A sensing layer containing self-assembled modified magnesium aluminum silicate, a sensor thereof and a preparation method thereof

Abstract

The application discloses a sensing layer containing self-assembled modified magnesium aluminum silicate, a sensor and a preparation method thereof. The sensing layer containing self-assembled modified magnesium aluminum silicate is formed by dropping and coating a sensing layer solution capable of self-assembling with a modified layer on the modified layer obtained by grafting a hydrophilic organic polymer on a working electrode of a flexible electrode. The sensing layer solution comprises a redox polymer, a tool enzyme, a crosslinking agent A and magnesium aluminum silicate. The mass ratio of the redox polymer, the tool enzyme, the crosslinking agent A and the magnesium aluminum silicate is 15-40:20-45:5-30:1-10. The hydrophilic organic polymer is grafted on the surface of the working electrode, and the hydrophilic organic polymer can be self-assembled with the magnesium aluminum silicate. The self-assembled and modified magnesium aluminum silicate has excellent gel performance and rheological property, has good film forming effect, and can improve the consistency, stability and accuracy of the sensor.

Description

Technical Field

[0001] This invention relates to the field of electrochemical sensor technology, specifically to a sensing layer containing self-assembled modified magnesium aluminum silicate, a sensor thereof, and a preparation method thereof. Background Technology

[0002] Currently, diabetes has become the third leading cause of death in my country after cardiovascular disease and cancer. China has nearly 130 million diabetes patients, ranking first in the world, and the prevalence of diabetes is increasing exponentially. Diabetes is a chronic, systemic, metabolic disease caused by the long-term combined effects of genetic and environmental factors, characterized by elevated plasma glucose levels. It is mainly caused by insufficient insulin secretion or impaired insulin action, leading to disorders in the metabolism of carbohydrates, fats, and proteins, thus affecting normal physiological activities.

[0003] Currently, the main method for testing blood glucose is to collect blood from the fingertip using in vitro glucose monitoring strips. Patients need to prick their fingers multiple times a day to know their blood glucose level at a given moment. This method cannot reflect real-time changes and trends in blood glucose concentration and can also cause harm to patients. Continuous glucose monitoring systems (CGMS), on the other hand, can display the user's blood glucose changes in real time during the usage period. They are quicker and more convenient to use, providing detailed blood glucose data support for patients and medical staff. The electrochemical reaction principle of CGMS involves a biological enzyme, such as glucose oxidase, fixed on a sensor and implanted in the subcutaneous tissue to measure the glucose concentration in the tissue fluid. The electrical signal measured by glucose oxidase is processed by the transmitter or receiver of the CGMS and algorithms to convert the electrical signal into glucose concentration, which is then displayed on a monitor or in software, forming a glucose monitoring graph.

[0004] Glucose electrochemical sensors require high accuracy to meet the needs of real-time blood glucose monitoring. This poses a significant challenge to the consistency and stability of the sensor's initial sensitivity. Therefore, ensuring the consistency of the sensing layer is crucial in the mass production of glucose electrochemical sensors. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned problems by providing a sensing layer with less agglomeration, high stability, high consistency, and better control of the droplet area, as well as a sensor thereof and its preparation method.

[0006] To achieve its objective, the present invention employs the following technical solution:

[0007] A first aspect of the present invention provides a sensing layer containing self-assembled modified magnesium aluminum silicate for preparing an electrochemical sensor, the electrochemical sensor including a flexible electrode, the sensing layer being formed by drop-coating a sensing layer solution capable of self-assembling and modifying the modified layer onto a modified layer obtained by grafting a hydrophilic organic polymer onto the working electrode of the flexible electrode.

[0008] The sensing layer solution comprises a redox polymer, a tool enzyme, a crosslinking agent A, and magnesium aluminum silicate, wherein the mass ratio of redox polymer: tool enzyme: crosslinking agent A: magnesium aluminum silicate is 15-40:20-45:5-30:1-10.

[0009] Preferably, the hydrophilic organic polymer is selected from one or more of polyallylamine, polyethylene glycol, polyethyleneimine, polyamide-amine dendritic polymer, and poly(L-lysine);

[0010] The redox polymer is a redox polymer containing transition metals, including ruthenium, rhodium, and osmium;

[0011] The tool enzymes are selected from glucose dehydrogenase, glucose oxidase, uricase, lactate oxidase, and cholesterol oxidase.

[0012] The crosslinking agent A is selected from one or more of 1,4-butanediol diglycidyl ether, poly(dimethylsiloxane)-diglycidyl ether, glutaraldehyde, polyethylene glycol diglycidyl ether, tetraglycidyl-4,4-diaminodiphenylmethane, or glycerol triglycidyl ether; preferably, crosslinking agent A is polyethylene glycol diglycidyl ether with MW = 200 Da-5000 Da, preferably with MW = 400 Da.

[0013] Preferably, the mass ratio of the redox polymer:tool enzyme:crosslinking agent A:magnesium aluminum silicate is 35-40:20-35:20-30:4-10, more preferably 35-40:25-35:25-30:5-10, and even more preferably 39:31:25:5;

[0014] The concentration of the redox polymer in the sensing layer solution is 2-5 mg / ml, preferably 2-4.5 mg / ml, and more preferably 3.0-4.5 mg / ml.

[0015] A second aspect of the present invention provides a method for preparing a sensing layer, wherein the sensing layer is a sensing layer containing self-assembled modified magnesium aluminum silicate as described in any of the preceding claims, and the preparation method includes the following steps:

[0016] S1. A modified layer is obtained by grafting a hydrophilic organic polymer onto the working electrode of a flexible electrode using plasma initiation.

[0017] S2. The sensing layer solution is drop-coated onto the modified layer, and the sensing layer is obtained after cross-linking and curing.

[0018] The above-mentioned method for preparing the sensing layer, wherein the modified layer is obtained by grafting a hydrophilic organic polymer onto the working electrode of the flexible electrode using plasma initiation, comprises:

[0019] After treating the flexible electrode with air plasma, the flexible electrode is immersed in an aqueous solution of a hydrophilic organic polymer with a concentration of 25-40 mg / mL and a temperature of 35-40°C for 14-20 hours. After immersing the electrode in purified water for 20-30 hours, it is taken out and air-dried for 30-40 minutes to form the modified layer on the working electrode of the flexible electrode.

[0020] The above-described method for preparing the sensing layer, wherein the sensing layer solution is drop-coated onto the modified layer and cross-linked and cured to obtain the sensing layer comprises the following steps:

[0021] S2.1 Preparation of the sensing layer solution: A mixed solution of redox polymer, tool enzyme, crosslinking agent A, and magnesium aluminum silicate is prepared using solvent A and thoroughly mixed to obtain the sensing layer solution; the solvent A is HEPES buffer or TES buffer; preferably, the concentration of the HEPES buffer is 5-20 mmol / L and the pH is 7.5-8.2.

[0022] S2.2 Preparation of the sensing layer: The prepared sensing layer solution is drop-coated onto the surface of the modified layer obtained in step S1, and the sensing layer is obtained after cross-linking and curing at a temperature of 20-30°C for 20-28 hours.

[0023] A third aspect of the present invention provides an electrochemical sensor, comprising: a flexible electrode, the flexible electrode comprising a substrate, a working electrode, a counter electrode, and a reference electrode, wherein,

[0024] The working electrode and the counter electrode are respectively disposed on both sides of the substrate. A dielectric layer is disposed on the side of the counter electrode away from the substrate. The reference electrode is disposed on the side of the working electrode away from the substrate. The dielectric layer is disposed between the working electrode and the reference electrode. The dielectric layer is disposed on the side of the reference electrode away from the working electrode.

[0025] The surface of the working electrode has an exposed portion without a dielectric layer and a reference electrode, and a modified layer and a sensing layer are sequentially coated on the surface of the exposed portion.

[0026] It also includes a diffusion-restricting layer that encloses the sensing layer, the dielectric layer, the reference electrode, and the exposed portions of the counter electrode;

[0027] The modified layer and the sensing layer are either the modified layer and the sensing layer described in any of the preceding claims, or the modified layer and the sensing layer prepared according to the preparation method described in any of the preceding claims.

[0028] Preferably, the above-mentioned electrochemical sensor,

[0029] The confined diffusion layer is derived from a polymer containing nitrogen-containing heterocyclic groups and crosslinking agent B;

[0030] The polymer containing nitrogen-containing heterocyclic groups is selected from one or more of polyvinylpyridine, polyvinylpyrrole, polyvinylpyridine-styrene copolymer, and sulfonic acid-modified polyvinylpyridine-styrene copolymer;

[0031] The crosslinking agent B is selected from one or more of triglycidyl-p-aminophenol, polyethylene glycol diglycidyl ether, tetraglycidyl-4,4'-diaminodiphenylmethane, tris(4-hydroxyphenyl)methane triglycidyl ether, and glycerol triglycidyl ether; preferably, the crosslinking agent B is polyethylene glycol diglycidyl ether with a MW of 200 Da to 5000 Da, preferably with a MW of 200 Da;

[0032] The mass ratio of the polymer containing nitrogen heterocyclic groups to crosslinking agent B is 7–9.9:0.1–3, preferably 9:1.

[0033] A fourth aspect of the present invention provides a method for preparing the above-mentioned electrochemical sensor, comprising the following steps:

[0034] S3. The flexible electrode with sensing layer obtained in step S2 is immersed in the outer membrane solution of the confined diffusion layer, and then cured at a temperature of 20-35°C to form the confined diffusion layer, thus obtaining the electrochemical sensor.

[0035] Preferably, step S3 includes the following steps:

[0036] S3.1 Preparation of the restricted diffusion layer outer membrane solution: Using solvent B as the solvent, a mixture of a polymer containing nitrogen heterocyclic groups and crosslinking agent B is prepared to obtain the restricted diffusion layer outer membrane solution; the solvent B is TES buffer, or a mixture of anhydrous ethanol and HEPES buffer, preferably solvent B is obtained by mixing anhydrous ethanol and HEPES buffer at a volume ratio of 2 to 10:1, the concentration of the HEPES buffer is 5 to 20 mmol / L, and the pH is 7.5 to 8.2; the concentration of the polymer containing nitrogen heterocyclic groups in the restricted diffusion layer outer membrane solution is 40 to 80 mg / ml, preferably 50 to 70 mg / ml;

[0037] S3.2 Preparation of the confined diffusion layer: The flexible electrode with the sensing layer obtained in step S2 is immersed in the outer membrane solution of the confined diffusion layer multiple times, preferably 2 to 5 times, with an interval of 6 to 20 minutes each time. After completion, it is cured at a temperature of 20 to 35°C for 40 to 55 hours to form the confined diffusion layer and obtain the electrochemical sensor.

[0038] The redox polymer is a compound with the chemical structural formula (I):

[0039]

[0040] In equation (I), R1 and R2 each represent independent connecting keys, and R1 and R2 may be the same or different;

[0041] M represents a transition metal;

[0042] L1, L2, L3, L4, L5, and L6 each independently represent a ligand, which is a heterocycle and is coordinated with M through a heteroatom of the heterocycle;

[0043] X represents a self-assembly group;

[0044] Y represents anion.

[0045] a represents an integer; b represents the number of anions, which is a natural number; where b > a + 1;

[0046] n, m, and r represent natural numbers, and the sum of n, m, and r is 20 or more;

[0047] e, g, and f represent integers;

[0048] The self-assembling group is selected from one of the following groups, substituted or unsubstituted:

[0049]

[0050] Where c and d are integers from 1 to 16;

[0051] The substituents of the self-assembled group are selected from benzyl bromide, benzyl chloride, -F, -Cl, -Br, -I, -NO2, -COOH, -SO3H, -NH2, -OH, alkoxy groups with 1-6 carbon atoms, or alkyl groups with 1-6 carbon atoms.

[0052] The beneficial effects of this invention are:

[0053] First, a hydrophilic organic polymer is grafted onto the surface of the working electrode. This hydrophilic organic polymer undergoes self-assembly with magnesium aluminum silicate, with the polymer adsorbed between the aluminum silicate layers and hydrogen bonds present between them. This self-assembly of the two components makes the surface of the aluminum silicate smoother. The modified magnesium aluminum silicate exhibits excellent gelation and rheological properties, allowing the sensing layer solution to have a consistent wetting angle when drop-coated onto the electrode surface. This effectively reduces aggregation between enzymes and redox polymers, preventing the formation of large particle aggregates. The sensing layer is evenly spread on the surface, ensuring consistent drop-coating area and resulting in a uniformly dispersed sensing layer with good film-forming effect. This reduces enzyme activity loss and improves the consistency, stability, and accuracy of the sensor. The confined diffusion layer on the sensor is biocompatible, broadening the sensor's monitoring range, reducing interference from non-specifically adsorbed proteins, and improving the sensor's usability and stability. Attached Figure Description

[0054] Figure 1 These are scanning electron microscope (SEM) images of the sensing layer solutions: (ab) SEM image of magnesium aluminum silicate, (c) SEM image of magnesium aluminum silicate mixed with polyethylene glycol, and (d) SEM image of self-assembled modified magnesium aluminum silicate.

[0055] Figure 2 Results of the wetting angle test of the sensing layer solution: (a) Wetting angle of the sensing layer solution with self-assembled modified magnesium aluminum silicate, (b) Wetting angle of the sensing layer solution with a mixture of magnesium aluminum silicate and polyethylene glycol, (c) Wetting angle of the sensing layer solution without polyethylene glycol, (d) Wetting angle of the sensing layer solution without magnesium aluminum silicate.

[0056] Figure 3 The following are electron microscope (EM) images of the sensor layer surface: (a) EM image of the modified sensor layer surface, (b) EM image of the sensor layer surface mixed with magnesium aluminum silicate and polyethylene glycol, (c) EM image of the sensor layer surface without magnesium aluminum silicate, and (d) EM image of the sensor layer surface without polyethylene glycol.

[0057] Figure 4 The results of the sensitivity consistency test of the sensing layer are as follows: (a) Sensitivity consistency of the sensing layer with self-assembled modified magnesium aluminum silicate, (b) Sensitivity consistency of the sensing layer with a mixture of magnesium aluminum silicate and polyethylene glycol, (c) Sensitivity consistency of the sensing layer without magnesium aluminum silicate, and (d) Sensitivity consistency of the sensing layer without polyethylene glycol.

[0058] Figure 5 The results of the 15-day stability test of the sensing layer are as follows: (a) 15-day stability of the modified sensing layer, (b) 15-day stability of the sensor with a mixture of magnesium aluminum silicate and polyethylene glycol, (c) 15-day stability of the sensor without magnesium aluminum silicate in the sensing layer, and (d) 15-day stability of the sensor without polyethylene glycol in the sensing layer.

[0059] Figure 6 It is the linear spectrum (0-25mM) of a sensor containing modified magnesium aluminum silicate in the sensing layer.

[0060] Figure 7 It is a linear spectrum (0-25mM) of a sensor composed of magnesium aluminum silicate and polyethylene glycol.

[0061] Figure 8 It is the linear spectrum (0-25mM) of a sensor whose sensing layer does not contain magnesium aluminum silicate.

[0062] Figure 9 It is a linear spectrum (0-25mM) of a sensor whose sensing layer does not contain polyethylene glycol.

[0063] Figure 10 This is a human trial map of a sensor containing modified magnesium aluminum silicate in the sensing layer in an implantable continuous glucose monitoring system.

[0064] Figure 11 This is a human trial map of a sensor composed of magnesium aluminum silicate and polyethylene glycol in an implantable continuous glucose monitoring system.

[0065] Figure 12 This is a human trial map of a sensor without magnesium aluminum silicate in the sensing layer in an implantable continuous glucose monitoring system.

[0066] Figure 13 This is a human trial map of a sensor without polyethylene glycol in the sensing layer in an implantable continuous glucose monitoring system.

[0067] Figure 14 This is a schematic diagram of the structure of a conventional flexible electrode (a) and the sensor of the present invention (b). Detailed Implementation

[0068] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0069] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0070] Main materials and reagents:

[0071] Glucose oxidase: CAS No.: 9001-37-0, 120U / mg.

[0072] Polyethylene glycol diglycidyl ether: CAS No.: 26403-72-5.

[0073] Magnesium aluminum silicate: a white composite colloidal substance, CAS number: 71205-22-6, with models including but not limited to HS-100, HV-101, HV-102, YK-R, YK-101, and YK-200. Model HV-101 is used in the embodiments and comparative examples of this invention.

[0074] Sulfonic acid-modified polyvinylpyridine-styrene copolymer: prepared according to the method in US Patent 6932894B2.

[0075] Redox polymers: The redox polymers used in Examples 1-5 and Comparative Examples 1-3 were all self-assembled redox polymers a prepared in Example 1 of Chinese Patent ZL 202210837308.6 (CN 115215953 B), and their chemical structural formulas are shown in Formula (II):

[0076]

[0077] Example 1

[0078] I. Electrode Pretreatment:

[0079] The flexible electrode was treated with air plasma (flow rate 150 mL / min, power 100 W, vacuum degree 50 Pa) for 5 minutes. Then, the electrode was immersed in a 30 mg / mL polyethylene glycol aqueous solution (37 °C) for 16 hours, followed by immersion in purified water for 24 hours. Afterward, it was removed and air-dried for 35 minutes, forming a modified layer on the flexible electrode, specifically on the working electrode of the flexible electrode.

[0080] II. Fabrication of the sensing layer:

[0081] Using 10 mmol / L HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) buffer (pH=8.0) as solvent, 5 mg / mL redox polymer, 40 mg / mL glucose oxidase, 25 mg / mL polyethylene glycol diglycidyl ether (MW=400Da) (as a crosslinking agent), and 5 mg / mL magnesium aluminum silicate were prepared separately. These four prepared solutions were mixed, with a mass ratio of redox polymer, glucose oxidase, polyethylene glycol diglycidyl ether, and magnesium aluminum silicate of 39%:31%:25%:5%. After vortexing for 30 minutes, a sensing layer solution was obtained, in which the concentration of the redox polymer was 3.69 mg / mL. 0.2 μL of this solution was uniformly drop-coated onto the modified layer on the working electrode surface of the flexible electrode using a microsyringe (1 μL). After crosslinking and curing at 25°C for 24 hours, the sensing layer was obtained, which is the flexible electrode with the sensing layer.

[0082] Magnesium aluminum silicate itself is a layered inorganic material. The hydroxyl groups (-OH) of hydrophilic grafted polymers (such as polyethylene glycol) within the spacers of this layered structure are bonded together by hydrogen bonds. Under certain conditions, it spontaneously organizes or aggregates to form a relatively ordered and stable structure. This process is usually called self-assembly, which is the process of spontaneous bonding due to various chemical bond forces.

[0083] III. Preparation of the outer membrane of the restricted diffusion layer:

[0084] Using anhydrous ethanol and 10 mmol / L HEPES buffer (pH 8.0) at a volume ratio of 4:1 as solvents, solutions of sulfonic acid-modified polyvinylpyridine-styrene copolymer (PVP-S) with a concentration of 90 mg / mL and polyethylene glycol diglycidyl ether (PEG = 200 Da) with a concentration of 20 mg / mL were prepared. The prepared PPVP-S solution and PEG diglycidyl ether (PEG = 200 Da) were then mixed to obtain the outer membrane solution for the confined diffusion layer. The mass ratio of PPVP-S to PEG diglycidyl ether in the mixture was 90%:10%, with the concentration of PPVP-S at 60 mg / mL. The flexible electrode with the sensing layer is immersed in the solution of the outer membrane of the confined diffusion layer for 10 seconds each time. After immersion, it is removed and air-dried for 10 minutes to evaporate the solvent. The immersion operation is then repeated for a total of 4 times. After completion, it is cured in an oven at 25°C for 48 hours to obtain the desired sensor.

[0085] Example 2

[0086] I. Electrode Pretreatment:

[0087] The flexible electrode was treated with air plasma (flow rate 150 mL / min, power 100 W, vacuum degree 50 Pa) for 5 minutes. Then, the electrode was immersed in a 25 mg / mL polyethylene glycol solution (36 °C) for 20 hours, followed by immersion in purified water for 20 hours. Afterward, it was removed and air-dried for 30 minutes, forming a modified layer on the flexible electrode, specifically on the working electrode.

[0088] II. Fabrication of the sensing layer:

[0089] Using 20 mmol / L HEPES buffer (pH 7.5) as solvent, 5 mg / mL redox polymer, 40 mg / mL glucose oxidase, 25 mg / mL polyethylene glycol diglycidyl ether (MW = 400 Da) (as a crosslinking agent), and 5 mg / mL magnesium aluminum silicate were prepared separately. These four solutions were mixed in a mass ratio of 40%:45%:14%:1% for the redox polymer, glucose oxidase, polyethylene glycol diglycidyl ether, and magnesium aluminum silicate. After vortexing for 30 minutes, a sensing layer solution was obtained, with a redox polymer concentration of 4.05 mg / mL. 0.2 μL of this solution was uniformly drop-coated onto the modified layer on the working electrode surface of the flexible electrode using a microsyringe (1 μL). After crosslinking and curing at 25°C for 24 hours, the sensing layer was obtained, which is the flexible electrode with the sensing layer.

[0090] III. Preparation of the outer membrane of the restricted diffusion layer:

[0091] Using anhydrous ethanol and 5 mmol / L HEPES buffer (pH 8.2) at a volume ratio of 4:1 as solvents, solutions of sulfonic acid-modified polyvinylpyridine-styrene copolymer (PVP-S) with a concentration of 90 mg / mL and polyethylene glycol diglycidyl ether (PEG = 200 Da) with a concentration of 20 mg / mL were prepared. The prepared PPVP-S solution and PEG diglycidyl ether (PEG = 200 Da) solution were then mixed to obtain the outer membrane solution for the confined diffusion layer. The mass ratio of PPVP-S to PEG diglycidyl ether in the mixture was 90%:10%, with the concentration of PPVP-S at 60 mg / mL. The flexible electrode with the sensing layer is immersed in the solution of the outer membrane of the confined diffusion layer for 10 seconds each time. After immersion, it is removed and air-dried for 10 minutes to evaporate the solvent. The immersion operation is then repeated for a total of 4 times. After completion, it is cured in an oven at 25°C for 48 hours to obtain the desired sensor.

[0092] Example 3

[0093] I. Electrode Pretreatment:

[0094] The flexible electrode was treated with air plasma (flow rate 150 mL / min, power 100 W, vacuum degree 50 Pa) for 5 minutes. Then, the electrode was immersed in a 40 mg / mL polyethylene glycol solution (39 °C) for 14 hours, followed by immersion in purified water for 30 hours. Afterward, it was removed and air-dried for 40 minutes, forming a modified layer on the flexible electrode, specifically on the working electrode.

[0095] II. Fabrication of the sensing layer:

[0096] Using 5 mmol / L HEPES buffer (pH 8.2) as solvent, 5 mg / mL redox polymer, 40 mg / mL glucose oxidase, 25 mg / mL polyethylene glycol diglycidyl ether (MW = 400 Da) (as a crosslinking agent), and 5 mg / mL magnesium aluminum silicate were prepared separately. These four solutions were mixed in a mass ratio of 15%:45%:30%:10% for the redox polymer, glucose oxidase, polyethylene glycol diglycidyl ether, and magnesium aluminum silicate. After vortexing for 30 minutes, a sensing layer solution was obtained, with a redox polymer concentration of 2.05 mg / mL. 0.2 μL of this solution was uniformly drop-coated onto the modified layer on the working electrode surface of the flexible electrode using a microsyringe (1 μL). After crosslinking and curing at 25°C for 24 hours, the sensing layer was obtained, which is the flexible electrode with the sensing layer.

[0097] III. Preparation of the outer membrane of the restricted diffusion layer:

[0098] Using anhydrous ethanol and 15 mmol / L HEPES buffer (pH 7.8) at a volume ratio of 4:1 as solvents, solutions of sulfonic acid-modified polyvinylpyridine-styrene copolymer (PVP-S) with a concentration of 90 mg / mL and polyethylene glycol diglycidyl ether (PEG = 200 Da) with a concentration of 20 mg / mL were prepared. The prepared PPVP-S solution and PEG diglycidyl ether (PEG = 200 Da) were then mixed to obtain the outer membrane solution for the confined diffusion layer. The mass ratio of PPVP-S to PEG diglycidyl ether in the mixture was 90%:10%, with the concentration of PPVP-S at 60 mg / mL. The flexible electrode with the sensing layer is immersed in the solution of the outer membrane of the confined diffusion layer for 10 seconds each time. After immersion, it is removed and air-dried for 10 minutes to evaporate the solvent. The immersion operation is then repeated for a total of 4 times. After completion, it is cured in an oven at 25°C for 48 hours to obtain the desired sensor.

[0099] Example 4

[0100] I. Electrode Pretreatment:

[0101] The flexible electrode was treated with air plasma (flow rate 150 mL / min, power 100 W, vacuum degree 50 Pa) for 5 minutes. Then, the electrode was immersed in a 35 mg / mL polyethylene glycol solution (40 °C) for 16 hours, followed by immersion in purified water for 26 hours. Afterward, it was removed and air-dried for 30 minutes, forming a modified layer on the flexible electrode, specifically on the working electrode.

[0102] II. Fabrication of the sensing layer:

[0103] Using 15 mmol / L HEPES buffer (pH 7.7) as solvent, 5 mg / mL redox polymer, 40 mg / mL glucose oxidase, 25 mg / mL polyethylene glycol diglycidyl ether (MW = 400 Da) (as a crosslinking agent), and 5 mg / mL magnesium aluminum silicate were prepared separately. These four solutions were mixed in a mass ratio of 40%:20%:30%:10% for the redox polymer, glucose oxidase, polyethylene glycol diglycidyl ether, and magnesium aluminum silicate. After vortexing for 30 minutes, a sensing layer solution was obtained, with a redox polymer concentration of 3.42 mg / mL. 0.2 μL of this solution was uniformly drop-coated onto the modified layer on the working electrode surface of the flexible electrode using a microsyringe (1 μL). After crosslinking and curing at 25°C for 24 hours, the sensing layer was obtained, which is the flexible electrode with the sensing layer.

[0104] III. Preparation of the outer membrane of the restricted diffusion layer:

[0105] Using anhydrous ethanol and 20 mmol / L HEPES buffer (pH 7.5) at a volume ratio of 4:1 as solvents, solutions of sulfonic acid-modified polyvinylpyridine-styrene copolymer (PVP-S) with a concentration of 90 mg / mL and polyethylene glycol diglycidyl ether (PEG = 200 Da) with a concentration of 20 mg / mL were prepared. The prepared PPVP-S solution and PEG diglycidyl ether (PEG = 200 Da) solution were then mixed to obtain the outer membrane solution for the confined diffusion layer. The mass ratio of PPVP-S to PEG diglycidyl ether in the mixture was 90%:10%, with the concentration of PPVP-S at 60 mg / mL. The flexible electrode with the sensing layer is immersed in the solution of the outer membrane of the confined diffusion layer for 10 seconds each time. After immersion, it is removed and air-dried for 10 minutes to evaporate the solvent. The immersion operation is then repeated for a total of 4 times. After completion, it is cured in an oven at 25°C for 48 hours to obtain the desired sensor.

[0106] Example 5

[0107] I. Electrode Pretreatment:

[0108] The flexible electrode was treated with air plasma (flow rate 150 mL / min, power 100 W, vacuum degree 50 Pa) for 5 minutes. Then, the electrode was immersed in a 32 mg / mL polyethylene glycol solution (35 °C) for 17 hours, followed by immersion in purified water for 26 hours. Afterward, it was removed and air-dried for 40 minutes, forming a modified layer on the flexible electrode, specifically on the working electrode.

[0109] II. Fabrication of the sensing layer:

[0110] Using 20 mmol / L HEPES buffer (pH 8.2) as solvent, 5 mg / mL redox polymer, 40 mg / mL glucose oxidase, 25 mg / mL polyethylene glycol diglycidyl ether (MW = 400 Da) (as a crosslinking agent), and 5 mg / mL magnesium aluminum silicate were prepared separately. These four solutions were mixed in a mass ratio of 40%:45%:5%:10% for the redox polymer, glucose oxidase, polyethylene glycol diglycidyl ether, and magnesium aluminum silicate. After vortexing for 30 minutes, a sensing layer solution was obtained, with a redox polymer concentration of 3.53 mg / mL. 0.2 μL of this solution was uniformly drop-coated onto the modified layer on the working electrode surface of the flexible electrode using a microsyringe (1 μL). After crosslinking and curing at 25°C for 24 hours, the sensing layer was obtained, which is the flexible electrode with the sensing layer.

[0111] III. Preparation of the outer membrane of the restricted diffusion layer:

[0112] Using anhydrous ethanol and 20 mmol / L HEPES buffer (pH 8.2) at a volume ratio of 4:1 as solvents, solutions of sulfonic acid-modified polyvinylpyridine-styrene copolymer (PVP-S) with a concentration of 90 mg / mL and polyethylene glycol diglycidyl ether (PEG = 200 Da) with a concentration of 20 mg / mL were prepared. The prepared PPVP-S solution and PEG diglycidyl ether (PEG = 200 Da) solution were then mixed to obtain the outer membrane solution for the confined diffusion layer. The mass ratio of PPVP-S to PEG diglycidyl ether in the mixture was 90%:10%, with the concentration of PPVP-S at 60 mg / mL. The flexible electrode with the sensing layer is immersed in the solution of the outer membrane of the confined diffusion layer for 10 seconds each time. After immersion, it is removed and air-dried for 10 minutes to evaporate the solvent. The immersion operation is then repeated for a total of 4 times. After completion, it is cured in an oven at 25°C for 48 hours to obtain the desired sensor.

[0113] Comparative Example 1

[0114] I. Electrode Pretreatment:

[0115] The flexible electrode was treated with air plasma (flow rate 150 mL / min, power 100 W, vacuum degree 50 Pa) for 5 minutes. Then, the electrode was immersed in a 30 mg / mL polyethylene glycol aqueous solution (37 °C) for 16 hours, followed by immersion in purified water for 24 hours. Afterward, it was removed and air-dried for 35 minutes, forming a modified layer on the flexible electrode, specifically on the working electrode of the flexible electrode.

[0116] II. Preparation of a sensing layer without magnesium aluminum silicate:

[0117] Using 8 mmol / L HEPES buffer (pH 7.5) as solvent, a solution of 5 mg / mL redox polymer, 40 mg / mL glucose oxidase, and 25 mg / mL polyethylene glycol diglycidyl ether (MW = 400 Da) was prepared. The prepared solution was mixed, with a mass ratio of redox polymer, glucose oxidase, polyethylene glycol diglycidyl ether, and magnesium aluminum silicate of 39%:31%:30%. After vortexing for 30 minutes, a sensing layer solution was obtained, in which the concentration of the redox polymer was 3.99 mg / mL. 0.2 μL of this solution was uniformly drop-coated onto the modified layer on the working electrode surface of the flexible electrode using a microsyringe (1 μL). After cross-linking and curing at 25°C for 24 hours, the sensing layer was obtained, which is the flexible electrode with the sensing layer.

[0118] III. Preparation of the outer membrane of the restricted diffusion layer:

[0119] Using anhydrous ethanol and 12 mmol / L HEPES buffer (pH 7.8) at a volume ratio of 4:1 as solvents, solutions of sulfonic acid-modified polyvinylpyridine-styrene copolymer (PVP-S) with a concentration of 90 mg / mL and polyethylene glycol diglycidyl ether (PEG = 200 Da) with a concentration of 20 mg / mL were prepared. The prepared PPVP-S solution and PEG diglycidyl ether (PEG = 200 Da) were then mixed to obtain the outer membrane solution for the confined diffusion layer. The mass ratio of PPVP-S to PEG diglycidyl ether in the mixture was 90%:10%, with the concentration of PPVP-S at 60 mg / mL. The flexible electrode with the sensing layer is immersed in the solution of the outer membrane of the confined diffusion layer for 10 seconds each time. After immersion, it is removed and air-dried for 10 minutes to evaporate the solvent. The immersion operation is then repeated for a total of 4 times. After completion, it is cured in an oven at 25°C for 48 hours to obtain the desired sensor.

[0120] Comparative Example 2

[0121] I. Preparation of a sensing layer without hydrophilic polymer grafting:

[0122] Using 10 mmol / L HEPES buffer (pH 8.0) as solvent, a mixture of 5 mg / mL redox polymer, 40 mg / mL glucose oxidase, 25 mg / mL polyethylene glycol diglycidyl ether (MW = 400 Da), and 5 mg / mL magnesium aluminum silicate was prepared. The four prepared solutions were mixed, with a mass ratio of redox polymer, glucose oxidase, polyethylene glycol diglycidyl ether, and magnesium aluminum silicate of 40%:45%:5%:10%. After vortexing for 30 minutes, a sensing layer solution was obtained, in which the concentration of the redox polymer was 3.53 mg / mL. 0.2 μL of this solution was uniformly drop-coated onto the working electrode surface of the flexible electrode using a microsyringe (1 μL). After cross-linking and curing at 25°C for 24 hours, the sensing layer was obtained, which is the flexible electrode with the sensing layer.

[0123] II. Preparation of the outer membrane of the restricted diffusion layer:

[0124] Using anhydrous ethanol and 10 mmol / L HEPES buffer (pH 8.0) at a volume ratio of 4:1 as solvents, solutions of sulfonic acid-modified polyvinylpyridine-styrene copolymer (PVP-S) with a concentration of 90 mg / mL and polyethylene glycol diglycidyl ether (PEG = 200 Da) with a concentration of 20 mg / mL were prepared. The prepared PPVP-S solution and PEG diglycidyl ether (PEG = 200 Da) were then mixed to obtain the outer membrane solution for the confined diffusion layer. The mass ratio of PPVP-S to PEG diglycidyl ether in the mixture was 90%:10%, with the concentration of PPVP-S at 60 mg / mL. The flexible electrode with the sensing layer is immersed in the solution of the outer membrane of the confined diffusion layer for 10 seconds each time. After immersion, it is removed and air-dried for 10 minutes to evaporate the solvent. The immersion operation is then repeated for a total of 4 times. After completion, it is cured in an oven at 25°C for 48 hours to obtain the desired sensor.

[0125] Comparative Example 3

[0126] I. Preparation of a sensing layer composed of magnesium aluminum silicate and polyethylene glycol:

[0127] A mixed solution of magnesium aluminum silicate and polyethylene glycol was prepared using 5 mmol / L HEPES buffer (pH 8.2) as the solvent. The concentration of magnesium aluminum silicate in the mixed solution was 5 mg / mL, and the concentration of polyethylene glycol was 30 mg / mL. Separately, a mixture of 5 mg / mL redox polymer, 40 mg / mL glucose oxidase, 25 mg / mL polyethylene glycol diglycidyl ether (MW = 400 Da), and the magnesium aluminum silicate-polyethylene glycol mixture was prepared. The mass ratio of redox polymer, glucose oxidase, polyethylene glycol diglycidyl ether, and magnesium aluminum silicate-polyethylene glycol in the mixed solution was 42%:45%:5%:8%. After vortexing for 30 minutes, the sensor layer solution was obtained, in which the concentration of redox polymer was 4.22 mg / mL. 0.2 μL was transferred using a micro-syringe (1 μL) and uniformly drop-coated onto the working electrode surface of the flexible electrode. After cross-linking and curing in an oven at 25 °C for 24 hours, the sensing layer was obtained, which is the flexible electrode with the sensing layer.

[0128] II. Preparation of the outer membrane of the restricted diffusion layer:

[0129] Using anhydrous ethanol and 5 mmol / L HEPES buffer (pH 8.2) at a volume ratio of 4:1 as solvents, solutions of sulfonic acid-modified polyvinylpyridine-styrene copolymer (PVP-S) with a concentration of 90 mg / mL and polyethylene glycol diglycidyl ether (PEG = 200 Da) with a concentration of 20 mg / mL were prepared. The prepared PPVP-S solution and PEG diglycidyl ether (PEG = 200 Da) solution were then mixed to obtain the outer membrane solution for the confined diffusion layer. The mass ratio of PPVP-S to PEG diglycidyl ether in the mixture was 90%:10%, with the concentration of PPVP-S at 60 mg / mL. The flexible electrode with the sensing layer is immersed in the solution of the outer membrane of the confined diffusion layer for 10 seconds each time. After immersion, it is removed and air-dried for 10 minutes to evaporate the solvent. The immersion operation is then repeated for a total of 4 times. After completion, it is cured in an oven at 25°C for 48 hours to obtain the desired sensor.

[0130] Comparison of Examples 1-5 and Comparative Examples 1-3

[0131] Except for the different mass ratios of the raw materials in the sensing layer (as shown in Table 1), the preparation methods in Examples 1-5 are completely the same. The mass ratios of Examples 1-5 and Comparative Examples 1-2 in Table 1 represent the mass ratio of redox polymer: glucose oxidase: polyethylene glycol diglycidyl ether: magnesium aluminum silicate in the sensing layer; the mass ratio of Comparative Example 3 represents the mass ratio of redox polymer: glucose oxidase: polyethylene glycol diglycidyl ether: magnesium aluminum silicate - polyethylene glycol in the sensing layer.

[0132] The difference between Comparative Example 1 and Example 1 is that magnesium aluminum silicate is not added when preparing the sensing layer; the other steps are exactly the same. The difference between Comparative Example 2 and Example 5 is that step "I. Electrode Pretreatment" of Example 5 is omitted; the sensing layer is directly prepared on the electrode; the other steps are exactly the same. The difference between Comparative Example 3 and Example 5 is that the hydrophilic organic polymer (polyethylene glycol) is not grafted separately, but rather polyethylene glycol is mixed with magnesium aluminum silicate during the preparation of the sensing layer, and the mass ratio of the substances is also different.

[0133] Table 1

[0134] Example The mass ratio of each substance in the sensing layer Example 1 39:31:25:5 Example 2 40:45:14:1 Example 3 15:45:30:10 Example 4 40:20:30:10 Example 5 40:45:5:10 Comparative Example 1 39:31:30:0 Comparative Example 2 40:45:5:10 Comparative Example 3 42:45:5:8

[0135] Example 6: Scanning electron microscopy observation and wetting angle test

[0136] The objects to be tested are as follows:

[0137] In this embodiment, the modified sensing layer solution refers to the sensing layer solution in Example 1. During testing, 2-3 drops of the sensing layer solution are applied to the electrode that has undergone electrode pretreatment in step one of Example 1, and the wetting angle of the droplets is measured using an SDC-BOS wetting angle tester. The sensing layer solution of magnesium aluminum silicate and polyethylene glycol in Table 3 is the sensing layer solution of Comparative Example 3; the non-hydrophilic polymer sensing layer solution in Table 4 is the sensing layer solution of Comparative Example 2; and the magnesium aluminum silicate-free sensing layer solution in Table 5 is the sensing layer solution of Comparative Example 1.

[0138] Scanning electron microscopy (SEM) observations of magnesium aluminum silicate solution, a mixed solution of magnesium aluminum silicate and polyethylene glycol, and the modified sensing layer solution revealed that the hydrophilic organic polymer polyethylene glycol of this invention grafts onto the surface of the working electrode and undergoes self-assembly with magnesium aluminum silicate. It can be seen that the magnesium aluminum silicate exhibits a layered structure. Figure 1 (ab), while magnesium aluminum silicate mixed with polyethylene glycol produces larger sheet-like blocks. Figure 1 c) The structure is more compact, but its gelation and rheological properties are poor. Figure 1 d indicates that polyethylene glycol is adsorbed between the self-assembled modified magnesium aluminum silicate sheet structure, and there is a hydrogen bond between the two. The self-assembly of the two components makes the surface of magnesium aluminum silicate smoother, thus exhibiting excellent gelation properties and rheological properties. It can effectively reduce the aggregation phenomenon between enzymes and redox polymers and reduce the loss of enzyme activity.

[0139] The excellent rheological properties allow the sensing layer solution to have a smaller wetting angle when dropped onto the electrode surface. The wetting angle of the solution dropped onto the electrode surface was measured using an SDC-BOS wetting angle meter. The results are as follows: Figure 2 As shown, the wetting angle formed by the modified sensing layer solution ( Figure 2 a) The wetting angle of the sensing layer solution, which is significantly smaller than that of the magnesium aluminum silicate and polyethylene glycol mixture, is ( Figure 2b) The wetting angle of the sensing layer solution without polyethylene glycol ( Figure 2 c) and the wetting angle of the sensing layer solution without magnesium aluminum silicate ( Figure 2 d). The results in Table 2 show that the wetting angle of the modified solution is highly consistent after multiple tests. However, comparing Tables 3, 4 and 5, it can be seen that the wetting angle of the sensor layer solution formed by self-assembly modification is better than that of the other three sensor layer solutions, which can produce a better film formation effect and thus improve the consistency and stability of the sensor.

[0140] Table 2 Wetting angle of the modified sensor layer solution

[0141] Number of experiments Wetting angle (mean) Immersion angle (left) Immersion angle (right) 1 45.747 45.434 46.06 2 45.4165 45.598 45.235 3 45.574 45.789 45.359 4 45.5225 45.821 45.224 5 45.473 45.198 45.748 6 45.3715 45.532 45.211 7 45.664 45.875 45.453 8 45.6165 45.321 45.912 9 45.5025 45.582 45.423 10 46.2545 46.638 45.871

[0142] Table 3. Wetting angles of the sensing layer solution composed of magnesium aluminum silicate and polyethylene glycol.

[0143] Number of experiments Wetting angle (mean) Immersion angle (left) Immersion angle (right) 1 54.169 54.236 54.102 2 56.7625 56.954 56.571 3 55.21 54.099 56.321 4 54.847 55.457 54.237 5 55.745 54.644 56.846 6 57.938 57.187 58.689 7 56.0715 55.798 56.345 8 54.343 54.741 53.945 9 57.994 57.663 58.325 10 57.3455 57.142 57.549

[0144] Table 4 Wetting angle of hydrophilic polymer sensing layer solution

[0145] Number of experiments Wetting angle (mean) Immersion angle (left) Immersion angle (right) 1 102.976 103.254 102.698 2 99.2605 98.045 100.476 3 104.2665 103.174 105.359 4 101.2605 102.157 100.364 5 100.3375 99.654 101.021 6 100.892 100.635 101.149 7 102.067 103.089 101.045 8 104.558 103.671 105.445 9 98.3355 99.112 97.559 10 103.3715 103.044 103.699

[0146] Table 5 Wetting angle of magnesium aluminum silicate-free sensing layer solution

[0147]

[0148]

[0149] The solutions in Tables 2-5 were drop-coated onto the working electrode surface of the electrode, and after cross-linking and curing (25°C) for 24 hours, an electrode with a sensing layer was obtained. The electrode was then observed under a scanning electron microscope: the modified sensing layer of this invention (…). Figure 3 a) The film-forming effect is better, the surface of the sensing layer is smooth and porous, and there are few cracks in the film layer; while the sensing layer of magnesium aluminum silicate and polyethylene glycol mixture ( Figure 3 b) Numerous surface cracks; and Figure 3 c and Figure 3 d proves that the sensing layer lacking magnesium aluminum silicate or polyethylene glycol exhibits severe agglomeration.

[0150] Example 7: Sensor Performance Testing

[0151] In this embodiment, the sensor containing modified magnesium aluminum silicate in the sensing layer refers to the sensor prepared in the above embodiments, the sensor without magnesium aluminum silicate in the sensing layer refers to the sensor prepared in Comparative Example 1, the sensor without polyethylene glycol in the sensing layer refers to the sensor prepared in Comparative Example 2, and the sensor with a mixture of magnesium aluminum silicate and polyethylene glycol refers to the sensor prepared in Comparative Example 3.

[0152] I. Sensitivity Consistency Detection of Sensing Layer

[0153] The sensors prepared in Example 1 and the comparative example were used for testing. A 5mm portion of the sensor covering the diffusion-limiting layer was immersed in a transparent sample vial containing 10mM glucose solution, and the solution was kept at a constant temperature of 37°C. The sensor was connected to a Chenhua 1000c electrochemical workstation, and the sensor sensitivity was tested using the chronoamperometry (it) method. The curve of the current value changing over time was measured. (See attached image.) Figure 4 c represents the sensitivity variation spectrum of sensors without magnesium aluminum silicate in the sensing layer. The sensor sensitivity range in 10 mM sugar solution is 0.78–1.85 nA / mM, with a coefficient of variation (CV) of 19.27%. Figure 4 b represents the sensor with a coefficient of variation (CV) of 14.08% for a mixture of magnesium aluminum silicate and polyethylene glycol. (Comparison) Figure 4 a) The sensor with modified magnesium aluminum silicate in the sensing layer exhibits a more concentrated sensitivity distribution, with a coefficient of variation (CV) of 9.81% within the range of 1.01-1.48 nA / mM. This demonstrates that modified magnesium aluminum silicate can effectively reduce aggregation between enzymes and redox polymers, contributing to a uniformly dispersed sensing layer and thus improving the consistency of biosensor sensitivity.

[0154] II. Sensor Layer Stability Testing

[0155] The sensors prepared in Example 2 and the comparative example were used for testing. A 5mm portion of the sensor covering the outer membrane of the diffusion-limiting layer was immersed in a transparent sample vial containing a 10mM glucose solution, and the solution was kept at a constant temperature of 37°C. The sensor was connected to a Chenhua 1000c electrochemical workstation, and the sensor stability was tested using the chronoamperometry (it) method, measuring the current value change over 15 days. Figure 5 The glucose sensor with modified magnesium aluminum silicate in the sensing layer was tested for stability in vitro for 15 days. Based on day one, the current decay was ≤13% within 15 days. The sensor with a mixture of magnesium aluminum silicate and polyethylene glycol showed a current decay of 23%. Furthermore, the glucose sensor without magnesium aluminum silicate or polyethylene glycol in the sensing layer showed current decays of 34% and 32% respectively after 15 days in in vitro stability tests. Magnesium aluminum silicate, with its unique crystal structure—silicon-oxygen tetrahedron—possesses excellent adsorption properties while exposing more enzyme active sites. The modified magnesium aluminum silicate also allows the sensing layer solution to have a consistent wetting angle when drop-coated onto the electrode surface, resulting in better film formation and thus improving the sensor's stability.

[0156] III. Sensor Linear Spectrum

[0157] The sensors prepared in Example 3 and the comparative example were used for testing. The portion of the sensor covering the diffusion-limiting layer (5 mm) was immersed in a transparent sample vial containing PBS buffer, and the solution was kept at a constant temperature of 37°C. The sensor was connected to a Chenhua 1000c electrochemical workstation, and chronoamperometry (it) was used to test the sensor's linearity. After each testing period, the solution was replaced with a glucose solution (0-25 mM) with an increasing concentration gradient. Figure 6 Linear spectrum of a sensor with a sensing layer containing modified magnesium aluminum silicate. Figure 7 Linear spectrum of a sensor containing a mixture of magnesium aluminum silicate and polyethylene glycol. Figure 8 and 9 The figures show the linear spectra of sensors with and without magnesium aluminum silicate and polyethylene glycol, respectively. As shown in the figure, the current value of the modified magnesium aluminum silicate sensor changes significantly with the solution concentration gradient in glucose solutions with concentrations of 0-25 mM, exhibiting excellent linearity with a linear fitting coefficient R0. 2 The linear fitting coefficient R of the sensor composed of magnesium aluminum silicate and polyethylene glycol reached 99.7%. 2 The linearity was 98.4%, while sensors without magnesium aluminum silicate or polyethylene glycol in the sensing layer showed poor linearity in glucose solutions with concentrations of 0-25 mM, with no significant change in current gradient and a low linear fitting coefficient R0. 2 Only 96.5% and 96.3%.

[0158] IV. Human Trial Map of Sensors in Implantable Continuous Glucose Monitoring Systems

[0159] The detection targets were the sensors prepared in Example 4 and the comparative example: biosensors covered with a restricted diffusion layer outer membrane were applied to implantable continuous glucose monitoring systems, such as... Figure 10 As shown, during a 14-day human trial, the sensor's sensitivity remained highly stable, and the monitored dynamic glucose concentration (curve) showed no decay, closely matching the results of fingertip blood glucose testing (dots). Furthermore, the human trial results were compared with those of a foreign brand of continuous glucose monitoring device; the blood glucose values ​​from the latter were very close to the dynamic glucose concentration (curve), indicating consistency between the two monitoring methods. Figure 11 The sensor made from a mixture of magnesium aluminum silicate and polyethylene glycol showed that only some data matched fingertip blood glucose levels in vivo, and it also lagged behind competing products. In contrast... Figure 12 and 13 Biosensors that do not contain magnesium aluminum silicate or polyethylene glycol showed significant differences in blood glucose levels detected in human trials compared to finger-prick blood glucose and data from a certain foreign brand of continuous glucose meter, and the monitoring time could only be maintained for 7-10 days.

[0160] Example 8: The structure of the electrochemical sensor of the present invention

[0161] Conventional flexible electrode structures, such as Figure 14 As shown in figure a, the electrode is fabricated using a stacked electrode method.

[0162] The biosensor for electrochemical detection of the present invention was prepared according to the methods of Examples 1-5. It is obtained by sequentially loading a modified layer, a sensing layer, and a diffusion-confining layer onto a conventional flexible electrode. The structure of the biosensor of the present invention is as follows: Figure 14 As shown in b, it includes: a flexible electrode, which includes a substrate, a working electrode, a counter electrode, and a reference electrode. The working electrode and the counter electrode are respectively disposed on both sides of the substrate. A dielectric layer is disposed on the side of the counter electrode away from the substrate. A reference electrode is disposed on the side of the working electrode away from the substrate. A dielectric layer is disposed between the working electrode and the reference electrode. A dielectric layer is disposed on the side of the reference electrode away from the working electrode. Furthermore, the surface of the working electrode has an exposed portion without a dielectric layer and a reference electrode. A modified layer and a sensing layer are sequentially loaded on the surface of the exposed portion from the inside to the outside. A diffusion-limiting layer is also disposed on the surface of the flexible electrode. The diffusion-limiting layer covers the sensing layer and the exposed portions of the dielectric layer, the reference electrode, and the counter electrode.

[0163] This invention does not impose particular limitations on the materials of the working electrode, counter electrode, and reference electrode; they can be working electrodes, counter electrodes, and reference electrodes commonly used in the field of electrochemical sensor technology. Specifically, the conductive materials of the working electrode, counter electrode, and reference electrode can be one or a combination of two or more of graphite, graphene, carbon nanotubes, carbon nanotubes, and silver / silver chloride.

Claims

1. A sensing layer containing self-assembled modified magnesium aluminum silicate for fabricating an electrochemical sensor, the electrochemical sensor comprising a flexible electrode, characterized in that, The sensing layer is formed by drop-coating a sensing layer solution that can self-assemble and modify the modified layer onto a modified layer obtained by grafting a hydrophilic organic polymer onto the working electrode of a flexible electrode. The sensing layer solution comprises a redox polymer, a tool enzyme, a crosslinking agent A, and magnesium aluminum silicate, wherein the mass ratio of redox polymer: tool enzyme: crosslinking agent A: magnesium aluminum silicate is 15~40: 20~45: 5~30: 1~10; The hydrophilic organic polymer is selected from one or more of polyallylamine, polyethylene glycol, polyethyleneimine, polyamide-amine dendritic polymer, and poly(L-lysine); The redox polymer is a redox polymer containing transition metals, including ruthenium, rhodium, and osmium; The tool enzymes are selected from glucose dehydrogenase, glucose oxidase, uricase, lactate oxidase, and cholesterol oxidase. The crosslinking agent A is selected from one or more of 1,4-butanediol diglycidyl ether, poly(dimethylsiloxane)-diglycidyl ether, glutaraldehyde, polyethylene glycol diglycidyl ether, tetraglycidyl-4,4-diaminodiphenylmethane, or glycerol triglycidyl ether.

2. The sensing layer according to claim 1, characterized in that, The crosslinking agent A is polyethylene glycol diglycidyl ether with a MW of 200 Da - 5000 Da.

3. The sensing layer according to claim 2, characterized in that, The crosslinking agent A is polyethylene glycol diglycidyl ether with an MW of 400 Da.

4. The sensing layer according to claim 1, characterized in that, The mass ratio of the redox polymer: tool enzyme: crosslinking agent A: magnesium aluminum silicate is 35~40: 20~35: 20~30: 4~10.

5. The sensing layer according to claim 4, characterized in that, The mass ratio of the redox polymer, tool enzyme, crosslinking agent A, and magnesium aluminum silicate is 35-40: 25-35: 25-30: 5-10.

6. The sensing layer according to claim 5, characterized in that, The mass ratio of the redox polymer, tool enzyme, crosslinking agent A, and magnesium aluminum silicate is 39:31:25:

5.

7. The sensing layer according to claim 1, characterized in that, The concentration of the redox polymer in the sensing layer solution is 2–5 mg / ml.

8. The sensing layer according to claim 7, characterized in that: The concentration of the redox polymer in the sensing layer solution is 2–4.5 mg / ml.

9. The sensing layer according to claim 8, characterized in that: The concentration of the redox polymer in the sensing layer solution is 3.0–4.5 mg / ml.

10. A method for preparing a sensing layer, wherein the sensing layer is the sensing layer containing self-assembled modified magnesium aluminum silicate as described in any one of claims 1 to 9, characterized in that, The preparation method includes the following steps: S1. A modified layer is obtained by grafting a hydrophilic organic polymer onto the working electrode of a flexible electrode using plasma initiation. S2. The sensing layer solution is drop-coated onto the modified layer, and the sensing layer is obtained after cross-linking and curing.

11. The preparation method according to claim 10, characterized in that, The modified layer obtained by grafting a hydrophilic organic polymer onto the working electrode of the flexible electrode using plasma initiation includes: After treating the flexible electrode with air plasma, the flexible electrode is immersed in an aqueous solution of a hydrophilic organic polymer with a concentration of 25-40 mg / mL and a temperature of 35-40 °C for 14-20 hours. After immersing the electrode in purified water for 20-30 hours, it is taken out and air-dried for 30-40 minutes to form the modified layer on the working electrode of the flexible electrode.

12. The preparation method according to claim 10, characterized in that, The step of drop-coating the sensing layer solution onto the modified layer and then cross-linking and curing it to obtain the sensing layer includes the following steps: S2.1 Preparation of the sensing layer solution: A mixed solution of redox polymer, tool enzyme, crosslinking agent A, and magnesium aluminum silicate is prepared using solvent A and thoroughly mixed to obtain the sensing layer solution; the solvent A is HEPES buffer or TES buffer. S2.2 Preparation of the sensing layer: The prepared sensing layer solution is drop-coated onto the surface of the modified layer obtained in step S1, and the sensing layer is obtained after cross-linking and curing at a temperature of 20~30 ℃ for 20~28 hours.

13. The preparation method according to claim 12, characterized in that, The concentration of the HEPES buffer is 5–20 mmol / L, and the pH is 7.5–8.

2.

14. An electrochemical sensor, characterized in that, include: The flexible electrode includes a substrate, a working electrode, a counter electrode, and a reference electrode, wherein... The working electrode and the counter electrode are respectively disposed on both sides of the substrate. A dielectric layer is disposed on the side of the counter electrode away from the substrate. The reference electrode is disposed on the side of the working electrode away from the substrate. The dielectric layer is disposed between the working electrode and the reference electrode. The dielectric layer is disposed on the side of the reference electrode away from the working electrode. The surface of the working electrode has an exposed portion without a dielectric layer and a reference electrode, and a modified layer and a sensing layer are sequentially coated on the surface of the exposed portion. It also includes a diffusion-restricting layer that encloses the sensing layer, the dielectric layer, the reference electrode, and the exposed portions of the counter electrode; The sensing layer is the sensing layer described in any one of claims 1 to 9, or the sensing layer prepared by the preparation method according to any one of claims 10 to 13.

15. The electrochemical sensor according to claim 14, characterized in that, The confined diffusion layer is derived from a polymer containing nitrogen-containing heterocyclic groups and crosslinking agent B; The polymer containing nitrogen-containing heterocyclic groups is selected from one or more of polyvinylpyridine, polyvinylpyrrole, polyvinylpyridine-styrene copolymer, and sulfonic acid-modified polyvinylpyridine-styrene copolymer; The crosslinking agent B is selected from one or more of the following: triglycidyl-p-aminophenol, polyethylene glycol diglycidyl ether, tetraglycidyl-4,4'-diaminodiphenylmethane, tris(4-hydroxyphenyl)methane triglycidyl ether, and glycerol triglycidyl ether. The mass ratio of the polymer containing nitrogen heterocyclic groups to crosslinking agent B is 7~9.9 : 0.1~3.

16. The electrochemical sensor according to claim 15, characterized in that, The crosslinking agent B is polyethylene glycol diglycidyl ether with a MW of 200 Da - 5000 Da.

17. The electrochemical sensor according to claim 16, characterized in that, The crosslinking agent B is polyethylene glycol diglycidyl ether with a MW of 200 Da.

18. A method for preparing an electrochemical sensor according to any one of claims 14 to 17, characterized in that, The preparation method according to any one of claims 10 to 13 further includes the following steps: S3. The flexible electrode with sensing layer obtained in step S2 is immersed in the outer membrane solution of the confined diffusion layer, and then cured at a temperature of 20~35 ℃ to form the confined diffusion layer, thus obtaining the electrochemical sensor.

19. The preparation method according to claim 18, characterized in that, Step S3 includes the following steps: S3.1 Preparation of the restricted diffusion layer outer membrane solution: Using solvent B as the solvent, prepare a mixture of a polymer containing nitrogen heterocyclic groups and crosslinking agent B to obtain the restricted diffusion layer outer membrane solution; the solvent B is TES buffer, or a mixture of anhydrous ethanol and HEPES buffer; the concentration of the polymer containing nitrogen heterocyclic groups in the restricted diffusion layer outer membrane solution is 40~80 mg / ml; S3.2 Preparation of confined diffusion layer: The flexible electrode with sensing layer obtained in step S2 is immersed in the outer film solution of confined diffusion layer multiple times. After completion, it is cured at 20~35 ℃ for 40~55 hours to form confined diffusion layer and obtain the electrochemical sensor.

20. The preparation method according to claim 19, characterized in that, Solvent B is obtained by mixing anhydrous ethanol and HEPES buffer at a volume ratio of 2 to 10:1, wherein the concentration of the HEPES buffer is 5 to 20 mmol / L and the pH is 7.5 to 8.

2.

21. The preparation method according to claim 19, characterized in that, The concentration of the polymer containing nitrogen-containing heterocyclic groups in the outer membrane solution of the restricted diffusion layer is 50–70 mg / ml.

22. The preparation method according to claim 19, characterized in that, In step S3.2, the flexible electrode with the sensing layer obtained in step S2 is immersed in the outer membrane solution of the confined diffusion layer 2 to 5 times, with an interval of 6 to 20 minutes each time.

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