Electrode and preparation method thereof, electrode assembly and biosensor

Abstract

The invention relates to the technical field of biosensors, in particular to an electrode and a preparation method thereof, an electrode assembly and a biosensor. The electrode comprises an electrode body and a composite layer formed on the surface of the electrode body. A biomimetic catalyst is loaded on the composite layer; the composite layer has a cross-linked network structure, materials for forming the cross-linked network structure comprise glucose oxidase, a conductive material and a cross-linking agent, and the conductive material contains amino functional groups. The electrode provided by the invention is simple and easy to obtain, and interference of reducing substances can be avoided, so that the detection sensitivity and precision of the biosensor can be improved, and double electrochemical response signals can be generated.

Description

Technical Field

[0001] The present application relates to the technical field of biosensors, and in particular to an electrode and a preparation method thereof, an electrode assembly and a biosensor. Background Art

[0002] Glucose is an important substance for clinical diagnosis and metabolic regulation, so the detection of glucose is particularly important. For example, diabetes is a long-term chronic disease, and how to effectively control the development of diabetes has become a research problem that has attracted much attention. Blood glucose test results are of great significance for assessing the degree of glucose metabolism disorders in diabetic patients, formulating blood glucose-lowering plans, reflecting the effects of blood glucose-lowering treatments, and guiding the adjustment of treatment plans. Therefore, the development of a blood glucose detection system with high precision and accuracy is of great significance for the diagnosis and treatment of diabetes.

[0003] In recent years, implantable continuous glucose monitoring systems (CGMS) have provided thousands of diabetic patients with a comprehensive tool for regulating blood sugar. Traditional CGMS are based on the high selectivity and efficient catalytic performance of glucose oxidase, and use electrochemical methods to detect glucose. However, traditional CGMS have high costs, high detection potentials, and the risk of heavy metal ion leakage, and are significantly interfered by reducing substances such as ascorbic acid, uric acid, and acetaminophen. Moreover, the CGMS currently used all have a single electrochemical signal output. During use, as body proteins, cells and other biomass are non-specifically adsorbed on the sensor surface, the sensor detection sensitivity and accuracy gradually decrease, and the service life is shortened. Summary of the invention

[0004] Based on this, it is necessary to provide an electrode and its preparation method, electrode assembly and biosensor that are simple and easy to obtain, can avoid the interference of reducing substances, improve detection sensitivity and accuracy, and can generate dual electrochemical response signals.

[0005] In the first aspect, the present application provides an electrode, comprising: an electrode body and a composite layer formed on the surface of the electrode body; the composite layer is loaded with a bionic catalyst; the composite layer has a cross-linked network structure, and the materials forming the cross-linked network structure include glucose oxidase, a conductive material and a cross-linking agent, and the conductive material contains an amino functional group.

[0006] In some embodiments, the method of loading the biomimetic catalyst includes: forming one or more of π-π stacking effect, electrostatic interaction and hydrogen bond between the conductive material and the biomimetic catalyst.

[0007] In some embodiments, the biomimetic catalyst includes one or more of metalloporphyrin and metalphthalocyanine.

[0008] In some embodiments, the cross-linking agent includes one or more of an aldehyde cross-linking agent, an epoxy group-containing cross-linking agent, N,N-methylenebisacrylamide, and genipin.

[0009] In some embodiments, the conductive material includes one or more of a conductive polymer and an amino-functionalized inorganic conductive material.

[0010] In some embodiments, the composite layer has at least one of the following features:

[0011] 1) The mass ratio of the bionic catalyst to the glucose oxidase is (0.5-2):1;

[0012] 2) The mass ratio of the glucose oxidase, the conductive material and the cross-linking agent is 1:(0.05-0.5):(0.05-0.5).

[0013] In some embodiments, the electrode further includes a diffusion limiting membrane, and the diffusion limiting membrane is disposed on a side of the composite layer away from the electrode body.

[0014] In a second aspect, the present application further provides a method for preparing the electrode as described in the first aspect, comprising the following steps:

[0015] A dispersion liquid including the bionic catalyst, the glucose oxidase, the conductive material and the cross-linking agent is applied to the surface of the electrode body to form the composite layer on the surface of the electrode body.

[0016] In some embodiments, the preparation method further comprises the step of forming a diffusion limiting film on a side of the composite layer facing away from the electrode body.

[0017] In some embodiments, the step of preparing the dispersion comprises:

[0018] Mixing the biomimetic catalyst and the conductive material in a solvent and performing ultrasonic treatment to prepare a mixed solution;

[0019] The mixed solution is mixed with the glucose oxidase and the cross-linking agent and reacted to form a cross-linked network structure between the conductive material, the glucose oxidase and the cross-linking agent to prepare the dispersion.

[0020] In some embodiments, the dispersion has at least one of the following characteristics:

[0021] 1) The concentration of the biomimetic catalyst is 0.5 mg / mL to 10 mg / mL;

[0022] 2) The concentration of the glucose oxidase is 1 mg / mL to 5 mg / mL;

[0023] 3) The concentration of the conductive material is 0.05 mg / mL to 2.5 mg / mL;

[0024] 4) The concentration of the cross-linking agent is 0.05 mg / mL to 2.5 mg / mL.

[0025] In a third aspect, the present application further provides an electrode assembly, comprising a working electrode, a counter electrode and a reference electrode, wherein the working electrode is the electrode described in the first aspect.

[0026] In a fourth aspect, the present application provides a biosensor comprising the electrode assembly described in the third aspect.

[0027] The electrode provided by the present application is modified with a composite layer on the electrode body, so that the concentration of glucose in the body can be detected. Glucose oxidase and amino-functionalized conductive materials form a cross-linked network structure under the cross-linking action of the cross-linking agent to ensure the formation of a uniform composite layer; the conductive material in the composite layer can form a non-covalent bond with the biomimetic catalyst and stably load on the composite layer, so as to avoid leakage of the biomimetic catalyst; the conductive material in the composite layer can accelerate the electron transfer rate between the biomimetic catalyst and the electrode body, and improve the sensor detection performance. The biomimetic catalyst can catalytically reduce oxygen in the body. As the concentration of glucose in the body increases, glucose is oxidized to gluconolactone under the catalytic action of glucose oxidase, and oxygen is reduced to hydrogen peroxide, which can be further catalytically reduced by the biomimetic catalyst. The present application found that the catalytic reduction of oxygen and hydrogen peroxide by the biomimetic catalyst can make the biosensor produce a dual electrochemical response signal to glucose, and the concentration of glucose in the body can be quantified by detecting the consumed oxygen and the generated hydrogen peroxide. Compared with the traditional single electrochemical signal detection mode, the ratiometric electrochemical biosensor constructed in this way can use dual electrochemical response signals as internal references for each other, and is therefore less affected by external environmental disturbances. It can avoid the problem of decreased sensitivity caused by body protein and cell contamination during long-term use of CGMS, thereby improving the accuracy and sensitivity of glucose detection and extending the service life of the biosensor.

[0028] Moreover, the biosensor provided by the present application can detect glucose at extremely low potentials, and can avoid interference from reducing substances such as ascorbic acid (AA), uric acid (UA) and acetaminophen (AP), thereby further improving the detection accuracy of the biosensor. Moreover, the upper limit of glucose detection can reach at least 30mM, which can widely meet the glucose detection concentration requirements required in various fields (such as blood sugar detection). BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 This is a process flow chart of electrode preparation in one embodiment of the present application;

[0031] Figure 2 The cyclic voltammetric response curve of the CoPc functionalized electrochemical biosensor prepared in Example 1 before and after the addition of glucose solution;

[0032] Figure 3 The differential pulse voltammetric response curve of the CoPc functionalized electrochemical biosensor prepared in Example 1 in glucose solutions with different concentrations added;

[0033] Figure 4 is a calibration curve of the CoPc functionalized electrochemical biosensor prepared in Example 1 in solutions with different concentrations of glucose added;

[0034] Figure 5 The dual electrochemical response curve of the CoPc functionalized electrochemical biosensor prepared in Example 1 with 10 mM glucose solution added;

[0035] Figure 6 Schematic diagram of the dual electrochemical response principle of the CoPc functionalized electrochemical biosensor prepared in Example 1 to glucose;

[0036] Figure 7 The cyclic voltammetric response curve of the hemin functionalized electrochemical biosensor prepared in Example 2 before and after the addition of glucose solution;

[0037] Figure 8 The differential pulse voltammetric response curve of the hemin functionalized electrochemical biosensor prepared in Example 2 in glucose solutions with different concentrations added;

[0038] Fig. 9 is a calibration curve of the hemin functionalized electrochemical biosensor prepared in Example 2 in solutions with different concentrations of glucose added;

[0039] Fig.10 The dual electrochemical response curve of the hemin functionalized electrochemical biosensor prepared in Example 2 for 10 mM glucose solution;

[0040] Fig.11The differential pulse voltammetric response curve of the CoPc functionalized electrochemical biosensor prepared in Example 5 in glucose solutions with different concentrations added;

[0041] Fig.12 is a calibration curve of the CoPc functionalized electrochemical biosensor prepared in Example 5 in solutions with different concentrations of glucose added;

[0042] Fig.13 The differential pulse voltammetric response curves of the CoPc functionalized electrochemical biosensor prepared in Example 5 in phosphate buffer solution (PBS), 5 mM glucose (Glu) solution, 5 mM ascorbic acid (AA) solution, 5 mM uric acid (UA) solution and 5 mM acetaminophen (AP) solution. DETAILED DESCRIPTION

[0043] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0045] Terms and Definitions:

[0046] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0047] The term "and / or" used herein includes any and all combinations of one or more of the related listed items. For example, "A and / or B" includes three parallel solutions of A, B and "a combination of A and B".

[0048] In this article, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other expressions that mean "one or more" are also understood in the same way unless otherwise specified.

[0049] In this document, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0050] In this article, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers in the numerical interval, including the two endpoint integers of the numerical range, and each integer between the two endpoints, is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0051] In this article, if there are multiple steps involved in the method flow, unless there is a clear different description in this article, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0052] The structural formulas of phthalocyanine and porphyrin are shown in Formula I and Formula II respectively:

[0053]

[0054] "Metal phthalocyanine" refers to coordination compounds containing metal ions and phthalocyanine molecules and their derivatives, that is, metal ions are embedded in the center of phthalocyanine molecules and their derivatives, usually presenting a typical planar structure.

[0055] "Metalloporphyrin" refers to coordination compounds containing metal ions and porphyrin molecules and their derivatives, that is, the nitrogen atom in the center of the structure of the porphyrin molecule and its derivatives is coordinated with the metal ion, usually presenting a typical planar structure.

[0056] "Bionic catalyst" refers to a catalyst that has both chemical catalytic and biological catalytic activities.

[0057] Traditional CGMS all output a single electrochemical signal, and the detection sensitivity and accuracy of the sensor are low. In addition, the detection potential is high, and is often significantly interfered by reducing substances such as ascorbic acid, uric acid and acetaminophen. To this end, the present application provides an electrode and a preparation method thereof, an electrode assembly and a biosensor that can improve the above problems.

[0058] In a first aspect, the present application provides an electrode, comprising: an electrode body and a composite layer formed on the surface of the electrode body; wherein the composite layer is loaded with a bionic catalyst; the composite layer has a cross-linked network structure, and the materials forming the cross-linked network structure include glucose oxidase, a conductive material and a cross-linking agent, and the conductive material contains an amino functional group.

[0059] The electrode provided by the present application is modified with a composite layer on the electrode body, so that the concentration of glucose in the body can be detected. Glucose oxidase and the conductive material containing amino groups form a cross-linked network structure under the cross-linking action of the cross-linking agent to ensure the formation of a uniform composite layer; the conductive material in the composite layer can form a non-covalent bond with the biomimetic catalyst and stably load on the composite layer, so as to avoid leakage of the biomimetic catalyst; the conductive material in the composite layer can accelerate the electron transfer rate between the biomimetic catalyst and the electrode body, and improve the sensor detection performance. The biomimetic catalyst can catalytically reduce oxygen in the body. As the concentration of glucose in the body increases, glucose is oxidized to gluconolactone under the catalytic action of glucose oxidase, and oxygen is reduced to hydrogen peroxide, which can be further catalytically reduced by the biomimetic catalyst. The present application found that the catalytic reduction of oxygen and hydrogen peroxide by the biomimetic catalyst can make the biosensor produce a dual electrochemical response signal to glucose, and the concentration of glucose in the body can be quantified by detecting the consumed oxygen and the generated hydrogen peroxide. Compared with the traditional single electrochemical signal detection mode, the ratiometric electrochemical biosensor constructed in this way can use dual electrochemical response signals as internal references for each other, and is therefore less affected by external environmental disturbances. It can avoid the problem of decreased sensitivity caused by body protein and cell contamination during long-term use of CGMS, thereby improving the accuracy and sensitivity of glucose detection and extending the service life of the biosensor.

[0060] In the present application, the thickness of the composite layer is not limited. In some embodiments, the thickness of the composite layer is 0.1 μm to 1 μm. By adjusting the thickness of the composite layer within the above range, the electron transfer rate between the composite layer and the electrode can be accelerated, thereby improving the sensor detection sensitivity.

[0061] In the present application, loading specifically refers to non-covalent interaction. In some embodiments, the method of loading the biomimetic catalyst includes: forming one or more of π-π stacking interaction, electrostatic interaction and hydrogen bonding between the conductive material and the biomimetic catalyst.

[0062] In some embodiments, the biomimetic catalyst includes one or more of metalloporphyrin and metal phthalocyanine, wherein the metal ions in the metalloporphyrin and metal phthalocyanine are independently selected from iron ions, cobalt ions, magnesium ions, copper ions, manganese ions, nickel ions or zinc ions.

[0063] In the present application, the choice of cross-linking agent is not limited, and the cross-linking agent commonly used in the field of biosensor preparation can be selected. In some embodiments, the cross-linking agent includes one or more of an aldehyde cross-linking agent, an epoxy group-containing cross-linking agent, N,N-methylenebisacrylamide and genipin. Among them, the aldehyde cross-linking agent can be specifically glutaraldehyde; the epoxy group-containing cross-linking agent includes polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, poly(dimethylsiloxane)-diglycidyl ether, tetracyclyl-4,4-diaminodiphenylmethane, 4-(2,3-epoxypropoxy)-N,N-di(2,3-epoxypropyl)aniline and diglycidyl ethyl ether.

[0064] In the present application, the selection of conductive materials is not limited, and any known conductive material can be selected. In some embodiments, the conductive material includes one or more of a conductive polymer and an amino-functionalized inorganic conductive material. Among them, the amino inorganic conductive material includes one or more of carbon nanotubes, graphene, graphyne, fullerene, Mxene and black phosphorus; the conductive polymer includes an amino-containing conductive polymer or an amino-functionalized conductive polymer, wherein the amino-containing conductive polymer includes one or more of polyaniline, polypyrrole, poly(N-phenylglycine) and their derivatives; the amino-functionalized conductive polymer includes one or more of amino-functionalized polythiophene, amino-functionalized polyacetylene and their derivatives.

[0065] In some embodiments, the mass ratio of the biomimetic catalyst to the glucose oxidase is (0.5-2):1, for example, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1; preferably, the mass ratio of the biomimetic catalyst to the glucose oxidase is (0.5-1.5):1; more preferably, the mass ratio of the biomimetic catalyst to the glucose oxidase is 1:1. By regulating the mass ratio of the biomimetic catalyst to the glucose oxidase within this range, a larger glucose response current can be obtained, thereby improving the sensitivity of the detection sensor.

[0066] In some embodiments, the mass ratio of glucose oxidase, conductive material and cross-linking agent is 1: (0.05-0.5): (0.05-0.5), for example, 1: 0.05: 0.05, 1: 0.1: 0.1, 1: 0.2: 0.2, 1: 0.3: 0.3, 1: 0.4: 0.4, 1: 0.5: 0.5; preferably, the mass ratio of glucose oxidase, conductive material and cross-linking agent is 1: (0.05-0.15): (0.05-0.15); more preferably, the mass ratio of glucose oxidase, conductive material and cross-linking agent is 1: 0.1: 0.1. By regulating the mass ratio of glucose oxidase, conductive material and cross-linking agent within the above range, the composite layer can be stably cross-linked and modified, so that the sensor has higher detection sensitivity and stability.

[0067] In some embodiments, the electrode further comprises a diffusion limiting membrane, which is disposed on the side of the composite layer away from the electrode body. The diffusion limiting membrane can reduce the glucose flux on the electrode surface, thereby achieving a good linear response of the electrode to glucose, accompanied by a reduction in reduction current and a negative shift in reduction peak potential, thereby increasing the upper limit of glucose detection.

[0068] It is understood that the diffusion limiting membrane is a porous polymer membrane. In some embodiments, the material of the diffusion limiting membrane includes one or more of polyurethane, polyvinyl pyridine, polyvinyl imidazole, polyacrylate, polyether urethane and silicone resin.

[0069] In the present application, the thickness of the diffusion limiting film is not limited, and a thickness value commonly used in the art may be selected. For example, the thickness of the diffusion limiting film may be 1 μm to 10 μm.

[0070] In a second aspect, the present application further provides a method for preparing the electrode as described in the first aspect, comprising the following steps:

[0071] A dispersion liquid including a bionic catalyst, glucose oxidase, a conductive material and a cross-linking agent is coated on the surface of the electrode body to form a composite layer on the surface of the electrode body.

[0072] In the present application, the specific coating process is not limited, for example, it can be screen printing, drip coating, blade coating, spin coating or spray coating.

[0073] In some embodiments, in the dispersion, the concentration of the biomimetic catalyst is 0.5 mg / mL to 10 mg / mL, for example, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL.

[0074] In some embodiments, in the dispersion, the concentration of glucose oxidase is 1 mg / mL to 5 mg / mL, for example, 2 mg / mL, 3 mg / mL, 4 mg / mL.

[0075] In some embodiments, in the dispersion, the concentration of the conductive material is 0.05 mg / mL to 2.5 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL, 2.2 mg / mL.

[0076] In some embodiments, in the dispersion, the concentration of the cross-linking agent is 0.05 mg / mL to 2.5 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL, 2.2 mg / mL.

[0077] It is understood that after the dispersion is applied to the surface of the electrode body, a drying step may be further included to solidify the composite layer. The drying conditions are not limited, for example, natural air drying may be performed.

[0078] In some embodiments, the step of preparing the dispersion comprises:

[0079] The biomimetic catalyst and the conductive material are mixed in a solvent and subjected to ultrasonic treatment to prepare a mixed solution;

[0080] The mixed solution is mixed with glucose oxidase and a cross-linking agent and reacted to form a cross-linked network structure between the conductive material, the glucose oxidase and the cross-linking agent to prepare a dispersion solution.

[0081] In the present application, the choice of solvent is not limited, and any solvent commonly used in the field of biosensor manufacturing can be selected. In some embodiments, the solvent is phosphate buffer solution (PBS), water, tris(hydroxymethyl)aminomethane buffer solution, phthalic acid buffer solution, acetic acid buffer solution, sodium chloride solution or an organic solvent; wherein the organic solvent includes one or more of methanol, ethanol, toluene and acetone.

[0082] In the present application, the specific process parameters of the ultrasonic treatment are not limited, and the biomimetic catalyst and the conductive material can be mixed evenly. In some embodiments, the parameters of the ultrasonic treatment include: ultrasonic power of 50W to 200W, ultrasonic time of 0.5h to 2h, and ultrasonic temperature of room temperature.

[0083] It is understood that in order to make the cross-linking reaction between the conductive material, glucose oxidase and the cross-linking agent more thorough, stirring can be performed during the mixing process. The stirring method and time are not limited, for example, mechanical stirring and / or magnetic stirring can be performed, and the stirring time can be 4h to 6h.

[0084] In the present application, the temperature for preparing the dispersion is room temperature.

[0085] It should be noted that, herein, the term "room temperature" generally refers to 4°C to 35°C, for example, 20°C ± 5°C. In some embodiments, "room temperature" refers to 10°C to 30°C. In some embodiments, "room temperature" refers to 20°C to 30°C.

[0086] In some embodiments, the preparation method further comprises the step of forming a diffusion limiting film on a side of the composite layer facing away from the electrode body.

[0087] In some embodiments, the steps of forming the diffusion limiting film are as follows:

[0088] Adding the polymer and the cross-linking agent to the solvent and stirring at room temperature to form a dispersion;

[0089] The dispersion is applied to the side of the composite layer away from the electrode body and cured.

[0090] See also Figure 1 According to a specific embodiment, the method for preparing an electrode comprises the following steps:

[0091] S100: mixing the biomimetic catalyst and the conductive material in a solvent and performing ultrasonic treatment to prepare a mixed solution;

[0092] S200: mixing the mixed solution with glucose oxidase and a cross-linking agent and reacting them to form a cross-linked network structure between the conductive material, the glucose oxidase and the cross-linking agent to prepare a dispersion;

[0093] S300: coating the dispersion on the surface of the electrode body and drying it to form a composite layer on the surface of the electrode body.

[0094] In a third aspect, the present application further provides an electrode assembly, comprising a working electrode, a counter electrode and a reference electrode, wherein the working electrode is the electrode described in the first aspect.

[0095] In the present application, there is no restriction on the selection of the counter electrode and the reference electrode, and electrode materials commonly used in the field of biosensors can be selected. For example, the counter electrode can be a platinum electrode or a carbon electrode, and the reference electrode can be a silver-silver chloride electrode or a mercury-mercury oxide electrode.

[0096] In a fourth aspect, the present application provides a biosensor comprising the electrode assembly described in the third aspect.

[0097] The biosensor provided by the present application can detect glucose at extremely low potentials, and can avoid interference from reducing substances such as ascorbic acid (AA), uric acid (UA) and acetaminophen (AP), thereby further improving the detection accuracy of the biosensor. Moreover, the upper limit of glucose detection can reach at least 30mM, which can widely meet the glucose detection concentration requirements required in various fields (such as blood sugar detection).

[0098] It should be noted that the detection potential of the biosensor provided in the present application for glucose can be as low as below -0.4V or below -0.6V.

[0099] The present application is further described in detail below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manuals or conventional conditions in the art, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0100] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0101] In the following examples, unless otherwise specified, "room temperature" is 20°C to 30°C, such as 25°C.

[0102] Example 1

[0103] Step 1: Cobalt phthalocyanine (CoPc) and amino-functionalized carbon nanotubes (CNTs) are mixed in phosphate buffer solution (PBS) and subjected to ultrasonic treatment to prepare a mixed solution; wherein the concentration of CoPc in the mixed solution is 2 mg / mL, and the concentration of amino-functionalized CNTs is 0.2 mg / mL;

[0104] Step 2: Under stirring conditions, a 2 mg / mL glucose oxidase solution and a 0.2 mg / mL polyethylene glycol diglycidyl ether (PEGDGE) solution are added dropwise to the mixed solution prepared in step 1, and stirring is continued for 4 hours to allow the epoxy functional groups in PEGDGE to fully react with the amino functionalized CNTs and the amino groups in the glucose oxidase to form a stable cross-linked network, thereby preparing a dispersion;

[0105] Step 3: Take 5 μL of the dispersion prepared in step 2 and drop it onto the surface of the working electrode body, and dry it naturally at room temperature to prepare a CoPc functionalized electrochemical biosensor.

[0106] The cyclic voltammetric response curves of the CoPc functionalized electrochemical biosensor prepared in this example in pure PBS solution and after adding 5 mM glucose solution are shown in FIG. Figure 1 As shown. Figure 1 It can be seen that the CoPc functionalized electrochemical biosensor shows an obvious reduction peak in the PBS solution, which is attributed to the electrocatalytic reduction process of CoPc to solution oxygen. After adding 5mM glucose, the reduction peak is significantly weakened. This is because glucose oxidase needs oxygen as a substrate to catalyze the oxidation of glucose, resulting in a decrease in the solution oxygen concentration. This result shows that the CoPc functionalized electrochemical biosensor has good electrocatalytic performance for glucose.

[0107] The differential pulse voltammetric response curves and calibration curves of the CoPc functionalized electrochemical biosensor prepared in this example in solutions with different concentrations of glucose (0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 5 mM, 7 mM, 10 mM) are shown in Figures 1 and 2. Figure 3 and 4 As shown. Among them, Figure 4 The formula of the calibration curve is y = 1.21x-14.62, and the correlation coefficient R 2 =0.9991. Differential pulse voltammetry can significantly reduce the non-Faraday current at the electrode interface and improve the detection sensitivity. Therefore, differential pulse voltammetry was further used to study the response of the sensor to different concentrations of glucose. Figure 3 and 4 It can be seen that as the glucose concentration increases, the reduction current gradually weakens, and the CoPc-functionalized electrochemical biosensor shows a good linear response to glucose concentrations of 0.2 mM to 3 mM.

[0108] like Figure 5 As shown, by expanding the potential detection range, the CoPc functionalized electrochemical biosensor prepared in this embodiment can present dual electrochemical response signals for glucose. Figure 5 It can be seen that the reduction peak near -0.4V is attributed to the catalytic reduction of oxygen by CoPc. With the addition of glucose, glucose oxidase consumes solution oxygen, resulting in a weakening of the reduction peak at -0.4V. There was no reduction peak near -0.7V, but an obvious reduction peak appeared after the addition of glucose. This may be because in the process of glucose oxidase catalyzing the oxidation of glucose, oxygen is reduced to a byproduct, hydrogen peroxide (H2O2), which is further catalytically reduced by CoPc. The specific reaction principle diagram is shown in the figure below. Figure 6This shows that the biosensor provided by the present application can be constructed as a ratiometric electrochemical sensing platform. Compared with the traditional single-signal detection mode, the dual-signal responses of the ratiometric sensor provided by the present application can serve as internal references to each other, and are therefore less affected by external environmental disturbances, which is of great significance for maintaining detection sensitivity and improving monitoring accuracy.

[0109] Example 2

[0110] Step 1: mixing hemin (porphyrin iron) and amino-functionalized graphene (Gr) in phosphate buffer solution (PBS) and performing ultrasonic treatment to prepare a mixed solution; wherein the concentration of hemin in the mixed solution is 2 mg / mL, and the concentration of amino-functionalized Gr is 0.2 mg / mL;

[0111] Step 2: Under stirring conditions, a 2 mg / mL glucose oxidase solution and a 0.2 mg / mL PEGDGE solution were added dropwise to the mixed solution prepared in step 1, and the stirring was continued for 4 hours to allow the epoxy functional groups in PEGDGE to fully react with the amino functionalized Gr and the amino groups in the glucose oxidase to form a stable cross-linked network, thereby preparing a dispersion;

[0112] Step 3: Take 5 μL of the dispersion prepared in step 2 and drop it onto the surface of the working electrode, and dry it naturally at room temperature to prepare a hemin functionalized electrochemical biosensor.

[0113] The cyclic voltammetric response curves of the hemin functionalized electrochemical biosensor prepared in this example in pure PBS solution and after adding 2 mM glucose solution are shown in FIG. Figure 7 As shown. Figure 7 It can be seen that the hemin functionalized electrochemical biosensor exhibits obvious solution oxygen electrocatalytic behavior in PBS solution, while the reduction peak current in glucose solution is significantly weakened, indicating that this hemin sensor has a good electrochemical response to glucose.

[0114] The differential pulse voltammetric response curves and calibration curves of the hemin functionalized electrochemical biosensor prepared in this example in solutions with different concentrations of glucose (0, 0.2 mM, 0.3 mM, 0.5 mM, 1 mM, 2 mM, 3 mM) are shown in Figures 2 and 3, respectively. Figure 8 and 9 As shown. Among them, Fig. 9 The formula of the calibration curve is y = 5.13x-16.65, and the correlation coefficient R 2 =0.9956. Figure 8 and 9 It can be seen that the hemin functionalized electrochemical biosensor shows a good linear response to glucose concentrations ranging from 0.2mM to 2mM.

[0115] like Fig.10 As shown, by expanding the potential detection range, the hemin functionalized electrochemical biosensor prepared in this embodiment can present a dual electrochemical response signal for glucose. Fig.10 It can be seen that the reduction peak at -0.4 V corresponds to the electrocatalytic reduction of solution oxygen by hemin, and the newly appeared reduction peak at -0.6 V is attributed to the electrocatalysis of hemin on the by-product of the enzymatic reaction, hydrogen peroxide.

[0116] Example 3

[0117] Step 1: Mixing manganese phthalocyanine tetrasulfonate (MnPcTS) and polyaniline (PANI) in a PBS solution and performing ultrasonic treatment to prepare a mixed solution; wherein the concentration of MnPcTS in the mixed solution is 1 mg / mL, and the concentration of PANI is 0.1 mg / mL;

[0118] Step 2: Under stirring conditions, a 2 mg / mL glucose oxidase solution and a 0.1 mg / mL glutaraldehyde solution were added dropwise to the mixed solution prepared in step 1, and the stirring was continued for 4 hours to allow the aldehyde group in the glutaraldehyde to fully react with the amino group in PANI and glucose oxidase to form a stable cross-linked network, thereby preparing a dispersion;

[0119] Step 3: Take 5 μL of the dispersion prepared in step 2 and drop it onto the surface of the working electrode, and dry it naturally at room temperature to prepare the MnPcTS functionalized electrochemical biosensor.

[0120] Example 4

[0121] Step 1: copper (II) meso-tetrakis (4-carboxyphenyl) porphyrin (CuTCPP) and amino-functionalized poly (3,4-ethylenedioxythiophene) (PEDOT) are mixed in a PBS solution and subjected to ultrasonic treatment to prepare a mixed solution; wherein the concentration of CuTCPP in the mixed solution is 4 mg / mL, and the concentration of amino-functionalized PEDOT is 1 mg / mL;

[0122] Step 2: Under stirring conditions, a 2 mg / mL glucose oxidase solution and a 1 mg / mL 1,4-butanediol diglycidyl ether solution were added dropwise to the mixed solution prepared in step 1, and the mixture was stirred for 4 hours to allow the epoxy functional groups in 1,4-butanediol diglycidyl ether to fully react with the amino functionalized PEDOT and the amino groups in glucose oxidase to form a stable cross-linked network, thereby preparing a dispersion;

[0123] Step 3: Take 5 μL of the dispersion prepared in step 2 and drop it onto the surface of the working electrode, and dry it naturally at room temperature to prepare a CuTCPP functionalized electrochemical biosensor.

[0124] Example 5

[0125] The preparation method of this embodiment is basically the same as that of embodiment 1, except that it further includes a diffusion limiting membrane. The steps for preparing the diffusion limiting membrane are as follows:

[0126] 30 mg of polyurethane and 3 mg of PEGDGE were dissolved in 1 mL of ethanol and stirred at room temperature for 4 hours to obtain a diffusion-limiting outer membrane dispersion with a concentration of 30 mg / mL; the dispersion was uniformly coated on the surface of the CoPc functionalized electrochemical biosensor using the immersion pulling technique, and cross-linked and cured at room temperature to form a polyurethane diffusion-limiting membrane on the surface of the CoPc functionalized electrochemical biosensor, thereby obtaining a CoPc functionalized electrochemical biosensor with a diffusion-limiting membrane.

[0127] The glucose concentration in different analytical environments varies greatly (can range from micromolar μM to tens of millimolar mM). By coating a diffusion limiting membrane, the detection range and usage scenarios can be broadened.

[0128] The differential pulse voltammetric response curves and calibration curves of the CoPc functionalized electrochemical biosensor coated with a diffusion limiting membrane prepared in this example in solutions with different concentrations of glucose (0, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM) are shown in Figures 1 and 2, respectively. Fig.11 and 12 As shown. Among them, Fig.12 The formula of the calibration curve is y = 0.19x-22.33, and the correlation coefficient R 2 =0.9980. Fig.11 and 12 It can be seen that after coating the diffusion limiting membrane, the reduction current of the electrochemical biosensor decreases and the reduction peak potential shifts negatively; as the glucose concentration increases, the current response gradually weakens, and the electrochemical biosensor has a good linear response to glucose concentrations of 0 to 30 mM. This is because the cross-linked diffusion limiting membrane is a porous structure, and modifying it on the sensor surface can effectively hinder the mass transfer process on the sensor surface and reduce the glucose flux, thereby broadening the linear detection range of the glucose sensor to widely meet the glucose detection concentration requirements required in various fields (such as blood sugar detection).

[0129] The anti-interference performance of the CoPc functionalized electrochemical biosensor coated with a diffusion limiting membrane prepared in Example 5 was tested by using a 5 mM glucose solution (Glu) and a 5 mM common interfering substance solution (ascorbic acid (AA), uric acid (UA), acetaminophen (AP)). The results are as follows: Fig.13 As shown. Fig.13It can be seen that when the sensor is placed in a glucose solution, the reduction current decreases significantly; and when the sensor is placed in different interfering solutions in turn, the current does not change significantly, indicating that this sensor has good detection selectivity for glucose and will not be affected by interfering substances. This is because the detection potential of the glucose sensor of the present application is extremely low (can be as low as below -0.4V or below -0.6V), and it is difficult for reducing interfering substances to undergo oxidation reactions at this potential. It is worth noting that the detection potentials of traditional CGMS are all positive potentials (such as Abbott FreeStyle Libre detection potential is usually +0.04V, Dekang G6 detection potential is usually +0.6V), and at these detection potentials, even if the concentration of interfering substances (especially ascorbic acid) is only 1 / 10 of the glucose concentration, it will still cause significant interference to the detection of glucose, thereby affecting the CGMS monitoring accuracy, and the sensor provided by the present application can avoid this problem.

[0130] Example 6

[0131] The preparation method of this embodiment is basically the same as that of embodiment 2, except that it further includes a diffusion limiting membrane. The steps for preparing the diffusion limiting membrane are as follows:

[0132] 30 mg of polyvinyl pyridine and 3 mg of PEGDGE were dissolved in 1 mL of ethanol and stirred at room temperature for 4 hours to prepare a diffusion-limiting outer membrane dispersion with a concentration of 30 mg / mL; the dispersion was uniformly coated on the surface of the hemin functionalized electrochemical biosensor using the immersion pulling technique, and cross-linked and cured at room temperature to form a polyvinyl pyridine diffusion-limiting membrane on the surface of the hemin functionalized electrochemical biosensor, thereby preparing a hemin functionalized electrochemical biosensor with a diffusion-limiting membrane.

[0133] Example 7

[0134] The preparation method of this embodiment is basically the same as that of embodiment 3, except that it further includes a diffusion limiting membrane. The steps for preparing the diffusion limiting membrane are as follows:

[0135] 30 mg of polyvinyl imidazole and 3 mg of PEGDGE were dissolved in 1 mL of ethanol and stirred at room temperature for 4 hours to obtain a diffusion-limiting outer membrane dispersion with a concentration of 30 mg / mL; the dispersion was uniformly coated on the surface of the MnPcTS functionalized electrochemical biosensor using the immersion pulling technique, and cross-linked and cured at room temperature to form a polyvinyl imidazole diffusion-limiting membrane on the surface of the MnPcTS functionalized electrochemical biosensor, thereby obtaining a MnPcTS functionalized electrochemical biosensor with a diffusion-limiting membrane.

[0136] Example 8

[0137] The preparation method of this embodiment is basically the same as that of embodiment 4, except that it further includes a diffusion limiting membrane. The steps for preparing the diffusion limiting membrane are as follows:

[0138] 30 mg of polyacrylate and 3 mg of PEGDGE were dissolved in 1 mL of ethanol and stirred at room temperature for 4 hours to prepare a diffusion-limiting outer membrane dispersion with a concentration of 30 mg / mL; the dispersion was uniformly coated on the surface of the CuTCPP functionalized electrochemical biosensor using the immersion pulling technique, and cross-linked and cured at room temperature to form a polyacrylate diffusion-limiting membrane on the surface of the CuTCPP functionalized electrochemical biosensor, thereby preparing a CuTCPP functionalized electrochemical biosensor with a diffusion-limiting membrane.

[0139] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0140] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the specification and drawings may be used to interpret the scope of the claims.

Claims

1. An electrode, characterized in that: include: An electrode body and a composite layer formed on the surface of the electrode body; the composite layer is loaded with a biomimetic catalyst; The composite layer has a cross-linked network structure. The materials forming the cross-linked network structure include glucose oxidase, a conductive material and a cross-linking agent. The conductive material contains an amino functional group.

2. The electrode according to claim 1, characterized in that The manner of loading the bionic catalyst includes: forming one or more of π-π stacking effect, electrostatic interaction and hydrogen bond between the conductive material and the bionic catalyst.

3. The electrode according to claim 1, characterized in that The biomimetic catalyst includes one or more of metal porphyrin and metal phthalocyanine.

4. The electrode according to claim 1, characterized in that The cross-linking agent includes one or more of an aldehyde cross-linking agent, an epoxy group-containing cross-linking agent, N,N-methylenebisacrylamide and genipin.

5. The electrode according to claim 1, characterized in that The conductive material includes one or more of a conductive polymer and an amino-functional inorganic conductive material.

6. The electrode according to any one of claims 1 to 5, characterized in that The composite layer has at least one of the following characteristics: 1) The mass ratio of the bionic catalyst to the glucose oxidase is (0.5-2):1; 2) The mass ratio of the glucose oxidase, the conductive material and the cross-linking agent is 1:(0.05-0.5):(0.05-0.5).

7. The electrode according to any one of claims 1 to 5, characterized in that It also includes a diffusion limiting film, which is arranged on a side of the composite layer away from the electrode body.

8. The method for preparing an electrode according to any one of claims 1 to 7, characterized in that: The following steps are involved: A dispersion liquid including the bionic catalyst, the glucose oxidase, the conductive material and the cross-linking agent is applied to the surface of the electrode body to form the composite layer on the surface of the electrode body.

9. The preparation method according to claim 8, characterized in that: The following steps are also included: A diffusion limiting film is formed on a side of the composite layer facing away from the electrode body.

10. The preparation method according to claim 8, characterized in that: The steps of preparing the dispersion include: Mixing the biomimetic catalyst and the conductive material in a solvent and performing ultrasonic treatment to prepare a mixed solution; The mixed solution is mixed with the glucose oxidase and the cross-linking agent and reacted to form a cross-linked network structure between the conductive material, the glucose oxidase and the cross-linking agent to prepare the dispersion.

11. The preparation method according to any one of claims 8 to 10, characterized in that: The dispersion has at least one of the following characteristics: 1) The concentration of the biomimetic catalyst is 0.5 mg / mL to 10 mg / mL; 2) The concentration of the glucose oxidase is 1 mg / mL to 5 mg / mL; 3) The concentration of the conductive material is 0.05 mg / mL to 2.5 mg / mL; 4) The concentration of the cross-linking agent is 0.05 mg / mL to 2.5 mg / mL.

12. An electrode assembly, characterized in that: It comprises a working electrode, a counter electrode and a reference electrode, wherein the working electrode is the electrode according to any one of claims 1 to 7.

13. A biosensor, characterized in that: Comprising the electrode assembly according to claim 12.

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