Biocompatible membrane, preparation method thereof and analyte sensor

Abstract

The invention provides a biocompatible membrane which is prepared from a membrane layer working solution through a membrane forming process, the membrane layer working solution comprises amphiphilic polyurethane, and the amphiphilic polyurethane is prepared by any one of the following methods: hydrophilic molecules and hydrophobic molecules form an amphiphilic polymer through chemical bonding, the amphiphilic polymer and an isocyanate substance are subjected to an addition polymerization reaction to prepare the polyurethane resin. Or one of hydrophilic molecules or hydrophobic molecules and isocyanate substances are subjected to an addition polymerization reaction to prepare polyurethane, and then the polyurethane and the other molecule are subjected to chemical bonding to form the amphiphilic polyurethane. The invention also provides a preparation method of the biocompatible membrane and an analyte sensor using the biocompatible membrane. The biocompatible membrane provided by the invention can simultaneously control the permeation ratio of two analytes, when the biocompatible membrane is used as an outer membrane of an analyte sensor, only one layer of biocompatible membrane is needed to meet the monitoring requirement, and the detection accuracy can be ensured.

Description

Technical Field

[0001] This invention relates to the field of medical diagnostic technology, and in particular to a biocompatible membrane, its preparation method, and an analyte sensor. Background Technology

[0002] Diabetes mellitus is a common endocrine disorder with complex etiologies and pathogenesis. Its main characteristic is the ineffective utilization and storage of glucose in the blood, resulting in persistently high blood sugar levels. Prolonged hyperglycemia can lead to damage to multiple organs, including the heart, kidneys, and eyes, and may even cause diabetic ketoacidosis (DK) or diabetic ketoacidosis (DKA) due to stress or improper treatment. Simultaneous monitoring of glucose and β-blockers in tissue fluid is crucial. The concentration of hydroxybutyric acid indirectly reflects the levels of glucose and β-hydroxybutyric acid. Changes in hydroxybutyrate (HbA1c) concentration can effectively indicate the risk of diabetic ketoacidosis or diabetic ketoacidosis, enabling timely and accurate treatment.

[0003] Furthermore, with rising living standards, people's dietary habits are changing. Among these changes, the ketogenic diet, by restricting carbohydrate intake, induces a ketogenic state, converting fat into ketones for energy. In the later stages, the body preferentially utilizes fat for energy, promoting fat oxidation metabolism. This dietary pattern can be used to treat diseases such as epilepsy and Alzheimer's disease. Monitoring long-term ketone body trends during the process can provide doctors or nutritionists with a basis for adjusting treatment plans. This can be achieved by simultaneously monitoring glucose and beta-ketone levels in tissue fluid. The concentration of hydroxybutyrate (HBB) can help ketogenic individuals adjust the proportion of macronutrients (such as fat intake and carbohydrate threshold) based on data, optimize the ketogenic effect, and prevent hypoglycemia caused by insufficient gluconeogenesis in the early stages of a low-carb diet by monitoring blood glucose. This method of monitoring metabolic conversion makes it easier to track the efficiency of the body's transition from glucose to fat for energy, and guides the gradual achievement of "metabolic flexibility".

[0004] Implantable monitoring systems can provide continuous, comprehensive, and reliable 24 / 7 monitoring information. For example, an implantable glucose monitoring system can reveal trends in blood glucose fluctuations and detect hyperglycemia and hypoglycemia that are difficult to detect using traditional methods. The detection principle of an implantable monitoring system is as follows: the sensor comes into contact with the analyte in the tissue fluid. Under the direct electrochemical action of the enzyme corresponding to the analyte embedded in the sensor's enzyme layer, a corresponding current is generated. The magnitude of the current is linearly correlated with the concentration of the analyte in the interstitial fluid. This current information is processed and converted into a signal, which is then sent to a smart device. Finally, the concentration information of the analyte is displayed in the smart device's application software. However, some interfering substances in the subcutaneous tissue fluid can affect the sensor's detection, including large proteins and some electroactive small molecules. The presence of interfering substances can lead to inaccurate sensor readings, affecting the doctor's or patient's interpretation of the data and the implementation of treatment plans. Therefore, an outer membrane layer needs to be added to the sensor to control the penetration of analytes and interfering substances to ensure detection accuracy.

[0005] Implantable monitoring systems can monitor two analytes by placing enzymes targeting different analytes on two working electrodes. For example, a glucose oxidoreductase sensing layer as the first working electrode and a β-hydroxybutyrate sensing layer as the second working electrode can simultaneously detect glucose and β-hydroxybutyrate in tissue fluid, resulting in higher efficiency and better patient compliance. For dual-analyte sensors, the design of the outer membrane layer must simultaneously meet the requirements of permeation control and biocompatibility for both analytes. This membrane layer must not only allow glucose and β-hydroxybutyrate to permeate simultaneously, ensuring that the enzyme layer can contact the analytes, but also control the permeation rate of the two analytes, keeping their concentrations within the linear range of their respective enzyme layer reactions to avoid response saturation. Current solutions use two different outer membrane layers to control the enzyme sensor responses of the two analytes and the working electrodes, respectively. Using two outer membrane layers presents challenges to the fabrication process of dual-analyte monitoring sensors, and the simultaneous control of two or more sensor outer membrane layers to reach the working electrode also poses challenges to detection accuracy. Summary of the Invention

[0006] To address the technical difficulties in manufacturing and detection caused by the need for two outer membrane layers in existing dual-analyte monitoring systems, this invention provides a biocompatible membrane; this invention also provides a method for preparing the aforementioned biocompatible membrane; and this invention further provides an analyte sensor using the aforementioned biocompatible membrane. The biocompatible membrane provided in this application can simultaneously control the permeation ratio of two analytes. When used as the outer membrane of an analyte sensor, only one layer is needed to meet the monitoring requirements, eliminating the need for two or more outer membrane layers, and ensuring detection accuracy.

[0007] A biocompatible membrane is prepared by a membrane working fluid via a film-forming process, wherein the membrane working fluid comprises an amphiphilic polyurethane, and the amphiphilic polyurethane is prepared by any of the following methods: Hydrophilic and hydrophobic molecules form amphiphilic polymers through chemical bonding, and these amphiphilic polymers are then synthesized through addition polymerization with isocyanates; or, Polyurethane is prepared by adding one of the hydrophilic or hydrophobic molecules to an isocyanate through an addition polymerization reaction, and then forming an amphiphilic polyurethane by chemical bonding with another molecule.

[0008] Preferably, the hydrophilic molecule is any one of polyethylene glycol or its derivatives, polypropylene glycol or its derivatives, polytetrahydrofuran ether glycol, dimethylolpropionic acid, choline derivatives containing hydroxyl groups, and hyaluronic acid. The hydrophobic molecule is any one of polydimethylsiloxane or its derivatives, polyvinylpyridine or its derivatives, or cholesterol or cholesterol derivatives. The isocyanate is any one of toluene diisocyanate, diphenylmethane diisocyanate, hexamethylene diisocyanate, isophorone diisocyanate, polymethylene polyphenyl polyisocyanate, toluene diisocyanate trimer, and hexamethylene diisocyanate trimer.

[0009] Preferably, the mass ratio of the hydrophilic molecules, hydrophobic molecules, and isocyanate substances is 1:(0.2~0.5):(0.3~1.9); and / or, The hydroxyl-containing choline derivative is any one of choline ethanolamine, glycerophosphate choline, or methacryloyloxyethyl phosphate choline; The cholesterol derivative is any one of amino-cholesterol ester, cholesterol chloroformate, cholesterol succinate, and cholesterol azide.

[0010] Preferably, the working solution of the membrane layer is prepared by dissolving amphiphilic polyurethane in an organic solvent of the working solution, and the mass-volume ratio of amphiphilic polyurethane to organic solvent of the working solution is 1g:(3~10)mL.

[0011] Preferably, the organic solvent of the working solution is ethanol, ethyl acetate, or N,N Any one or more of dimethylformamide and tetrahydrofuran.

[0012] Preferably, a crosslinking agent with a mass fraction of 5% to 8% is also added to the working fluid of the membrane layer.

[0013] Preferably, the crosslinking agent is any one or more of glutaraldehyde, polyethylene glycol diglycidyl ether, triethylamine, and triethanolamine.

[0014] A method for preparing a biocompatible membrane according to any one of the above claims, comprising preparing an amphiphilic polyurethane and dissolving it in a working solution organic solvent to obtain a membrane working solution, and then forming a biocompatible membrane through a film-forming process, wherein the amphiphilic polyurethane is prepared by any one of the following methods: The first method, S1, involves dissolving hydrophilic and hydrophobic molecules separately in an organic solvent, mixing and reacting them at 20-40°C for 24-48 h, purifying the mixture after the reaction, and freeze-drying it to obtain an amphiphilic polymer; S2 involves dissolving the amphiphilic polymer and isocyanate in an organic solvent, mixing them under a protective atmosphere, adding an organotin or organobismuth catalyst, reacting them at 20-40°C for 24-48 h, terminating the reaction, collecting the precipitate, washing and purifying it, and freeze-drying it to obtain an amphiphilic polyurethane. The second method, S1, involves dissolving hydrophilic molecules and isocyanates separately in an organic solvent, adding organotin or organobismuth catalysts, and reacting under a protective atmosphere at 20–40°C for 24–48 h. After the reaction is complete, the mixture is purified and freeze-dried to obtain hydrophilic polyurethane. S2, involves dissolving hydrophilic polyurethane and hydrophobic molecules separately in an organic solvent, mixing them under a protective atmosphere, and reacting under 20–40°C for 24–48 h to obtain amphiphilic polyurethane. The third method, S1, involves dissolving hydrophobic molecules and isocyanates separately in an organic solvent, adding organotin or organobismuth catalysts, and reacting under a protective atmosphere at 20-40°C for 24-48 hours. After the reaction is complete, the mixture is purified and freeze-dried to obtain hydrophobic polyurethane. S2, involves dissolving hydrophobic polyurethane and hydrophilic molecules separately in an organic solvent, mixing them under a protective atmosphere, and reacting at 20-40°C for 24-48 hours to obtain amphiphilic polyurethane.

[0015] Preferably, the mass of the organotin or organobismuth catalyst is 0.05~0.1% of the mass of the isocyanate; and / or, The organotin catalyst is any one of dibutyltin dilaurate and stannous octanoate; the organobismuth catalyst is any one of bismuth isooctanoate and bismuth neodecanoate. When any two liquids react, one liquid is added dropwise to the other liquid over a period of 30 to 60 minutes. Freeze-drying specifically involves freeze-drying at -30℃ to -60℃ for 12 to 72 hours. The protective atmosphere is nitrogen or argon; The organic solvent is any one or more of dimethyl sulfoxide, N,N-dimethylformamide, ethanol, ethylene glycol, and tetrahydrofuran.

[0016] Preferably, the film-forming process is any one of dip coating, stretch coating, or spin coating; The working fluid of the membrane layer is used to form 1 to 10 stackable membrane layers with a thickness of 5 to 20 μm through a membrane formation process, which serve as biocompatible membranes.

[0017] Preferably, the film-forming process is dip coating, with a descent speed of 1000~6000um / s, a lifting speed of 400~3000um / s, and lifting repeated 3~8 times. After the lifting is repeated, the film is dried at room temperature or under vacuum for 1~24 hours to obtain an outer film layer with a thickness of 5~20μm, thus obtaining a coated sample.

[0018] Preferably, during the dip coating process, the descent speed is 3000~4000um / s, the lifting speed is 1500~2000um / s, the lifting is repeated 4~5 times, and vacuum drying is carried out for 1~2 hours to obtain the final product.

[0019] An analyte sensor includes a substrate and an enzyme layer disposed above the substrate. The substrate includes at least a reference electrode and a counter electrode. The enzyme layer has at least a first working electrode and a second working electrode. The first working electrode has an enzyme targeting any one of lactic acid, glucose, β-hydroxybutyric acid, alcohol, uric acid, cortisol, and ascorbic acid. The second working electrode has an enzyme targeting the other one of lactic acid, glucose, β-hydroxybutyric acid, alcohol, uric acid, cortisol, and ascorbic acid. An outer membrane layer covering the first and second working electrodes is also disposed above the enzyme layer. The outer membrane layer is a biocompatible membrane as described above, or a biocompatible membrane prepared by any one of the preparation methods described above.

[0020] This application first provides a biocompatible membrane, fabricated from a membrane working fluid via a film-forming process. The membrane working fluid includes amphiphilic polyurethane, which is prepared by any of the following methods: hydrophilic and hydrophobic molecules chemically bond to form an amphiphilic polymer, which is then reacted with an isocyanate through an addition polymerization reaction; or, one of the hydrophilic or hydrophobic molecules reacts with an isocyanate through an addition polymerization reaction to form polyurethane, which is then chemically bonded with another molecule to form the amphiphilic polyurethane. The biocompatible membrane provided by this application can meet the control requirements for a first analyte and a second analyte, simultaneously controlling the permeation ratio of the two analytes. When the above-mentioned biocompatible membrane is used as the outer membrane of an analyte sensor, only one layer is needed to meet the monitoring requirements, eliminating the need for two or more outer membranes, thus simplifying the preparation process of the analyte sensor; furthermore, using one layer of the above-mentioned biocompatible membrane ensures that the permeation ratio of both analytes meets the detection requirements, guaranteeing detection accuracy.

[0021] The biocompatible membrane provided in this application uses amphiphilic polyurethane, which is prepared by sequentially reacting hydrophilic molecules, hydrophobic molecules, and isocyanate-based substances in pairs. In the final amphiphilic polyurethane, the hydrophilic and hydrophobic molecules are chemically bonded. Compared to traditional membrane solutions formed by physical blending of hydrophilic and hydrophobic polymers or cross-linking through cross-linking agents, the membrane formed in this application is more uniform and stable, which is beneficial for improving the accuracy of sensor monitoring. Furthermore, this application uses isocyanate-based substances to polymerize hydrophilic and hydrophobic molecules into polyurethane. The elasticity and flexibility of polyurethane are superior to those of hydrophilic polymers formed by hydrophilic and hydrophobic molecules, making it easier to control the permeate ratio of two analytes (such as glucose and β-hydroxybutyric acid, or lactic acid and alcohol; analytes with molecular weights comparable to glucose can achieve simultaneous detection of two analytes).

[0022] In this application, the working fluid for the membrane layer is prepared by dissolving amphiphilic polyurethane in an organic solvent. Preferably, a crosslinking agent is added for crosslinking; the crosslinking agent is used here to form a polymer network structure, which is a form of intramolecular crosslinking.

[0023] This application also provides a method for preparing the above-mentioned biocompatible membrane.

[0024] This application also provides an analyte sensor using the aforementioned biocompatible membrane, comprising a substrate, the substrate including at least a reference electrode and a counter electrode; an enzyme layer disposed above the substrate, the enzyme layer having at least a first working electrode and a second working electrode, the first working electrode having an enzyme targeting any one of lactic acid, glucose, β-hydroxybutyric acid, alcohol, uric acid, cortisol, and ascorbic acid; the second working electrode having an enzyme targeting the other one of lactic acid, glucose, β-hydroxybutyric acid, alcohol, uric acid, cortisol, and ascorbic acid; and an outer membrane layer covering the first and second working electrodes, the outer membrane layer being the aforementioned biocompatible membrane. Compared with the prior art, this application uses only a single hydrophilic / hydrophobic sensor outer membrane to simultaneously meet the control requirements of the first and second analytes, without the need for two or more outer membranes, thus not only meeting the biocompatibility requirements of the sensor outer membrane but also simplifying the fabrication process of the dual analyte monitoring sensor. Furthermore, applying the same or different voltages less than 100mV (such as 5, 10, 20, 30, 70, 90mV, etc.) to the two working electrodes can meet the redox potential requirements of the first and second analytes, enabling accurate detection of the analytes.

[0025] In this application, the enzyme layer is provided with at least a first working electrode and a second working electrode, and a third working electrode, a fourth working electrode, etc., may also be provided as needed. When multiple working electrodes are provided, the biocompatible membrane provided in this application serves as an outer membrane covering at least two of the working electrodes, or it may cover all of the working electrodes. The biocompatible membrane provided in this application is also suitable for use as an outer membrane in existing single-working-electrode analyte sensors.

[0026] In this application, the hydrophilic molecule is any one of polyethylene glycol or its derivatives, polypropylene glycol or its derivatives, polytetrahydrofuran ether glycol, dimethylolpropionic acid, choline derivatives containing hydroxyl groups, and hyaluronic acid. Hyaluronic acid requires the addition of an activator for reaction, while other hydrophilic molecules can react directly. The activator can be one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), hydroxybenzotriazole (HOBT), triethylamine, and 4-dimethylaminopyridine (DMAP).

[0027] In this application, the temperature is controlled below 40°C during the reaction of isocyanate substances to prevent local overheating during the reaction. Simultaneously, the reaction solution is preferably added dropwise, with the addition time controlled within 30-60 minutes to ensure a complete and smooth reaction. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The results of the cyclic voltammetry test of the electrode of the dual analyte sensor without an outer membrane layer in Experimental Example 1 of this invention; Figure 2 The results of enzyme electrode response current testing for the dual analyte sensor without an outer membrane layer in Experimental Example 1 of this invention; Figure 3 The results of membrane electrode tests performed on the coated sample (a dual analyte sensor with an outer membrane layer) under the first applied voltage in Example 1 of this invention; Figure 4 The results of membrane electrode tests performed on the coated sample in Example 1 of this invention under the second applied voltage; Figure 5 The results of the membrane electrode test performed on the coated sample in Example 1 of this invention under the third applied voltage; Figure 6The results of the membrane electrode test performed on the coated sample in Example 1 of this invention under the fourth applied voltage; Figure 7 The results of the stability test of the coating sample in Example 1 of this invention under the applied voltage in the first group; Figure 8 The results of the stability test of the coating sample in Example 1 of this invention under the applied voltage in Group 2; Figure 9 The results of the stability test of the coating sample in Example 1 of this invention under the applied voltage in Group 3; Figure 10 The results of the stability test of the coating sample in Example 1 of this invention under the applied voltage in Group 4 are shown. Detailed Implementation

[0030] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0031] Example 1: Fabrication and Performance Testing of a Dual-Analyte Sensor Without an Outer Membrane Layer I. Fabrication of a dual analyte sensor without an outer membrane layer The dual analyte sensor includes a substrate and an enzyme layer disposed on the substrate, wherein the enzyme layer is provided with a first working electrode and a second working electrode; the substrate includes a reference electrode and a counter electrode.

[0032] The first working electrode is the glucose enzyme sensing layer sensor layer; The second working electrode is the β-hydroxybutyrate sensor layer.

[0033] Preparation of the first working electrode (Examples 1 and 2 of prior patent CN114152656A) Glucose dehydrogenase (1-10 mg / mL, optimal: 5) was incubated in PBS buffer containing 1-6 mol / L urea (optimal: 3) at 4°C for 8-24 hours (optimal: 12) to fully develop the glucose dehydrogenase. Then, a ruthenium or osmium complex with a free amino group (1-10 mg / mL, optimal: 5) was thoroughly mixed with 1-10 mg / mL glucose dehydrogenase (optimal: 5), followed by the sequential addition of 0.2-10 mmol / L carbodiimide (optimal: 1) and 0.1-1 mmol / L N-hydroxysuccinimide (optimal: 0.5). After thorough mixing, the mixture was incubated at 4°C for 8-24 hours (optimal: 12). The modified glucose dehydrogenase was then separated and purified by ultrafiltration (molecular weight cutoff: 1000-30000, optimal: 10000). The purified glucose dehydrogenase can now directly and effectively exchange electrons with the electrode; To further enhance the rapid electron exchange between glucose dehydrogenase and the electrode, the purified glucose dehydrogenase can be incubated again in PBS buffer containing 1-6 mol / L urea (optimal: 3) at 4°C for 8-24 hours (optimal: 12) to fully develop the glucose dehydrogenase once more. Then, 1-10 mg / mL (optimal: 5) of a ruthenium or osmium complex with a free carboxyl group, 0.2-10 mmol / L of carbodiimide (optimal: 1), and 0.1-1 mmol / L of N-hydroxysuccinimide (optimal: 0.5) are added sequentially to the solution. After thorough mixing, the mixture is reacted again at 4°C for 8-24 hours (optimal: 12). After the reaction, the modified glucose dehydrogenase is separated and purified again by ultrafiltration (molecular weight cutoff: 1000-30000, optimal: 10000).

[0034] 2-20 mg / mL (optimal: 5) of modified glucose dehydrogenase was thoroughly mixed with 0.1-5% (optimal: 1) glutaraldehyde solution in PBS buffer solution. After 30-180 minutes (optimal: 60 minutes), the chemically cross-linked glucose dehydrogenase was coated onto the electrode surface using the dip-coating method to form a glucose sensing membrane.

[0035] Preparation of the second working electrode (Examples 1 and 3 of prior patent CN119861124A) Synthetic N 6 -(2-(CH2)2NH2)-NAD + S1. Dissolve 1.2 g of bromoethylamine hydrobromide in 2 mL of methanol, and dissolve 4.8 g of NAD in 5 mL of water. Add the bromoethylamine hydrobromide solution dropwise to a flask containing the NAD solution. Under nitrogen protection, react at 25 °C for 20 hours. After the reaction is complete, remove the solvent by vacuum rotary evaporation, wash three times with 5 mL of ethanol, centrifuge the precipitate, and dry it by vacuum rotary evaporation to obtain a white powder product. After ion exchange chromatography, concentrate the collected fraction by rotary evaporation to obtain N. 1 -(2-(CH2)2NH2)-NAD + Yield 47.8% (2.3g); S2, the N obtained in step S1 1 -(2-(CH2)2NH2)-NAD + 2.0 g of the solution was dissolved in 5 mL of deionized water and added to a 1 mM LiOH solution. The pH was adjusted to 6.5, and the reaction was carried out at 50 °C for 5 h. After the reaction was completed, the product was dried under vacuum by rotary evaporation to obtain a white powder. The collected fraction was concentrated by rotary evaporation after ion exchange chromatography to obtain N. 6 -(2-(CH2)2NH2)-NAD + Yield: 62% (1.24g).

[0036] N 6 -(2-(CH2)2NH2)-NAD + Fixed Take N prepared in Example 1 6 -(2-(CH2)2NH2)-NAD + The glutaraldehyde was used as a coupling agent to fix the glutaraldehyde onto a silicon dioxide nanocarrier material with a size of 300-500 nm modified with polyethyleneimine.

[0037] Take the above fixed N 6 -(2-(CH2)2NH2)-NAD + A β-hydroxybutyrate (BHT) sensitive layer solution was prepared using silica nanocarrier material, along with β-hydroxybutyrate dehydrogenase, myocardial flavin enzyme, electron mediator, cross-linking agent, and HEPES buffer solution. The BHT sensitive layer solution was then deposited onto an electrode layer to obtain an enzyme-sensing membrane, which serves as the second working electrode.

[0038] The first working electrode and the second working electrode prepared in the above steps are combined with the substrate to obtain a dual analyte sensor without an outer membrane layer.

[0039] II. Performance Testing of Dual Analyte Sensors Without an Outer Membrane Layer The dual analyte sensors without an outer membrane layer were used to perform electrochemical activity tests in corresponding glucose or β-hydroxybutyric acid solutions, respectively. The results are as follows: Figure 1 As shown.

[0040] from Figure 1 It can be seen that the first working electrode and the second working electrode are in An oxidation peak is observed at approximately 20 mV. This indicates that the enzyme sensing membrane, utilizing the first and second working electrodes described above, can perform electrochemical reactions at relatively low potentials, with reaction potentials potentially less than 100 mV.

[0041] To test the sensitivity of the sample in vitro and to ensure the accuracy of the test data, the first and second working electrodes were tested once in solution using the same applied voltage of 100 mV. The results are as follows. Figure 2 As shown.

[0042] from Figure 2 It can be seen that the experimental sample has good linearity in the glucose test solution, with a linear range of 0~5 mmol / L; the experimental sample has a linear range of 0~4 mmol / L in the β-hydroxybutyric acid test solution.

[0043] The in vitro sensitivity test results show that the linear range of the enzyme electrode tests of the first and second working electrodes is relatively narrow. For the glucose enzyme sensing layer, the linear range is only 0~5 mmol / L; for the β-hydroxybutyrate sensing layer, the linear range is only 0~4 mmol / L, both of which are lower than the detection limit required by the human body. Generally, the linear range requirement for implantable glucose monitoring systems is 0~25 mmol / L, and the linear range requirement for β-hydroxybutyrate monitoring systems is 0~8 mmol / L.

[0044] Furthermore, a biocompatible outer membrane layer is added to the enzyme sensing layer of the first and second working electrodes, while controlling the permeation ratio of glucose and β-hydroxybutyric acid to improve the detection range of the sensor.

[0045] Example 1: Preparation of biocompatible membrane, setup of a dual analyte sensor with a biocompatible membrane as the outer membrane layer, and performance testing. I. Preparation of Biocompatible Membranes 3.1 Synthesis of amphiphilic polyurethane: S1. Dissolve 5g of hyaluronic acid in 25 mL of anhydrous dimethyl sulfoxide (DMSO) until fully dissolved. Then, add 125 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 60 mg of N-hydroxysuccinimide (NHS), 120 mg of hydroxybenzotriazole (HOBT), 120 μL of triethylamine, and 12.5 mg of 4-dimethylaminopyridine (DMAP) to the solution in sequence until fully dissolved. Stir at 30°C for 4 hours under nitrogen protection to obtain the reaction solution. S2. Slowly add 25 mL of anhydrous DMSO containing amino-cholesterol ester (0.2-0.3 times the mass of hyaluronic acid) dropwise to the above reaction solution, and continue stirring at 30°C for 36 h. After the reaction is completed, treat the reaction solution in ultrapure water at room temperature for 3 days using a dialysis bag with a molecular weight cutoff of 3.5 kDa, changing the water at least three times during the period. After freeze-drying at -40°C for 24 h, the cholesterol ester-hyaluronic acid polymer (i.e., amphiphilic polymer) can be obtained. S3. Dissolve 5g of diphenylmethane diisocyanate (MDI) in 20 mL of DMSO, add 50 μg of dibutyltin dilaurate catalyst, and protect under nitrogen atmosphere. Add the cholesterol ester-hyaluronic acid polymer dropwise to the MDI / DMSO solution (slowly over 30-60 minutes), continue stirring for 24 h, and terminate the reaction by adding 10 mL of anhydrous ethanol. Add the reaction mixture dropwise to 100 mL of acetone to precipitate the product. Centrifuge (8000 rpm, 10 min) to collect the precipitate, and wash three times with acetone. Redissolve in 20 mL of PBS (pH 7.4), treat with a dialysis bag with a molecular weight cutoff of 3.5 kDa in ultrapure water at room temperature for 3 days, changing the water at least three times during this period, and freeze-dry at -35°C for 20 h to obtain an amphiphilic polyurethane.

[0046] 3.2 Preparation of the outer membrane solution: Take 2g of the above-mentioned amphiphilic polyurethane polymer, dissolve it in 10mL of tetrahydrofuran, add 5% polyethylene glycol diglycidyl ether crosslinking agent to crosslink, and obtain the sensor outer membrane solution.

[0047] 3.3 Coating of the outer membrane: The above-mentioned sensor outer membrane solution was applied to the electrode of the dual analyte sensor without an outer membrane layer to form an outer membrane layer by dip coating. The specific parameters were: descent speed 3000 μm / s, lifting speed 1500 μm / s, lifting repetitions 4 times, vacuum drying for 1 hour, and the outer membrane layer thickness was 5~20 μm to obtain the coated sample.

[0048] II. Performance Testing of Coating Samples Applied voltage: The applied voltage is less than 100 mV. The first working electrode and the second working electrode can use the same applied voltage or different applied voltages. Preferably, 100 mV and 50 mV are used, as shown in Table 1. Table 1 Applied Voltage

[0049] In the linearity test, the concentrations of glucose were 0 mmol / L, 2.0 mmol / L, 5.0 mmol / L, 10.0 mmol / L, 15.0 mmol / L, 20.0 mmol / L, and 25.0 mmol / L; the concentrations of β-hydroxybutyric acid were 0 mmol / L, 0.5 mmol / L, 1.0 mmol / L, 2.0 mmol / L, 4.0 mmol / L, 6.0 mmol / L, and 8.0 mmol / L. The combinations are shown in Table 2 below, and tests were conducted at seven test points.

[0050] Table 2 shows the test solution concentrations and combinations for the dual analyte sensor.

[0051] Referring to the applied voltage in Table 1, the same coated samples were subjected to linearity tests at seven test concentrations, and the test results are as follows: Figures 3-6 , and as shown in Table 3.

[0052] Table 3. Sensitivity results of linearity test for dual analyte sensor

[0053] from Figures 3-6 As shown in Table 3, under the four applied voltages, the sensitivity of the prepared coated glucose sensor was 0.67–0.69 nA / mmol / L, and the coated samples exhibited good linearity in glucose solutions of 0–25 mmol / L. The sensitivity of the β-hydroxybutyric acid sensor was 1.48–1.52 nA / mmol / L, and the coated samples exhibited good linearity in β-hydroxybutyric acid solutions of 0–8 mmol / L. This indicates that both the same and different applied voltages are suitable for the dual-analyte sensor.

[0054] Table 4. Permeation ratio of dual analyte sensor before and after coating

[0055] It is preferred to use the same applied voltage of 100 mV for comparison. Under the condition of ensuring the sensitivity and linearity of the glucose sensor and β-hydroxybutyric acid sensor after coating, it can be seen from Table 4 that the permeation ratio of glucose can reach 51:1 and the permeation ratio of β-hydroxybutyric acid can reach 9:1 before and after coating. This further illustrates that a single layer of sensor outer membrane with hydrophilic and hydrophobic properties can simultaneously meet the control requirements of the first analyte and the second analyte.

[0056] Stability test: Referring to the applied voltage in Table 1, the coated dual analyte sensor was subjected to a 15-day stability test in a mixed solution of 20 mM glucose and 8 mM β-hydroxybutyric acid. The test results are as follows: Figures 7-10 As shown.

[0057] from Figures 7-10 It can be seen that, under the four sets of applied voltages, the prepared coated dual analyte sensor can maintain stability for 15 days in a mixed solution of 20 mM glucose and 8 mM 3-hydroxybutyric acid. This further demonstrates that, under the same applied voltage and different applied voltages, a single layer of sensor outer membrane with hydrophilic and hydrophobic properties can achieve effective monitoring of glucose and 3-hydroxybutyric acid for 15 days, meeting the lifespan requirements of the dual analyte sensor.

[0058] Example 2: Preparation of biocompatible membrane 3.1 Synthesis of amphiphilic polyurethane: S1. Dissolve 5g of poly(4-vinylpyridine) (P4VP) thoroughly in anhydrous dimethyl sulfoxide (DMSO) to obtain a clear or slightly turbid solution. Under nitrogen protection, add 12.5g of 2-bromoethylamine hydrobromide (approximately 2.5 times the mass of P4VP) to the solution to ensure the reaction proceeds fully.

[0059] Next, the reaction system was heated to 70-80°C in an oil bath and stirred continuously at this temperature for 24-48 hours. After the reaction was complete, the reaction solution was cooled to room temperature. Under vigorous stirring, the reaction solution was slowly poured into 500 mL of ice-cold diethyl ether. After standing for a short while, and once the solid had completely precipitated, the supernatant was carefully discarded. The obtained crude product was transferred to a centrifuge tube and washed three times with diethyl ether (each time adding approximately 50 mL of diethyl ether, shaking, centrifuging, and discarding the supernatant) to remove unreacted bromoethylamine, solvent, and byproducts. To completely remove bromide ions (Br... - The solid can be redispersed in a small amount of methanol, precipitated again with diethyl ether, and washed once by centrifugation. After the reaction is complete, the reaction solution is treated in ultrapure water at room temperature for 3 days using a dialysis bag with a molecular weight cutoff of 3.5 kDa, with the water changed at least three times during this period. The PVP-NH2 polymer is then obtained after freeze-drying.

[0060] S2. Dissolve 5g of diphenylmethane diisocyanate (MDI) in 20 mL of DMSO, add 4mg of dibutyltin dilaurate catalyst, and purge with nitrogen. Raise the reaction temperature to 35°C and react for 2 h under nitrogen protection and continuous stirring. Add 15.0g of polytetrahydrofuran ether diol (PTMG), raise the reaction temperature to 30°C, and react for 3 h under nitrogen protection and continuous stirring.

[0061] 4 g of the PVP-NH2 obtained in step S1 was dissolved in 20 mL of anhydrous DMSO. Under vigorous stirring, the PVP-NH2 DMSO solution was slowly added dropwise to the prepolymer (PTMG+MDI) solution using a constant-pressure dropping funnel. The dropping rate was controlled to be completed within 30-60 minutes, and the reaction temperature was maintained between 25-30°C. After the reaction was complete, the reaction solution was treated in ultrapure water at room temperature for 3 days using a dialysis bag with a molecular weight cutoff of 3.5 kDa, with the water changed at least three times during this period. After freeze-drying at -55°C for 12 hours, a poly(4-vinylpyridine) (P4VP) and polytetrahydrofuran ether diol (PTMG)-based polyurethane polymer was obtained.

[0062] 3.2 Preparation of the outer membrane solution: Take 3g of the above amphiphilic polyurethane, dissolve it in 10mL of ethanol, add 8% triethanolamine for crosslinking, and obtain the sensor outer membrane solution.

[0063] 3.3 Coating of the outer membrane: The above-mentioned sensor outer membrane solution was applied to the electrodes of a dual analyte sensor without an outer membrane layer to form an outer membrane layer by dip coating. The specific parameters were: descent speed 2000 μm / s, lifting speed 1000 μm / s, lifting repetitions 6 times, and drying at room temperature for 10 h to obtain an outer membrane layer thickness of 5~20 μm, thus obtaining a coated sample.

[0064] Example 3: Preparation of Biocompatible Membranes 3.1 Synthesis of amphiphilic polyurethane: S1. Dissolve 5g of hyaluronic acid in 25 mL of anhydrous tetrahydrofuran until fully dissolved. Then, add 125 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 60 mg of N-hydroxysuccinimide (NHS), 120 mg of hydroxybenzotriazole (HOBT), 120 μL of triethylamine and 12.5 mg of 4-dimethylaminopyridine (DMAP) to the solution in sequence until fully dissolved. Stir at 25°C for 4 h under nitrogen protection to obtain the reaction solution. S2. Slowly add 25 mL of anhydrous tetrahydrofuran containing α,ω-bis(3-aminopropyl)polydimethylsiloxane (0.2-0.3 times the mass of hyaluronic acid) dropwise to the above reaction solution, and continue stirring at 25°C for 40 h. After the reaction is completed, treat the reaction solution in ultrapure water at room temperature for 3 days using a dialysis bag with a molecular weight cutoff of 3.5 kDa, changing the water at least three times during the period. After freeze-drying at -30°C for 60 h, the α,ω-bis(3-aminopropyl)polydimethylsiloxane-hyaluronic acid polymer (i.e., amphiphilic polymer) can be obtained. S3. Dissolve 8 g of hexamethylene diisocyanate in 20 mL of tetrahydrofuran, add 4 mg of dibutyltin dilaurate catalyst, and purge with nitrogen. Add α,ω-bis(3-aminopropyl)polydimethylsiloxane-hyaluronic acid polymer dropwise to the hexamethylene diisocyanate / DMSO solution (slowly, over 30-60 minutes). Continue stirring for 24 hours, then add 10 mL of anhydrous ethanol to terminate the reaction. Add the reaction solution dropwise to 100 mL of acetone to precipitate the product. Centrifuge (8000 rpm, 10 minutes) to collect the precipitate, and wash three times with acetone. Redissolve in 20 mL of pure water, treat with a dialysis bag with a molecular weight cutoff of 3.5 kDa in ultrapure water at room temperature for 3 days, changing the water at least three times during this period, and freeze-dry at -45°C for 20 hours to obtain amphiphilic polyurethane.

[0065] 3.2 Preparation of the outer membrane solution: Take 3g of the above-mentioned amphiphilic polyurethane polymer and dissolve it in 25mL of N,N To dimethylformamide, 6% glutaraldehyde is added for cross-linking to obtain the sensor outer membrane solution.

[0066] 3.3 Coating of the outer membrane: The above-mentioned sensor outer membrane solution was applied to the electrodes of a dual analyte sensor without an outer membrane layer to form an outer membrane layer by dip coating. The specific parameters were: descent speed 600 μm / s, lifting speed 3000 μm / s, lifting repeated 3 times, and drying at room temperature for 20 h to obtain an outer membrane layer with a thickness of 5~20 μm, thus obtaining a coated sample.

[0067] Example 4: Preparation of Biocompatible Membranes 3.1 Synthesis of amphiphilic polyurethane: S1. Dissolve 5g of polyethylene glycol dicarboxylic acid in 25 mL of anhydrous dimethyl sulfoxide (DMSO) until fully dissolved; S2. Slowly add 25 mL of anhydrous DMSO containing α,ω-bis(3-aminopropyl)polydimethylsiloxane (0.2-0.3 times the mass of polyethylene glycol dicarboxylic acid) dropwise to the above reaction solution, and continue stirring at 25°C for 30 h. After the reaction is completed, treat the reaction solution in ultrapure water at room temperature for 3 days using a dialysis bag with a molecular weight cutoff of 3.5 kDa, changing the water at least three times during the period. After freeze-drying at -40°C for 72 h, the α,ω-bis(3-aminopropyl)polydimethylsiloxane-polyethylene glycol dicarboxylic acid polymer (i.e., amphiphilic polymer) can be obtained. S3. Dissolve 5g of toluene diisocyanate trimer in 20 mL of DMSO. Add 4mg of stannous octoate catalyst and purge with nitrogen. Add 5g of α,ω-bis(3-aminopropyl)polydimethylsiloxane-polyethylene glycol dicarboxylic acid polymer dropwise to the toluene diisocyanate trimer / DMSO solution (slowly, over 30-60 minutes). Heat to 30℃ and continue stirring for 30h. Add 10 mL of anhydrous ethanol to terminate the reaction. Add the reaction solution dropwise to 100 mL of acetone to precipitate the product. Centrifuge (8000 rpm, 10 min) to collect the precipitate and wash three times with acetone. Redissolve in 20 mL of PBS (pH 7.4). Treat the reaction solution in ultrapure water using a dialysis bag with a molecular weight cutoff of 3.5 kDa at room temperature for 3 days, changing the water at least three times during this period. Freeze-dry at -40℃ for 40h to obtain an amphiphilic polyurethane.

[0068] 3.2 Preparation of the outer membrane solution: Take 5g of the above-mentioned amphiphilic polyurethane polymer, dissolve it in 30mL of ethyl acetate, add 5% triethylamine for cross-linking, and obtain the sensor outer membrane solution.

[0069] 3.3 Coating of the outer membrane: The above-mentioned sensor outer membrane solution was applied to the electrodes of a dual analyte sensor without an outer membrane layer to form an outer membrane layer by dip coating. The specific parameters were: descent speed 1800 μm / s, lifting speed 2000 μm / s, lifting repetitions 4 times, vacuum drying for 8 hours, and the outer membrane layer thickness was 5~20 μm to obtain the coated sample.

[0070] Example 5: Preparation of Biocompatible Membranes 3.1 Synthesis of amphiphilic polyurethane: S1. Dissolve 5g of diphenylmethane diisocyanate in 20 mL of DMSO. Add 4mg of bismuth isooctanoate catalyst and purge with nitrogen. Raise the reaction temperature to 25°C and react for 2 h under nitrogen protection and continuous stirring. Add 10g of polyethylene glycol, raise the reaction temperature to 25°C, and react for 3 h under nitrogen protection and continuous stirring.

[0071] Dissolve 5g of the prepolymer (polyethylene glycol + MDI) in 20 mL of anhydrous DMSO. Under vigorous stirring, slowly add the 5g choline ethanolamine solution in DMSO to the prepolymer (polyethylene glycol + MDI) solution using a constant-pressure dropping funnel. Control the dropping rate to complete the process within 30-60 minutes, maintaining the reaction temperature between 25-30°C. After the reaction is complete, treat the reaction solution in ultrapure water at room temperature for 3 days using a dialysis bag with a molecular weight cutoff of 3.5 kDa, changing the water at least three times during this period. Freeze-dry at -45°C for 40 hours to obtain the poly(diphenylmethane diisocyanate)-polyethylene glycol-choline ethanolamine-based polyurethane polymer.

[0072] 3.2 Preparation of the outer membrane solution: Take 4g of the above-mentioned amphiphilic polyurethane polymer, dissolve it in 15mL of tetrahydrofuran, add 8% pentanediol for cross-linking, and obtain the sensor outer membrane solution.

[0073] 3.3 Coating of the outer membrane: The above-mentioned sensor outer membrane solution was applied to the electrodes of a dual analyte sensor without an outer membrane layer to form an outer membrane layer by dip coating. The specific parameters were: descent speed 3000 μm / s, lifting speed 1800 μm / s, lifting repetitions 4 times, and vacuum drying for 2 hours to obtain an outer membrane layer thickness of 5~20 μm, thus obtaining a coated sample.

[0074] Example 6: This embodiment refers to Embodiment 1. The dual analyte sensor includes a substrate electrode and an enzyme layer disposed on the substrate, wherein the enzyme layer is provided with a first working electrode and a second working electrode; the substrate includes a reference electrode and a counter electrode.

[0075] The first working electrode is the glucose enzyme sensing layer. The second working electrode is the alcoholase sensing layer. Table 5. Permeation ratio of dual analyte sensor before and after coating.

[0076] Using the same applied voltage of 100 mV for comparison, under the premise of ensuring the sensitivity and linearity requirements of the glucose sensor and ethanol sensor after coating, it can be seen from Table 5 that before and after coating, the permeation ratio of glucose can reach 50:1 and the permeation ratio of ethanol can reach 25:1. This further illustrates that a single layer of sensor outer membrane with hydrophilic and hydrophobic properties can simultaneously meet the control requirements of the first and second analytes.

[0077] Example 7: This embodiment refers to Embodiment 2. The dual analyte sensor includes a substrate and an enzyme layer disposed on the substrate, wherein the enzyme layer is provided with a first working electrode and a second working electrode; the substrate includes a reference electrode and a counter electrode.

[0078] The first working electrode is the glucose enzyme sensing layer sensor layer; The second working electrode is the β-hydroxybutyrate sensor layer.

[0079] Table 6. Permeation ratio of dual analyte sensor before and after coating.

[0080] Using the same applied voltage of 100 mV for comparison, under the requirement of ensuring the sensitivity and linearity of the glucose sensor and β-hydroxybutyric acid sensor after coating, it can be seen from Table 6 that before and after coating, the permeation ratio of glucose can reach 50:1, and the permeation ratio of β-hydroxybutyric acid can reach 9:1. This further illustrates that a single layer of sensor outer membrane with hydrophilic and hydrophobic properties can simultaneously satisfy the restriction function of the first analyte and the second analyte.

[0081] Example 8: This embodiment refers to Embodiment 3. The dual analyte sensor includes a substrate and an enzyme layer disposed on the substrate, wherein the enzyme layer is provided with a first working electrode and a second working electrode; the substrate includes a reference electrode and a counter electrode.

[0082] The first working electrode is the glucose enzyme sensing layer sensor layer; The second working electrode is the β-hydroxybutyrate sensor layer.

[0083] Table 7. Permeation ratio of dual analyte sensor before and after coating.

[0084] Using the same applied voltage of 100 mV for comparison, under the requirement of ensuring the sensitivity and linearity of the glucose sensor and β-hydroxybutyric acid sensor after coating, it can be seen from Table 7 that before and after coating, the permeation ratio of glucose can reach 47:1, and the permeation ratio of β-hydroxybutyric acid can reach 10:1. This further illustrates that a single layer of sensor outer membrane with hydrophilic and hydrophobic properties can simultaneously satisfy the restriction function of the first and second analytes.

[0085] Example 9: This embodiment refers to Embodiment 4. The dual analyte sensor includes a substrate and an enzyme layer disposed on the substrate, wherein the enzyme layer is provided with a first working electrode and a second working electrode; the substrate includes a reference electrode and a counter electrode.

[0086] The first working electrode is the glucose enzyme sensing layer sensor layer; The second working electrode is the β-hydroxybutyrate sensor layer.

[0087] Table 8. Permeation ratio of dual analyte sensor before and after coating.

[0088] Using the same applied voltage of 100 mV for comparison, under the requirement of ensuring the sensitivity and linearity of the glucose sensor and β-hydroxybutyric acid sensor after coating, it can be seen from Table 8 that before and after coating, the permeation ratio of glucose can reach 59:1 and the permeation ratio of β-hydroxybutyric acid can reach 11:1. This further illustrates that a single layer of sensor outer membrane with hydrophilic and hydrophobic properties can simultaneously satisfy the restriction function of the first analyte and the second analyte.

[0089] Example 10: This embodiment refers to Embodiment 5. The dual analyte sensor includes a substrate and an enzyme layer disposed on the substrate, wherein the enzyme layer is provided with a first working electrode and a second working electrode; the substrate includes a reference electrode and a counter electrode.

[0090] The first working electrode is the glucose enzyme sensing layer sensor layer; The second working electrode is the β-hydroxybutyrate sensor layer.

[0091] Table 9. Permeation ratio of dual analyte sensor before and after coating.

[0092] Using the same applied voltage of 100 mV for comparison, under the requirement of ensuring the sensitivity and linearity of the glucose sensor and β-hydroxybutyric acid sensor after coating, it can be seen from Table 9 that before and after coating, the permeation ratio of glucose can reach 57:1 and the permeation ratio of β-hydroxybutyric acid can reach 10:1. This further illustrates that a single layer of sensor outer membrane with hydrophilic and hydrophobic properties can simultaneously satisfy the restriction function of the first analyte and the second analyte.

[0093] The above description is merely an embodiment of the present invention. It should be noted that those skilled in the art can make improvements without departing from the inventive concept of the present invention, but these improvements all fall within the protection scope of the present invention.

Claims

1. A biocompatible membrane, formed from a membrane working fluid through a film-forming process, characterized in that, The working fluid of the membrane layer includes amphiphilic polyurethane, which is prepared by any of the following methods: Hydrophilic and hydrophobic molecules form amphiphilic polymers through chemical bonding, and these amphiphilic polymers are then synthesized through addition polymerization with isocyanates; or, Polyurethane is prepared by adding one of the hydrophilic or hydrophobic molecules to an isocyanate through an addition polymerization reaction, and then forming an amphiphilic polyurethane by chemical bonding with another molecule.

2. The biocompatible membrane according to claim 1, characterized in that, The hydrophilic molecule is any one of polyethylene glycol or its derivatives, polypropylene glycol or its derivatives, polytetrahydrofuran ether glycol, dimethylolpropionic acid, choline derivatives containing hydroxyl groups, and hyaluronic acid. The hydrophobic molecule is any one of polydimethylsiloxane or its derivatives, polyvinylpyridine or its derivatives, or cholesterol or cholesterol derivatives. The isocyanate is any one of toluene diisocyanate, diphenylmethane diisocyanate, hexamethylene diisocyanate, isophorone diisocyanate, polymethylene polyphenyl polyisocyanate, toluene diisocyanate trimer, and hexamethylene diisocyanate trimer.

3. The biocompatible membrane according to claim 2, characterized in that, The mass ratio of the hydrophilic molecules, hydrophobic molecules, and isocyanate substances is 1:(0.2~0.5):(0.3~1.9); and / or, The hydroxyl-containing choline derivative is any one of choline ethanolamine, glycerophosphate choline, or methacryloyloxyethyl phosphate choline; The cholesterol derivative is any one of amino-cholesterol ester, cholesterol chloroformate, cholesterol succinate, and cholesterol azide.

4. The biocompatible membrane according to claim 1, characterized in that, The membrane working solution is prepared by dissolving amphiphilic polyurethane in an organic solvent of the working solution, and the mass-volume ratio of amphiphilic polyurethane to organic solvent of the working solution is 1g:(3~10)mL.

5. The biocompatible membrane according to claim 4, characterized in that, The working solution uses ethanol, ethyl acetate, or N,N-methyl as the organic solvent. Any one or more of dimethylformamide and tetrahydrofuran.

6. The biocompatible membrane according to any one of claims 4-5, characterized in that, A crosslinking agent with a mass fraction of 5% to 8% is also added to the working fluid of the membrane layer.

7. The biocompatible membrane according to claim 6, characterized in that, The crosslinking agent is any one or more of glutaraldehyde, polyethylene glycol diglycidyl ether, triethylamine, and triethanolamine.

8. A method for preparing a biocompatible membrane according to any one of claims 1-7, characterized in that, An amphiphilic polyurethane was prepared and dissolved in an organic solvent to obtain a membrane working solution, which was then used to form a biocompatible membrane via a film-forming process. The amphiphilic polyurethane was prepared using any of the following methods: The first method, S1, involves dissolving hydrophilic and hydrophobic molecules separately in an organic solvent, mixing and reacting them at 20-40°C for 24-48 h, purifying the mixture after the reaction, and freeze-drying it to obtain an amphiphilic polymer; S2 involves dissolving the amphiphilic polymer and isocyanate in an organic solvent, mixing them under a protective atmosphere, adding an organotin or organobismuth catalyst, reacting them at 20-40°C for 24-48 h, terminating the reaction, collecting the precipitate, washing and purifying it, and freeze-drying it to obtain an amphiphilic polyurethane. The second method, S1, involves dissolving hydrophilic molecules and isocyanates separately in an organic solvent, adding organotin or organobismuth catalysts, and reacting under a protective atmosphere at 20–40°C for 24–48 h. After the reaction is complete, the mixture is purified and freeze-dried to obtain hydrophilic polyurethane. S2, involves dissolving hydrophilic polyurethane and hydrophobic molecules separately in an organic solvent, mixing them under a protective atmosphere, and reacting under 20–40°C for 24–48 h to obtain amphiphilic polyurethane. The third method, S1, involves dissolving hydrophobic molecules and isocyanates separately in an organic solvent, adding organotin or organobismuth catalysts, and reacting under a protective atmosphere at 20-40°C for 24-48 hours. After the reaction is complete, the mixture is purified and freeze-dried to obtain hydrophobic polyurethane. S2, involves dissolving hydrophobic polyurethane and hydrophilic molecules separately in an organic solvent, mixing them under a protective atmosphere, and reacting at 20-40°C for 24-48 hours to obtain amphiphilic polyurethane.

9. The preparation method according to claim 8, characterized in that, The mass of the organotin or organobismuth catalyst is 0.05–0.1% of the mass of the isocyanate; and / or, The organotin catalyst is any one of dibutyltin dilaurate and stannous octanoate; the organobismuth catalyst is any one of bismuth isooctanoate and bismuth neodecanoate. When any two liquids react, one liquid is added dropwise to the other liquid over a period of 30 to 60 minutes. Freeze-drying specifically involves freeze-drying at -30℃ to -60℃ for 12 to 72 hours. The protective atmosphere is nitrogen or argon; The organic solvent is any one or more of dimethyl sulfoxide, N,N-dimethylformamide, ethanol, ethylene glycol, and tetrahydrofuran.

10. The preparation method according to claim 8, characterized in that, The film-forming process can be any one of dip coating, stretch coating, or spin coating; The working fluid of the membrane layer is used to form 1 to 10 stackable membrane layers with a thickness of 5 to 20 μm through a membrane formation process, which serve as biocompatible membranes.

11. The preparation method according to claim 10, characterized in that, The film formation process is dip coating, with a descent speed of 1000~6000um / s and a lifting speed of 400~3000um / s. The lifting is repeated 3~8 times. After the lifting is repeated, the film is dried at room temperature or under vacuum for 1~24 hours to obtain an outer film layer with a thickness of 5~20 μm, thus obtaining a coated sample.

12. The preparation method according to claim 11, characterized in that, During the dip coating process, the descent speed is 3000~4000um / s, the lifting speed is 1500~2000um / s, the lifting is repeated 4~5 times, and vacuum drying is carried out for 1~2 hours to obtain the final product.

13. An analyte sensor, comprising a substrate, an enzyme layer disposed above the substrate, the substrate comprising at least a reference electrode and a counter electrode; the enzyme layer comprising at least a first working electrode and a second working electrode, the first working electrode comprising an enzyme targeting any one of lactic acid, glucose, β-hydroxybutyric acid, alcohol, uric acid, cortisol, and ascorbic acid; the second working electrode comprising an enzyme targeting the other one of lactic acid, glucose, β-hydroxybutyric acid, alcohol, uric acid, cortisol, and ascorbic acid; characterized in that, Above the enzyme layer, there is an outer membrane layer covering the first working electrode and the second working electrode. The outer membrane layer is a biocompatible membrane according to any one of claims 1-7, or a biocompatible membrane prepared by the preparation method according to any one of claims 8-12.

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