrode as described in claim 3, characterized in that, The content of the chitosan accounts for 0.1wt%-5wt% of the total amount of the molecularly imprinted polymer film.

5. The bioenzyme electrode as described in claim 2, characterized in that, The molecularly imprinted polymer film further comprises cellulose acetate.

6. The bioenzyme electrode as described in claim 5, characterized in that, The content of the cellulose acetate accounts for 0.05wt%-3wt% of the total amount of the molecularly imprinted polymer film.

7. The bio-enzyme electrode according to any one of claims 1 to 6, wherein The thickness of the anti-interference layer is 10μm-30μm.

8. The bio-enzyme electrode according to any one of claims 1 to 6, wherein A hydrophilic layer is further arranged between the electrode body and the biological enzyme recognition layer.

9. A method for preparing a bio-enzyme electrode, characterized by, The method comprises the following steps: Providing an electrode body, a biological enzyme solution, a high polymer material, a target interference substance, and a solvent; Using the biological enzyme solution to prepare a biological enzyme recognition layer on the surface of the electrode body; Mixing the high polymer material, the target interference substance, and the solvent to obtain a polymer mixed solution after uniform stirring; Using the polymer mixed solution to prepare a polymer film layer on the surface of the biological enzyme recognition layer; Using an eluent to elute and remove the target interference substance in the polymer film layer to form an anti-interference layer, thereby obtaining a biological enzyme electrode.

10. The method of claim 9, 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 high polymer material comprises at least one of a sulfobetaine polymer monomer, a carboxybetaine polymer monomer, a phosphocholine polymer monomer, and a methacrylic acid polymer monomer, and a crosslinking agent and an initiator, and the step of using the polymer mixed solution to prepare a polymer film layer on the surface of the biological enzyme recognition layer comprises: Coating the polymer mixed solution on the surface of the biological enzyme recognition layer, and forming a polymer film layer after solidification; or The high polymer material comprises a conductive polymer monomer, and the step of using the polymer mixed solution to prepare a polymer film layer on the surface of the biological enzyme recognition layer comprises: Using an electrodeposition method to deposit the polymer mixed solution on the surface of the biological enzyme recognition layer to form a polymer film layer.

11. The method of claim 10, wherein the enzyme electrode is prepared by the steps of: (a) mixing the enzyme, the electron mediator, and the polymer to form a mixture; (b) adding the mixture to the electrode; and (c) drying the electrode. The amount of the target interference substance accounts for 0.1wt%-10wt% of the total amount of the high polymer material.

12. The method for preparing the bioenzyme electrode according to any one of claims 9 to 11, characterized in that, The step of mixing the high polymer material, the target interference substance, and the solvent to obtain a polymer mixed solution after uniform stirring comprises: Mixing the high polymer material, the target interference substance, chitosan and / or cellulose acetate, and the solvent to obtain a polymer mixed solution after uniform stirring.

13. The method for preparing the bioenzyme electrode according to any one of claims 9 to 11, characterized in that, The step of using the biological enzyme solution to prepare a biological enzyme recognition layer on the surface of the electrode body comprises: Using a hydrophilic material to prepare a hydrophilic layer on the surface of the electrode body; The bio-enzyme solution is used to form a bio-enzyme recognition layer on the surface of the hydrophilic layer.

14. A wearable electrochemical biosensor, characterized in that, The working electrode is a bio-enzyme electrode as claimed in any one of claims 1 to 8, or is a bio-enzyme electrode prepared by the bio-enzyme electrode preparation method as claimed in any one of claims 9 to 13.

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