Working electrode for continuous glucose monitoring and method of making same

Abstract

The application relates to the field of medical devices, and discloses a CGM working electrode and a preparation method thereof. The working electrode comprises a flexible base layer, a metal conductive layer, a catalytic reaction layer, a surface modification layer, a biological enzyme sensing layer and a hydrophilic-hydrophobic semi-permeable membrane layer which are sequentially stacked; the surface modification layer forms a three-dimensional network structure on the surface of the catalytic reaction layer through silanization treatment, and is used for enhancing biological enzyme fixation; the semi-permeable membrane layer is composed of a biocompatible polymer, and is used for selectively controlling the permeation of glucose molecules. The technical scheme of the application not only improves the biocompatibility and measurement accuracy of the electrode, but also prolongs the service life of the electrode through special structural design, and the preparation process is simple, the cost is controllable, and the application is suitable for industrialization and popularization.

Description

Technical Field

[0001] This application relates to the field of medical device technology, and in particular to an electrochemical sensor technology for continuous blood glucose monitoring. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by elevated blood glucose levels, caused by insufficient insulin secretion and / or impaired insulin utilization. According to the latest epidemiological data, based on WHO diagnostic criteria, the prevalence of diabetes in my country has risen to 11.2%. Currently, there is no cure for diabetes; patients need to control their blood glucose levels to reduce their dependence on insulin. Therefore, blood glucose monitoring has become a crucial part of the daily management of diabetes patients.

[0003] Currently, there are two main methods for blood glucose monitoring in clinical practice: one is the traditional finger-prick blood glucose method, which uses a blood glucose meter (BGM); the other is the emerging implantable continuous glucose monitoring system (CGM). Because finger-prick blood glucose testing has drawbacks such as pain and a high risk of infection, patients typically only test 2-3 times a day, making it difficult to comprehensively reflect changes in blood glucose levels. In contrast, the CGM system requires only a single implantation and can continuously monitor for 14 days, providing real-time and continuous blood glucose data, thus gradually becoming the preferred monitoring method for diabetic patients.

[0004] However, existing CGM products still have the following technical problems that urgently need to be solved:

[0005] Biocompatibility issues: Most existing working electrodes use rigid substrate materials, which can cause significant foreign body sensation and pain after implantation in the human body, affecting patient compliance;

[0006] Signal stability issues: The complex physiological environment inside the human body can easily cause electrochemical interference, leading to deviations in measurement data; and byproducts such as H2O2 can reduce the activity of biological enzymes, affecting the accuracy of long-term monitoring.

[0007] Issues with bio-enzyme immobilization: Some products lack a surface modification layer or use a conductive polymer mesh structure, which makes it easy for bio-enzymes to detach and results in insufficient loading, thus shortening the electrode's lifespan; some products using conductive polymer structures also reduce their anti-interference capabilities, causing excessive data deviations.

[0008] Selectivity issues: Existing semipermeable membrane structures are not ideal in their selective permeation of glucose molecules and their blocking effect on interfering substances, and they are easily blocked by impurities, affecting detection performance.

[0009] Cost issues: Some products employ complex structural designs and expensive materials, resulting in high manufacturing costs, which hinders their widespread adoption.

[0010] Therefore, there is an urgent need to develop a CGM working electrode with good biocompatibility, accurate measurement, long service life and simple preparation process to meet the needs of clinical applications. Summary of the Invention

[0011] The purpose of this application is to provide a working electrode for continuous glucose monitoring and a method for preparing the same, so as to solve the problems mentioned in the background art.

[0012] This application discloses a working electrode for continuous glucose monitoring, comprising the following sequentially stacked components:

[0013] Flexible base layer;

[0014] A conductive metal layer is disposed on the surface of the flexible substrate;

[0015] A catalytic reaction layer is disposed on the surface of the metal conductive layer and is used to catalyze the glucose reaction;

[0016] A surface modification layer, formed on the surface of the catalytic reaction layer and having a three-dimensional network structure, is used to enhance the immobilization of biological enzymes;

[0017] The bio-enzyme sensing layer is chemically bonded to the surface of the surface modification layer;

[0018] A hydrophilic and hydrophobic semi-permeable membrane layer is disposed on the surface of the bio-enzyme sensing layer to selectively control the permeation of glucose molecules;

[0019] The flexible base layer has a thickness of 0.075-0.15 mm and a width of 0.35-0.45 mm; the semi-permeable membrane layer has a thickness of 10-40 μm.

[0020] In a preferred embodiment, the surface-modified layer is formed by silanization.

[0021] In a preferred embodiment, the flexible base layer is any one of polyimide (PI), polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), and polydimethylsiloxane (PDMS).

[0022] In a preferred embodiment, the metal conductive layer is made of any one of gold, graphite, silver, titanium, iridium, and ruthenium, and the thickness of the metal conductive layer is 2-30 μm.

[0023] In a preferred embodiment, the catalytic reaction layer is composed of platinum nanoparticles.

[0024] In a preferred embodiment, the surface-modified layer is obtained via a silanization process.

[0025] In a preferred embodiment, the silane compound used in the silanization treatment is selected from any one of vinyltrichlorosilane, vinyltriethoxysilane, vinyltri(β-methoxyethoxy)silane, γ-glycidylpropyltrimethoxysilane, γ-methacryloyloxypropyltrimethoxysilane, and γ-chloropropyltrimethoxysilane.

[0026] In a preferred embodiment, the bio-enzyme sensing layer uses glucose oxidase or glucose dehydrogenase as the bio-enzyme, and the thickness of the bio-enzyme sensing layer is 2-15 μm.

[0027] In a preferred embodiment, the hydrophilic-hydrophobic semipermeable membrane layer is composed of at least one material selected from polyvinylpyridine, polyurethane, polyvinylpyrrolidone, and polyvinyl butyral.

[0028] In a preferred embodiment, the working electrode has a lifespan of 1-28 days after implantation in the body.

[0029] In a preferred embodiment, the following steps are included:

[0030] Step 1 involves surface treatment of the flexible substrate, including sequential cleaning, aging, and plasma treatment.

[0031] Step 2: Form a conductive metal layer on the surface of the treated flexible substrate;

[0032] Step 3: A catalytic reaction layer is formed on the surface of the metal conductive layer by electroplating;

[0033] Step 4 involves surface modification treatment on the surface of the catalytic reaction layer to form a surface modified layer with a three-dimensional network structure.

[0034] Step 5 uses a chemical cross-linking method to fix the bio-enzyme on the surface modified layer to form a bio-enzyme sensing layer, wherein the cross-linking temperature is 30-50℃ and the cross-linking time is 20-60min;

[0035] Step 6: Form a hydrophilic-hydrophobic semi-permeable membrane layer on the surface of the bio-enzyme sensing layer.

[0036] In a preferred embodiment, step 1 involves cleaning the flexible substrate with deionized water, an organic solvent, or a combination thereof.

[0037] In a preferred embodiment, the metal conductive layer is formed on the surface of the flexible substrate in step 2 by screen printing, sputtering, or vapor deposition.

[0038] In a preferred embodiment, the electroplating process conditions in step 3 are: current density 10-50 mA / cm². 2 Electroplating time: 1-10 min; electroplating solution temperature: 30-70℃.

[0039] In a preferred embodiment, step 4 employs an immersion coating method for surface silanization modification, specifically including pre-degreasing, degreasing, water washing, and surface film formation processes, wherein:

[0040] The mass concentration of the silane compound is 0.1-10%, and the impregnation time is 10-60 min;

[0041] The surface film formation process involves drying at 25-45℃ for 20-90 minutes.

[0042] In a preferred embodiment, the crosslinking agent in step 5 is selected from any one of glutaraldehyde, adipaldehyde, chitosan, and ethylene carbonate, and the volume concentration of the crosslinking agent is 5-10 ml / dm³. 3 .

[0043] In a preferred embodiment, step 6 involves forming the semi-permeable membrane layer using a spin coating or spray coating process, employing tetrahydrofuran or chloroform as the solvent, and using a polymer material with a mass concentration of 1-20%, under the following conditions:

[0044] Film formation times: 1-15 times;

[0045] The drying time after each film formation is 30-90 minutes.

[0046] The embodiments of this application have the following technical effects:

[0047] This application provides a working electrode for continuous glucose monitoring. By sequentially setting a flexible base layer, a metal conductive layer, a catalytic reaction layer, a surface modification layer, a bio-enzyme sensing layer, and a hydrophilic-phobic semi-permeable membrane layer, it achieves continuous glucose monitoring with high sensitivity, high stability, and high anti-interference.

[0048] The flexible base layer, with a thickness of 0.075-0.15mm and a width of 0.35-0.45mm, reduces the wearer's foreign body sensation and pain, and improves wearing comfort.

[0049] Forming a surface modification layer with a three-dimensional network structure on the surface of the catalytic reaction layer can not only improve the surface state of the catalytic reaction layer, but also enhance the immobilization effect of the bio-enzyme sensing layer, and increase the amount and stability of the enzyme immobilized.

[0050] The surface-modified layer formed by silanization can form strong covalent bonds with the surface of the catalytic reaction layer, and form a three-dimensional network structure through intermolecular cross-linking of silane molecules, which greatly enhances the loading capacity and immobilization stability of biological enzymes.

[0051] The bio-enzyme sensing layer uses glucose oxidase or glucose dehydrogenase and is chemically cross-linked onto the surface modified layer, ensuring sufficient enzyme quantity and activity to meet the continuous monitoring needs for 1-28 days.

[0052] By setting a 10-40 μm thick hydrophilic-hydrophobic semi-permeable membrane on the surface of the bio-enzyme sensing layer, the diffusion rate of glucose molecules can be effectively controlled, while blocking other macromolecules and interfering substances, thus significantly improving the selectivity and anti-interference ability of glucose monitoring.

[0053] This application also provides a method for preparing the above-mentioned working electrode. By sequentially performing key steps such as substrate surface treatment, preparation of metal conductive layer, electroplating of catalytic reaction layer, surface silanization modification, bio-enzyme crosslinking fixation and semi-permeable membrane formation, the preparation parameters and performance of each functional layer can be precisely controlled, ensuring the overall performance and batch stability of the working electrode.

[0054] The surface silanization modification was carried out by the impregnation coating method, forming a uniform and dense silanized layer on the surface of the catalytic reaction layer. By controlling the concentration of silane compounds, impregnation time and drying conditions, the three-dimensional network structure of the surface modified layer was optimized, providing a large number of bonding sites for the immobilization of biological enzymes.

[0055] By controlling the type, concentration, temperature, and time of the crosslinking agent, efficient immobilization of biological enzymes on the surface-modified layer was achieved, and the activity and stability of the immobilized enzymes were guaranteed, providing enzymatic support for long-term continuous monitoring.

[0056] Semi-permeable membranes were prepared by spin coating or spray coating processes. By optimizing the concentration of polymer materials, the number of film formations, and the drying time, a stable, uniform, and dense semi-permeable membrane was obtained, achieving precise control over the diffusion of glucose molecules and the blocking of interfering substances.

[0057] In summary, the working electrode and preparation method of this application, through the rational design of the electrode structure and the optimization of the preparation process of each functional layer, achieve highly sensitive, long-term, and interference-resistant continuous monitoring of glucose. It can be widely used in the self-management and clinical diagnosis and treatment monitoring of chronic diseases such as diabetes, and has significant technical advantages and application value.

[0058] The specification of this application contains numerous technical features distributed across various technical solutions. Listing all possible combinations of these technical features (i.e., technical solutions) would make the specification excessively lengthy. To avoid this problem, the various technical features disclosed in the above-described invention, the various technical features disclosed in the following embodiments and examples, and the various technical features disclosed in the accompanying drawings can be freely combined to form various new technical solutions (all of which are considered to have been described in this specification), unless such a combination of technical features is technically infeasible. For example, one example discloses feature A+B+C, and another example discloses feature A+B+D+E. Features C and D are equivalent technical means that serve the same function, and technically only one needs to be used; they cannot be used simultaneously. Feature E can technically be combined with feature C. Therefore, the solution A+B+C+D should not be considered as described because it is technically infeasible, while the solution A+B+C+E should be considered as described. Attached Figure Description

[0059] Figure 1 This is a schematic diagram of the structure of a working electrode for continuous glucose monitoring according to the first embodiment of this application.

[0060] Figure 2 This is a schematic flowchart of a method for preparing a working electrode for continuous glucose monitoring according to the second embodiment of this application. Detailed Implementation

[0061] In the following description, many technical details are presented to help the reader better understand this application. However, those skilled in the art will understand that the technical solutions claimed in this application can be implemented even without these technical details and various variations and modifications based on the following embodiments.

[0062] Explanation of some concepts:

[0063] Continuous glucose monitoring (CGM) is a technology that can continuously, automatically, and in real time measure the glucose concentration in subcutaneous interstitial fluid. It typically uses an implantable electrochemical sensor to reflect the dynamic trend of blood glucose changes, providing more comprehensive information for blood glucose management in diabetic patients.

[0064] Flexible base layer: refers to the electrode base layer made of flexible polymer materials, such as polyimide (PI), polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polydimethylsiloxane (PDMS), etc., which can reduce the stimulation and damage of implanted electrodes to human tissues and improve wearing comfort.

[0065] Metal conductive layer: refers to a thin metal film deposited on the surface of a flexible substrate, such as gold, graphite, silver, titanium, iridium, ruthenium, etc., used to conduct electrochemical signals. It is the core conductive component of the electrode and needs to have excellent conductivity and stability.

[0066] Catalytic reaction layer: refers to a layer of catalyst such as platinum nanoparticles modified on the surface of a metal conductive layer, used for electrocatalytic oxidation of glucose, accelerating electron transfer and improving current response sensitivity and specificity.

[0067] Surface modification layer: refers to the introduction of special chemical groups or the formation of a three-dimensional porous structure on the surface of the catalytic reaction layer. It is usually achieved by methods such as silanization treatment, such as using vinyltrichlorosilane, γ-aminopropyltriethoxysilane, etc., to improve the physicochemical properties of the electrode surface, increase the specific surface area, and provide a large number of active sites for immobilized biological enzymes.

[0068] Bio-enzyme sensing layer: refers to the core functional layer for realizing biosensing, in which oxidoreductases such as glucose oxidase (GOx) or glucose dehydrogenase (GDH) are immobilized on a surface-modified layer. By utilizing the specific recognition reaction between the enzyme and glucose, the glucose concentration is converted into a detectable electrochemical signal.

[0069] Hydrophilic-hydrophobic semipermeable membrane: refers to a polymer membrane with both hydrophilic and hydrophobic properties, such as polyvinylpyridine and polyurethane, that is coated on the surface of the enzyme sensing layer. By utilizing the selective permeability of the semipermeable membrane, small molecules such as glucose can diffuse freely in and out, while blocking large molecular interferences such as blood cells and proteins, thereby improving sensing selectivity and anti-interference ability.

[0070] Silanization: This refers to the chemical modification of material surfaces using silane coupling agents. Silane molecules form Si-OM bonds through dehydration condensation with the metal oxide surface via their siloxane groups, simultaneously exposing the organic functional groups at the other end, thus forming a three-dimensional self-assembled film. Commonly used silanizing agents include γ-aminopropyltriethoxysilane (APTES) and vinyltrichlorosilane (VTCS).

[0071] Chemical cross-linking immobilization refers to the method of covalently bonding enzyme molecules to a surface modification layer using bifunctional reagents such as glutaraldehyde (GA) to form a stable enzyme immobilization modification layer. The cross-linking process is usually carried out under mild conditions to ensure that the enzyme molecule conformation and activity are not affected.

[0072] Current density: refers to the magnitude of the current per unit electrode area, measured in mA / cm². 2 Catalytic reaction rate is an important parameter for evaluating electrochemical reaction rate and sensing sensitivity. Optimizing electroplating process conditions such as current density, time, and temperature is crucial for obtaining a high-performance catalytic reaction layer.

[0073] The following is a brief summary of some of the innovative aspects of this application:

[0074] To address the key technical problems existing in the clinical application of CGM products, including poor biocompatibility, low signal stability, poor immobilization of biological enzymes, poor selectivity, and high cost, this application proposes a novel working electrode and its preparation method, which achieves a comprehensive improvement in performance through innovative structural design and process optimization.

[0075] The core innovation of this application lies in the adoption of a meticulously designed multi-layered composite structure. First, the selection of a flexible base material within a specific size range (thickness 0.075-0.15 mm, width 0.35-0.45 mm) significantly improves the biocompatibility and wearing comfort of the electrodes. Second, a unique three-dimensional network structure is formed on the surface of the catalytic reaction layer through silanization treatment. This structure not only firmly binds to the substrate through Si-OM covalent bonds but also forms a large number of active sites through intermolecular cross-linking, thereby greatly improving the loading capacity and immobilization stability of biological enzymes.

[0076] In terms of performance control, this application achieves effective regulation of electron transfer channels and precise control of substrate diffusion by optimizing the interface structure between the surface-modified layer and the catalytic layer, resulting in excellent sensitivity (1.9-3.1 nA / mmol) and linearity (deviation 1.72-11.21%). Simultaneously, the hydrophilic-hydrophobic semi-permeable membrane layer designed on the surface of the bioenzyme sensing layer selectively controls the permeation of glucose molecules, effectively suppressing the influence of interfering substances, and maintaining a stable recovery rate within the range of 92.7-105.5%.

[0077] Of particular note is that the synergistic effect of this multi-layered structure significantly extends the electrode's lifespan, enabling it to operate stably in vivo for 1-28 days, meeting the needs of continuous clinical monitoring. In terms of manufacturing process, this application employs conventional preparation methods such as impregnation coating, resulting in a simple and easily controllable process with good batch stability and cost advantages, laying the foundation for industrial-scale production.

[0078] In summary, this application, through innovative electrode structure design and optimized functional layer fabrication process, successfully achieves the comprehensive performance requirements of high sensitivity, low deviation, strong anti-interference and long lifespan, providing a feasible solution to the technical bottlenecks of existing CGM products, and has significant technical innovation and practical value.

[0079] Compared with the prior art, the technical solution of this application has achieved significant improvements and innovations in the following aspects:

[0080] Advantages of the surface-modified layer structure: A unique three-dimensional network structure is formed on the surface of the catalytic reaction layer through silanization treatment. This not only creates strong Si-OM covalent bonds but also forms numerous active sites through cross-linking between silane molecules. Compared with materials such as m-phenylenediamine and chitosan used in comparative experiments, the surface-modified layer of this application exhibits higher chemical stability and biocompatibility. Testing showed that the enzyme activity of this application (280-310 nA / mmol) was approximately 50% higher than that of the control group (182-196 nA / mmol), indicating that the modified layer significantly enhances the enzyme loading and stability.

[0081] Improved biocompatibility: By selecting a flexible base material and optimizing its thickness (0.075-0.15 mm) and width (0.35-0.45 mm) parameters, the electrode exhibits good flexibility and conformability. Experimental results show that this structural design can effectively reduce pain and foreign body sensation during implantation.

[0082] Improved Measurement Accuracy: This application achieves precise detection of glucose molecules through the special structure of the surface-modified layer and the selective control of the hydrophilic-hydrophobic semi-permeable membrane. Test data shows that the linear deviation is controlled within 11.21%, with an optimal deviation of 1.72%, significantly better than the control group's 12.97-15.73%; the temperature response deviation is reduced to below 1.5%, with an optimal deviation of 0.6%, while the control group is 3.0-4.2%, demonstrating higher measurement accuracy.

[0083] Extended service life: The multilayer composite structure design of this application significantly improves the stability and durability of the electrode. Among them, the surface modification layer firmly fixes the biological enzyme through covalent bonding, and the semi-permeable membrane layer effectively blocks interfering substances. The synergistic effect enables the electrode to work stably and continuously in vivo for 1-28 days.

[0084] Enhanced anti-interference performance: Utilizing a special surface-modified structure and selective semi-permeable membrane, the working electrode of this application exhibits excellent anti-interference performance. Test results show that, in the presence of interfering substances such as ascorbic acid (0.06 g / L) and acetaminophen (0.02 g / L), the recovery rate in the anti-interference test remains between 92.7% and 105.5%, while the control group is 112.1% to 116.5%, indicating that this application can more effectively suppress the influence of interfering substances.

[0085] Advantages of the preparation process: This application uses the impregnation coating method for surface modification and chemical cross-linking to fix biological enzymes, which has the characteristics of simple process, convenient operation and controllable cost, which is conducive to industrialization and promotion.

[0086] The above-mentioned technical effects have been verified through examples and comparative experiments, showing that the present application has achieved significant improvements in all performance indicators and can effectively solve the key technical problems existing in the current CGM products.

[0087] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0088] The first embodiment of this application relates to a working electrode for continuous glucose monitoring, the process of which is as follows: Figure 1 As shown, it includes the following stacked elements:

[0089] Flexible base layer;

[0090] A conductive metal layer is disposed on the surface of the flexible substrate;

[0091] A catalytic reaction layer is disposed on the surface of the metal conductive layer and is used to catalyze the glucose reaction;

[0092] A surface modification layer, formed on the surface of the catalytic reaction layer and having a three-dimensional network structure, is used to enhance the immobilization of biological enzymes;

[0093] The bio-enzyme sensing layer is chemically bonded to the surface of the surface modification layer;

[0094] A hydrophilic and hydrophobic semi-permeable membrane layer is disposed on the surface of the bio-enzyme sensing layer to selectively control the permeation of glucose molecules;

[0095] The flexible base layer has a thickness of 0.075-0.15 mm and a width of 0.35-0.45 mm; the semi-permeable membrane layer has a thickness of 10-40 μm.

[0096] Optionally, the surface modification layer is formed by silanization.

[0097] Optionally, the flexible base layer is made of any one of polyimide (PI), polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), or polydimethylsiloxane (PDMS).

[0098] Optionally, the metal conductive layer is made of any one of gold, graphite, silver, titanium, iridium, and ruthenium, and the thickness of the metal conductive layer is 2-30 μm.

[0099] Optionally, the catalytic reaction layer is composed of platinum nanoparticles.

[0100] Optionally, the surface-modified layer is prepared by silanization.

[0101] Optionally, the silane compound used in the silanization treatment is selected from any one of vinyltrichlorosilane, vinyltriethoxysilane, vinyltri(β-methoxyethoxy)silane, γ-glycidylpropyltrimethoxysilane, γ-methacryloyloxypropyltrimethoxysilane, and γ-chloropropyltrimethoxysilane.

[0102] Optionally, the bio-enzyme sensing layer uses glucose oxidase or glucose dehydrogenase as the bio-enzyme, and the thickness of the bio-enzyme sensing layer is 2-15 μm.

[0103] Optionally, the hydrophilic-hydrophobic semi-permeable membrane layer is composed of at least one material selected from polyvinylpyridine, polyurethane, polyvinylpyrrolidone, and polyvinyl butyral.

[0104] Optionally, the working electrode has a lifespan of 1-28 days after implantation in the body.

[0105] The second embodiment of this application relates to a method for preparing a working electrode for continuous glucose monitoring, the process of which is as follows: Figure 2 As shown, it includes the following steps:

[0106] Step 100: Perform surface treatment on the flexible substrate, including cleaning, aging and plasma treatment in sequence;

[0107] Step 200: Form a conductive metal layer on the surface of the treated flexible substrate;

[0108] Step 300: A catalytic reaction layer is formed on the surface of the metal conductive layer by electroplating;

[0109] Step 400: Perform surface modification treatment on the surface of the catalytic reaction layer to form a surface modified layer with a three-dimensional network structure;

[0110] Step 500: Immobilize the bio-enzyme on the surface-modified layer using a chemical cross-linking method to form a bio-enzyme sensing layer, wherein the cross-linking temperature is 30-50℃ and the cross-linking time is 20-60min;

[0111] Step 600: Form a hydrophilic-hydrophobic semi-permeable membrane layer on the surface of the bio-enzyme sensing layer.

[0112] Optionally, in step 100, deionized water, organic solvents, or a combination thereof are used to clean the flexible substrate.

[0113] Optionally, in step 200, the metal conductive layer is formed on the surface of the flexible substrate by screen printing, sputtering, or vapor deposition.

[0114] Optionally, the electroplating process conditions in step 300 are: current density 10-50 mA / cm². 2Electroplating time: 1-10 min; electroplating solution temperature: 30-70℃.

[0115] Optionally, step 400 employs an immersion coating method for surface silanization modification, specifically including pre-degreasing, degreasing, water washing, and surface film formation processes, wherein:

[0116] The mass concentration of the silane compound is 0.1-10%, and the impregnation time is 10-60 min;

[0117] The surface film formation process involves drying at 25-45℃ for 20-90 minutes.

[0118] Optionally, the crosslinking agent in step 500 is selected from any one of glutaraldehyde, adipaldehyde, chitosan, and ethylene carbonate, and the volume concentration of the crosslinking agent is 5-10 ml / dm³. 3 .

[0119] Optionally, in step 600, the semi-permeable membrane layer is formed using a spin coating or spray coating process, with tetrahydrofuran or chloroform as the solvent, and the polymer material has a mass concentration of 1-20%, and is carried out under the following conditions:

[0120] Film formation times: 1-15 times;

[0121] The drying time after each film formation is 30-90 minutes.

[0122] To better understand the technical solution of this application, a specific example is provided below. The details listed in this example are mainly for ease of understanding and are not intended to limit the scope of protection of this application.

[0123] This example provides a CGM working electrode and its preparation method, including the following steps:

[0124] 1. Prepare a flexible substrate.

[0125] A metal layer is set on a flexible substrate, a catalytic reaction layer is set on the metal layer, a surface modification layer is deposited on the catalytic reaction layer, a bio-enzyme sensing layer is fixed on the surface modification layer, and a semi-permeable membrane layer is coated on the bio-enzyme sensing layer.

[0126] 2. Selection of the base: A flexible base is selected. Unlike a rigid base, the human body has a stronger tolerance for flexible bases, resulting in less foreign body sensation and pain when wearing it. The preferred base is 0.075-0.15mm thick and 0.35-0.45mm wide. This is a limit for the human body to tolerate pain. The pain will increase significantly when the value exceeds this.

[0127] 3. Substrate treatment includes steps such as cutting, cleaning, aging, and plasma bombardment. After simple cleaning, the substrate enters the aging step to allow for sufficient dimensional changes during the initial process, stabilizing it and ensuring it does not affect subsequent processes. Then, the substrate undergoes plasma bombardment: plasma is a form of matter; when charged particles move, they cause the concentration of positive and negative charges, forming an electric field; the movement of charges forms a magnetic field; the entire process involves strong thermal radiation and conduction; the plasma collides with the substrate surface, impacting and removing contaminants, achieving surface cleaning; the plasma also reacts chemically with the substrate surface, generating volatile substances, further removing contaminants; simultaneously, the plasma reaction alters the microstructure of the substrate surface, either creating etching and roughening, forming a dense cross-linked layer, or introducing oxygen-containing polar groups, improving surface hydrophilicity and adhesion.

[0128] 4. The metal layer can be selected from one of gold, graphite, silver, titanium, iridium, etc. This application preferably selects gold as the metal layer. In addition to having good conductivity, gold also has excellent stability due to its properties. It can suppress the electrochemical reactions that occur in the complex environment of the human body and ensure the accuracy of the output signal.

[0129] 5. The preferred catalytic reaction layer is platinum nanoparticles, which are attached to the metal layer via electroplating. Electroplated platinum catalytic layers are gray or black in color and have a porous surface, providing a larger surface area for subsequent surface modification and bioenzyme preparation, thus accommodating more bioenzymes. Simultaneously, the adhesion of the modified layer and the bioenzyme sensing layer is enhanced, making them less prone to detachment. Catalytic reaction layers can also be prepared using screen printing or magnetic co-sputtering, but these methods produce platinum layers with smooth, silvery-white surfaces, making subsequent processes more difficult and less effective. The catalytic reaction layer also catalyzes the decomposition of H2O2 generated from the reaction between bioenzymes and glucose; this product reduces the activity of bioenzymes, thus decreasing the ability to monitor human blood glucose levels.

[0130] 6. The preferred surface modification layer is silanization surface treatment. After hydrolysis, silane rapidly adsorbs onto the metal surface through a condensation reaction between its SiOH groups and the MeOH groups (M represents metal) on the metal surface. On one hand, silane forms Si-O-Me covalent bonds at the metal interface. On the other hand, the remaining silane molecules form a silane film with a Si-O-Si three-dimensional network structure on the metal surface through a condensation reaction between SiOH groups. After drying, this silane film combines with subsequent bio-enzymes through a cross-linking reaction, forming strong chemical bonds. This surface modification layer has a highly positive auxiliary effect on the preparation of the subsequent bio-enzyme sensing layer, promoting the immobilization of bio-enzymes. Currently, some working electrodes do not use surface modification layers to assist in the immobilization of bio-enzymes, which leads to instability and easy detachment of the bio-enzyme sensing layer, as well as reduced working electrode life due to insufficient enzyme quantity. Other working electrodes use a conductive polymer three-dimensional network structure, but this also leads to a decrease in anti-interference performance, inaccurate current collection data, and excessive MAD deviation.

[0131] 7. The bio-enzyme sensing layer preferably uses either glucose oxidase or glucose dehydrogenase, as both have the same function; preferably, it uses one of triethylamine, triethanolamine, or glutaraldehyde, with a crosslinking dosage of 5-10 ml / dm³. 3 The cross-linking temperature is 30-50℃, the cross-linking time is 20-60min, and the cross-linking fixation time and dosage should not be too large.

[0132] 8. The semi-permeable membrane is preferably made of a biocompatible polymer with hydrophilic and hydrophobic properties. Its main functions are diffusion control, interference resistance, and self-cleaning. Diffusion control regulates the diffusion of glucose molecules, while interference resistance prevents the diffusion of non-glucose substances. First, it reduces the amount of tissue fluid or blood entering the semi-permeable membrane. Then, it prevents the entry of other macromolecules besides glucose while simultaneously removing impurities and macromolecule buildup on the membrane, thus avoiding affecting the lifespan of the working electrode. (For details on the self-cleaning function, please refer to the patent description of the self-cleaning membrane.)

[0133] Furthermore, optionally, the surface modification layer may be selected from one of vinyltrichlorosilane, vinyltriethoxysilane, vinyltri(β-methoxyethoxy)silane, γ-glycidylpropyltrimethoxysilane, γ-methacryloyloxypropyltrimethoxysilane, γ-chloropropyltrimethoxysilane, etc.

[0134] Optionally, the base layer is a flexible material, which is one of polyimide (PI), polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), and polydimethylsiloxane (PDMS); the thickness of the flexible base material can be one of 0.05mm, 0.075mm, 0.1mm, 0.125mm, 0.15mm, and 0.2mm; the width of the flexible base material can be between 0.3-0.5mm.

[0135] Optionally, the metal layer can be prepared by screen printing, welding, electrolytic deposition, or vapor deposition on the substrate; the thickness of the metal layer can be between 2-30 μm, which saves costs while ensuring its conductivity, stability, and adhesion of the catalyst layer to its surface.

[0136] Optionally, the catalytic reaction layer can be prepared by electroplating, electrolysis, sputtering, screen printing, or other methods.

[0137] Optionally, the surface modified layer needs to undergo processes such as pre-degreasing, degreasing, water washing, and surface film formation; the surface film of the surface modified layer can be dried at 25-45℃ for 20-90 minutes;

[0138] Optionally, the bio-enzyme used in the bio-enzyme sensing layer is either glucose oxidase or glucose dehydrogenase; the thickness of the bio-enzyme sensing layer can be between 2-15 μm, ensuring that the amount of enzyme can meet the requirements for long-term continuous monitoring.

[0139] Optionally, the semipermeable membrane may be composed of a biocompatible polymer with hydrophilic and hydrophobic properties, wherein the hydrophilic and hydrophobic polymer may be one or more of polyvinylpyridine, polyurethane, polyvinylpyrrolidone, and polyvinyl butyral; the solvent of the semipermeable membrane may be one of tetrachloroethylene and tetrahydrofuran, wherein the hydrophilic and hydrophobic polymers are blended therein.

[0140] Optionally, the semipermeable membrane can be formed by coating, dip-extraction, spin coating, or vapor deposition; the thickness of the semipermeable membrane can be between 10-40 μm, and the number of film formations can be stacked from 1 to 15; the drying time between film formations is 30 min to 90 min.

[0141] Optionally, the lifespan of the CGM working electrode is 1-28 days.

[0142] The first embodiment is a device embodiment corresponding to this embodiment. The technical details in the first embodiment can be applied to this embodiment, and the technical details in this embodiment can also be applied to the first embodiment.

[0143] Test data analysis

[0144] To comprehensively evaluate the performance advantages of the working electrode in this application, a series of comparative experiments were conducted. The specific comparison scheme is as follows:

[0145] Comparative Example 1 used m-phenylenediamine to prepare the surface-modified layer, and the remaining process conditions were the same as in Example 1;

[0146] Comparative Example 2 used chitosan to prepare the surface-modified layer, and the remaining process conditions were the same as in Example 1;

[0147] Comparative Example 3 was prepared by mixing m-phenylenediamine and chitosan, with the remaining process conditions being the same as in Example 1.

[0148] Through detailed analysis of the test data, this application demonstrates significant advantages in all performance indicators:

[0149] 1. Mechanism of bioenzyme activity enhancement:

[0150] The surface-modified layer of this application, formed through silanization, possesses a three-dimensional network structure that provides numerous active sites for immobilizing biological enzymes. Experimental data show that the enzyme activity of the silanized modification examples reaches 280-310 nA / mmol, while the comparative examples only reach 182-196 nA / mmol, representing an increase of approximately 50%. This is mainly attributed to:

[0151] *The Si-O-Si network formed by silanization exhibits excellent mechanical strength and chemical stability.

[0152] *The functional groups introduced on the surface form stable covalent bonds with enzyme molecules.

[0153] The three-dimensional network structure provides a larger specific surface area, increasing enzyme loading.

[0154] In contrast, the conductive polymer structure in the comparative example is prone to degradation and relies mainly on physical adsorption with enzyme molecules, resulting in significant loss of enzyme activity. This substantial improvement in bioenzyme activity directly extends the lifespan of the working electrode.

[0155] 2. Sensitivity control mechanism:

[0156] This application achieves precise sensitivity control within the ideal range of 1.9–3.1 nA / mmol, while the comparative ratio is higher (4.2–4.7 nA / mmol). This significant difference stems from:

[0157] *The uniform microporous structure formed by the silanization-modified layer effectively regulates the substrate diffusion rate.

[0158] The stable interface formed between the surface modification layer and the catalyst layer provides a controllable electron transfer channel.

[0159] *Directional immobilization of biological enzymes improves the contact efficiency between enzymes and substrates.

[0160] The conductive polymer structures used in comparative studies often suffer from problems such as uneven conductivity and unstable interfacial contact, making it difficult to achieve ideal sensitivity control. Precise sensitivity control ensures the reliability of measurement results and is of great significance for improving the accuracy of monitoring data.

[0161] 3. Principle of Linear Deviation Improvement:

[0162] The embodiments of this application exhibit excellent linearity (deviation 1.72-11.21%), significantly better than the comparative examples (12.97-15.73%). This improvement is attributed to:

[0163] The uniform microenvironment provided by the surface modification layer ensures stable enzyme catalytic kinetics.

[0164] *The selective control effect of the semi-permeable membrane layer maintains the linear response range.

[0165] *The synergistic effect of the multi-layer structure reduces signal fluctuations.

[0166] The non-uniform structure of the modified layer in the comparative example can easily lead to differences in local reactivity, affecting the linearity of the response. The improvement in linearity significantly enhances the measurement accuracy across different concentration ranges, making the electrode more suitable for clinical applications.

[0167] 4. Mechanism for improved temperature response and anti-interference performance:

[0168] This application exhibits excellent temperature stability (response deviation 0.6-1.5%) and interference immunity (recovery rate 92.7-105.5%). This performance improvement comes from:

[0169] *The silanization network structure protects enzyme molecules and reduces the effects of temperature.

[0170] *The selective shielding effect of the surface-modified layer blocks interfering substances.

[0171] * Multi-layered structure provides synergistic protection and a stable microenvironment.

[0172] In contrast, the modified layer structure in the comparative example was relatively simple, lacking effective protection and selective shielding mechanisms, resulting in unsatisfactory temperature response deviation (3.0-4.2%) and anti-interference performance (recovery rate 112.1-116.5%). Improved temperature response and anti-interference performance make the electrode more suitable for practical application environments, thus enhancing the reliability of monitoring results.

[0173] In summary, this application achieves significant improvements in various performance indicators through a meticulously designed surface modification layer structure. The unique three-dimensional network structure formed by silanization not only provides an ideal environment for the immobilization of biological enzymes but also ensures the stability and selectivity of the electrode through multiple mechanisms. These advantages have been fully verified in experimental data, demonstrating that the technical solution of this application has significant innovation and practical value, and providing important technical support for the development of a new generation of high-performance CGM products.

[0174] To further verify the overall performance of the working electrode of this application, a series of supplementary tests were conducted on a specific embodiment using γ-methacryloyloxypropyl-trimethoxysilane:

[0175] I. Reliability Verification Test

[0176] Electrode performance was tested under the following conditions, with 10 samples tested in each group:

[0177] (1) Temperature cycling test: The test was conducted under cycling conditions of 25℃, 32℃, 37℃ and 40℃, and each temperature point was maintained for 4 hours.

[0178] - Linear deviation variation range: 1.72 ± 0.31%

[0179] - Sensitivity variation range: 2.0 ± 0.15 nA / mmol

[0180] - Bioenzyme activity retention rate: ≥95%

[0181] (2) Mechanical bending test: Test after 500 cycles of 90° bending.

[0182] - No cracking observed in the flexible substrate

[0183] - Each functional layer is structurally intact, with no delamination or peeling.

[0184] -Performance deviation <3%

[0185] (3) Wet-dry cycle test: 50 cycles of PBS buffer immersion-air drying.

[0186] - No significant degradation of the semi-permeable membrane layer

[0187] - Bioenzyme activity retention rate >92%

[0188] -Sensitivity variation <5%

[0189] II. Repeatability Test Data

[0190] Three batches (10 samples per batch) of samples were prepared for performance testing. The results showed that the relative standard deviation (RSD) of the core performance indicators between batches was <5%, indicating that the process has good repeatability and controllability.

[0191] III. Long-term stability testing

[0192] Performance was monitored continuously for 28 days in a physiological environment at 37℃, with performance tests conducted every 3-4 days.

[0193] (1) Bioenzyme activity decay curve:

[0194] Initial value: 310 nA / mmol

[0195] -7 days: 298 nA / mmol (retention rate 96%)

[0196] -14 days: 285 nA / mmol (retention rate 92%)

[0197] -21 days: 273 nA / mmol (retention rate 88%)

[0198] -28 days: 260 nA / mmol (retention rate 84%)

[0199] (2) Changes in measurement accuracy:

[0200] -Sensitivity drift <8%

[0201] - Linearity deviation is always <5%

[0202] - Temperature response change <2%

[0203] - Interference immunity remains within the range of 90-105%.

[0204] (3) Structural stability:

[0205] - The surface modification layer maintains a complete three-dimensional network structure.

[0206] - The bio-enzyme layer is firmly fixed with no obvious peeling.

[0207] - The semi-permeable membrane layer has good permeability and no clogging phenomenon.

[0208] The supplementary test data above indicates that:

[0209] 1. The working electrode of this application has excellent environmental adaptability and mechanical stability, meeting the requirements under actual use conditions.

[0210] 2. The preparation process exhibits good repeatability and high batch-to-batch consistency, which is beneficial for industrial-scale production.

[0211] 3. It maintains stable performance indicators during 28 days of long-term use, meeting the needs of continuous clinical monitoring.

[0212] 4. The structural stability of each functional layer ensures long-lasting and effective synergy, guaranteeing reliable monitoring results.

[0213] These results not only further confirm the reliability and practical value of this application in real-world applications, but also provide ample data support for clinical use. Furthermore, the excellent batch stability and process controllability lay the foundation for large-scale production of the product.

[0214] It should be noted that in this patent application, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this patent application, if it refers to performing an action according to an element, it means performing the action at least according to that element, including two cases: performing the action only according to that element, and performing the action according to that element and other elements. Expressions such as "multiple," "repeatedly," and "various" include two, two times, two kinds, and more than two, more than two times, and more than two kinds.

[0215] All documents mentioned in this application are considered to be incorporated in their entirety into the disclosure of this application so that they can serve as a basis for modifications if necessary. Furthermore, it should be understood that after reading the foregoing disclosure of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. A working electrode for continuous glucose monitoring, characterized in that, The application relates to a flexible glucose sensor, which comprises the following layers which are sequentially stacked: a flexible base layer; a metal conductive layer arranged on the surface of the flexible base layer; a catalytic reaction layer arranged on the surface of the metal conductive layer and composed of platinum nanoparticles, which is used for catalyzing glucose reaction; a surface modification layer formed on the surface of the catalytic reaction layer through silanization treatment and in a three-dimensional network structure, which is not only firmly combined with the platinum nanoparticles through Si-O-M covalent bonds but also forms active sites through intermolecular cross-linking, and is used for enhancing the fixation of biological enzymes; a biological enzyme sensing layer fixed on the surface of the surface modification layer through chemical bonding by using a chemical cross-linking method; a hydrophilic-hydrophobic semi-permeable membrane layer arranged on the surface of the biological enzyme sensing layer and used for selectively controlling the permeation of glucose molecules.

2. The working electrode of claim 1, wherein, The flexible base layer is made of any one of polyimide (PI), polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP) and polydimethylsiloxane (PDMS).

3. The working electrode of claim 1, wherein, The metal conductive layer is made of any one of gold, graphite, silver, titanium, iridium and ruthenium, and the thickness of the metal conductive layer is 2-30 mu m.

4. A method of preparing the working electrode of claim 1, wherein, The application further discloses a preparation method of the flexible glucose sensor, which comprises the following steps: Step 1: performing surface treatment on the flexible base layer, including sequentially performing cleaning, aging and plasma treatment; Step 2: forming the metal conductive layer on the surface of the treated flexible base layer; Step 3: forming the catalytic reaction layer on the surface of the metal conductive layer through electroplating; Step 4: performing surface modification treatment on the surface of the catalytic reaction layer to form the surface modification layer with a three-dimensional network structure; Step 5: fixing the biological enzyme on the surface modification layer by using a chemical cross-linking method to form the biological enzyme sensing layer, wherein the cross-linking temperature is 30-50 DEG C, and the cross-linking time is 20-60 min; Step 6: forming the hydrophilic-hydrophobic semi-permeable membrane layer on the surface of the biological enzyme sensing layer.

5. The preparation method according to claim 4, characterized in that, In step 1, the flexible base layer is cleaned by using deionized water, an organic solvent or a combination thereof.

6. The preparation method according to claim 4, characterized in that, In step 2, the metal conductive layer is formed on the surface of the flexible base layer by using a silk screen printing, sputtering or vapor deposition process.

7. The preparation method according to claim 4, characterized in that, The electroplating process conditions in step 3 are: current density 10-50 mA / cm 2 , plating time 1-10 min, plating solution temperature 30-70℃.

8. The preparation method according to claim 4, characterized in that, In step 4, surface silanization modification is performed by using an immersion coating method, and the surface silanization modification specifically comprises the following procedures of pre-degreasing, degreasing, water washing and surface film forming, wherein: The mass concentration of the silane compound is 0.1-10%, and the immersion treatment time is 10-60 min; The surface film forming procedure is subjected to drying treatment at 25-45 DEG C, and the drying time is 20-90 min.

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