Polymer and preparation method therefor, porous membrane comprising polymer, and use of membrane

Abstract

The present application provides a polymer and a preparation method therefor, a porous membrane comprising the polymer, and a use of the membrane. The polymer comprises three or more structural units, and the polymer comprises at least one of the following: a structural unit of a compound represented by general formula I; a structural unit of a styrenic compound; and a structural unit of a (meth)acrylic compound. The polymer comprises optionally a structure represented by formula II, wherein "iii" represents a bond to N on a pyridine ring or an imidazole ring. N on a heterocyclyl-substituted vinyl monomer represented by formula I of the present application can react with a halide or an active ester to obtain a porous polymer membrane material made of an amphoteric compound or a cationic compound.

Description

A polymer, a method for preparing the polymer, a porous membrane comprising the polymer, and applications of the membrane.

[0001] This application claims priority to an earlier application filed on July 8, 2024, with patent application number 2024109047308, entitled "A polymer, a method for preparing the same, a porous membrane comprising the polymer, and an application of the membrane". The entire contents of the earlier application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biosensor technology, specifically relating to a polymer, its preparation method, a porous membrane containing the polymer, and the application of the membrane. Background Technology

[0003] A biosensor is an instrument that detects biological substances by converting their concentration into an electrical signal. It is an analytical tool or system consisting of immobilized biologically sensitive materials as recognition elements (including bioactive substances such as enzymes, antibodies, antigens, microorganisms, cells, tissues, and nucleic acids), appropriate physicochemical transducers (such as oxygen electrodes, phototubes, field-effect transistors, piezoelectric crystals, etc.), and signal amplification devices. Biosensors function as both receivers and transducers, and are typically used to rapidly detect specific chemical substances in the human body, such as glucose, uric acid, lactic acid, blood ketones, and a range of other compounds. Current-analytical biosensors usually employ two or three electrodes, including at least one measuring or working electrode and one reference electrode. In a two-electrode system, the reference electrode also acts as the counter electrode. In a three-electrode system, the third electrode is the reverse electrode. The measuring or working electrode is composed of corrosion-resistant carbon or a metallic conductor and is connected to the reference electrode via a circuit (e.g., a potentiostat).

[0004] In 1967, SJ Updick et al. developed the first biosensor, a glucose sensor. This was achieved by encapsulating glucose oxidase in a polyacrylamide colloidal membrane, solidifying it, and then fixing this membrane to the tip of a membrane oxygen electrode. By using other enzymes or microorganisms to solidify the membrane, other sensors that detect their corresponding substances can be developed.

[0005] In enzyme-based biosensors, polymer semi-permeable membranes are an important component. Hydrophilic polymer membrane materials can serve as an electrochemical reaction layer immobilizing enzymes and mediators, and can also control the diffusion rate of analytes from the external environment to the sensor electrode surface.

[0006] In implantable current-type sensors, polymeric semi-permeable membranes are often beneficial or necessary for regulating or limiting the flow of analyte to the sensing layer. In sensors without membranes, the flux of analyte to the sensing layer increases linearly with analyte concentration. When all analyte reaching the sensing layer is consumed, the measured output signal is linearly proportional to the flux, and thus linearly proportional to the analyte concentration. However, when analyte consumption is kinetically limited by chemical or electrochemical activity in the sensing layer, the measured output signal is no longer controlled by the analyte flux and is no longer linearly proportional to the flux or concentration. In this case, only a small fraction of the analyte reaching the sensing layer is consumed before the sensor saturates, so the measured signal stops increasing or increases only slightly with analyte concentration. On the other hand, in sensors equipped with diffusion-limiting membranes, the membrane reduces the flux of analyte to the sensing layer, preventing the sensor from saturating and thus allowing for efficient operation over a wider concentration range.

[0007] In implantable current sensors, the polymer semipermeable membrane needs to be in direct contact with the human body and immersed in the physiological environment for a long time. This requires the membrane material to have good biocompatibility to prevent rejection reactions that the organism cannot accept. At the same time, implantable biosensors will rub against surrounding tissues such as muscles during use. If the adhesion of the hydrophilic polymer semipermeable membrane to the hydrophobic biosensor surface is insufficient to overcome this friction, it will greatly reduce the stability of the sensor, thus causing potential problems such as membrane material peeling.

[0008] Currently, much research has been conducted on the outer membrane of biosensors, mainly focusing on how to obtain membrane materials with excellent performance in various aspects and their application scenarios. Many researchers have focused on using polymers with single monomers or diblock polymers for subsequent research and applications, but no examples of using polymers with more than three blocks in membrane materials have yet been found. Summary of the Invention

[0009] The purpose of this application is to provide a polymer, a method for preparing the polymer, a porous polymer membrane material containing the polymer and the method for preparing the polymer, and the application of the porous polymer membrane material on the outer membrane of a biosensor. The polymer has good physical properties and good biocompatibility, making it suitable for biosensors.

[0010] The technical solution of this application is as follows:

[0011] A polymer comprising three or more structural units:

[0012] The polymer comprises at least one structural unit of a compound represented by the following general formula I:

[0013] In formula I, Ra R b and R c Whether they are the same or different, they are independently selected from H and C. 1-6 Alkyl, sulfonic acid, ether, or ester substituents;

[0014] R d The ring is a substituted or unsubstituted imidazole or pyridine ring; the substitution or unsubstituent refers to the presence or absence of substituents on the carbon atoms of the imidazole or pyridine ring, wherein the substituents are selected from C14. 1-6 Alkyl, C 1-6 Alkoxy, sulfonic acid, sulfonic acid amino, amino, methylamino, dimethylamino, ethylamino, propylamino, or butylamino;

[0015] The structural unit of styrene compounds;

[0016] And, the structural units of (meth)acrylic acid compounds;

[0017] The polymer may optionally include the structure shown in Formula II:

[0018] Where A is O, N, or C; and B is O, N, or C;

[0019] R3 and R4 may be the same or different, and are independently selected from H, CH3, OMe, and SO3. 2- CO2 2- PO3 2- NR5R6; R5 and R6 may be the same or different, and are independently selected from H and C. 1-6 alkyl;

[0020] x is a number between 0 and 10; y is a number between 0 and 10;

[0021] This represents the bond connecting to the N atom on the pyridine or imidazole ring.

[0022] According to an embodiment of this application, preferably, the monomer comprising the structural unit shown in Formula I is selected from at least one of 4-vinylpyridine, 2-vinylpyridine and 3-vinylpyridine, 1-vinylimidazole, 2-vinylimidazole and 4-vinylimidazole.

[0023] According to the embodiments of this application, preferably, the styrene compound is selected from at least one of styrene, 2-methylstyrene, 3-methylstyrene, 4-methylstyrene, o-methoxystyrene, m-methoxystyrene, p-methoxystyrene, and m-sulfonated styrene.

[0024] According to an embodiment of this application, preferably, the (meth)acrylic acid compound is selected from at least one of (meth)acrylates, (meth)acrylic acid, (meth)acrylamide, (meth)propenol, (meth)acrylate ethoxylate, 2-methacryloyloxyethyl phosphocholine, and (meth)acrylate hydroxyalkyl esters. The (meth)acrylate may be selected from (meth)acrylate C 1-6 Alkyl esters, such as methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, n-propyl acrylate, n-propyl methacrylate, isopropyl acrylate, isopropyl methacrylate, n-butyl acrylate, n-butyl methacrylate, isobutyl acrylate, isobutyl methacrylate, n-pentyl acrylate, n-pentyl methacrylate, isopentyl acrylate, isopentyl methacrylate, n-hexyl acrylate, n-hexyl methacrylate, etc. (Meth)acrylate hydroxyalkyl esters can be selected from (meth)acrylate C. 1-6 Hydroxyalkyl esters, such as hydroxyethyl acrylate, hydroxyethyl methacrylate, hydroxypropyl acrylate, hydroxypropyl methacrylate, etc.

[0025] According to the embodiments of this application, the sulfonic acid substituent is selected from one of methanesulfonic acid group, ethylsulfonic acid group, propylsulfonic acid group, isopropylsulfonic acid group or butylsulfonic acid group.

[0026] According to the embodiments of this application, the ether substituent is selected from one of methoxyether, ethoxyether, phenyl ether, benzyl ether, methyl thioether or ethyl thioether.

[0027] According to an embodiment of this application, the ester substituents are selected from C 1-6 Alkyl ester group, such as methoxymethyl ester, ethoxymethyl ester, n-propoxymethyl ester, and isopropoxymethyl ester.

[0028] According to an embodiment of this application, the polymer is selected from one of the following structural formulas:

[0029] Where R is the structure shown in Equation II; x represents the degree of polymerization, which is a number between 100 and 10000;

[0030] a, b, c, and d are all integers between 1 and 1000.

[0031] The polymers used in this application may be random copolymers, block copolymers, or alternating copolymers.

[0032] The weight-average molecular weight of the polymers in this application can be 300,000 to 800,000, preferably 350,000 to 700,000. The number-average molecular weight of the polymers in this application can be 150,000 to 400,000, preferably 200,000 to 400,000. The molecular weight distribution of the polymers in this application can be 1.5 to 2.5.

[0033] This application also provides a method for preparing the above-mentioned polymer, wherein the method may optionally include:

[0034] Step (1) involves mixing and reacting the above-mentioned at least three monomers and initiator in a solvent to prepare the polymer.

[0035] According to the embodiments of this application, the method may optionally include step (2): reacting the polymer obtained in step (1) with a compound with the structure shown in Formula III to prepare a polymer containing the group shown in Formula II;

[0036] Where A is O, N, or C; and B is O, N, or C;

[0037] R3 and R4 may be the same or different, and are independently selected from H, CH3, OMe, and SO3. 2- CO2 2- PO3 2- NR5R6; R5 and R6 may be the same or different, and are independently selected from H and C1-C6 alkyl groups;

[0038] x is a number between 0 and 10; y is an integer between 0 and 10;

[0039] R is a leaving group, such as Cl, Br, I, methanesulfonyl (OMs) or toluenesulfonyl (OTs), etc.

[0040] According to embodiments of this application, the compound with the structure shown in Formula III is selected from 2-(2-methoxyethoxy) halogens or sulfonates or haloalkanes or halogenated carbonates or halogenated phosphates, such as 2-(2-methoxyethoxy) halogens or sulfonates or haloalkanes or halogenated carbonates or halogenated phosphates, such as 1,3-propanesulfonate lactone, diethylene glycol monotoluenesulfonate or 1-bromo-2-(2-methoxyethoxy)ethane, methyl 1-bromo-2-(2-methoxyethoxy)acetate or trimethyl 1-chloro-2-(2-methoxyethoxy)phosphoate.

[0041] According to the embodiments of this application, the initiator is selected from at least one of azobisisobutyronitrile, benzoyl peroxide, or potassium persulfide.

[0042] According to the embodiments of this application, the solvent is selected from at least one of ethanol, isopropanol, toluene, ethyl acetate, or N,N-dimethylformamide and water. The content of the solvent is not particularly limited, as long as it is sufficient to ensure uniform mixing of the raw materials.

[0043] According to an embodiment of this application, the initiator has a mass of 0.1-1% of the total mass of at least three monomers.

[0044] According to the implementation scheme of this application, in step (1), the reaction temperature is 35-110℃; the reaction time is 6-48h, preferably 6-24h.

[0045] According to the implementation scheme of this application, in step (2), the temperature of the reaction is 35-110℃; the reaction time is 6-48h, preferably 6-24h.

[0046] According to the implementation scheme of this application, in step (2), the mass ratio of the polymer and the compound with the structure shown in formula III in step (1) is 5-100:1.

[0047] According to the embodiments of this application, the method further includes a post-processing step, such as dissolving, filtering, and drying the prepared product.

[0048] As an exemplary embodiment of this application, the method for preparing the polymer specifically includes:

[0049] Mix the above-mentioned at least three monomers with a solvent, then add an initiator at a mass ratio of 0.1-1% based on the at least three monomers, purge with nitrogen three times under reduced pressure, heat to 35-110°C, and mechanically stir to react; after the reaction is complete, cool to room temperature, dilute with 5-30 times the amount of ethanol, and add the resulting solution dropwise to methyl tert-butyl ether to precipitate a solid. Filter the solid through a 200-mesh filter cloth, collect the solid, and dry it at 50°C for 48 hours. Dissolve the collected dry material again with ethanol, add it dropwise to pure water, filter, and then dry it at 50°C for 48 hours. After that, pulverize it with a pulverizer, filter it through a 200-mesh sieve, and vacuum dry the resulting powder at 50°C for 48 hours. Collect the polymer.

[0050] This application also provides a porous polymer membrane material containing the above-mentioned polymer.

[0051] According to the embodiments of this application, the polymer membrane material further includes a low molecular weight polymer.

[0052] According to the embodiments of this application, the low molecular weight polymer is selected from polyoxypropylene-polyoxyethylene block copolymer (Planic), carbomer (carboxyethylene copolymer), polyetheramine, polydimethylsiloxane (PDMS), polyethylene glycol diglycidyl ether, polyacrylate, hydroxypropyl methylcellulose, povidone, Dowsil 57 non-reactive ethylene glycol copolymer surfactant, and Dowsil 205SL 50% silicone polyether copolymer.

[0053] According to the embodiments of this application, the mass of the low molecular weight polymer is 0.005-20 wt% of the mass of the porous polymer membrane material.

[0054] Preferably, the specific information of the low molecular weight polymer is shown in the table below:

[0055] According to the embodiments of this application, the low molecular weight polymer is preferably a mixture of polyetheramine and polyethylene glycol diglycidyl ether, with a mass ratio of 4-10:1.

[0056] This application also provides a method for preparing the above-mentioned porous polymer membrane material, the method comprising:

[0057] The polymer is mixed and reacted with a low molecular weight polymer in a solvent to obtain the porous polymer membrane material.

[0058] According to the embodiments of this application, the reaction temperature is 60℃-120℃; the reaction time is 2-24h.

[0059] According to the embodiments of this application, the method specifically involves: mixing and reacting a low molecular weight polymer with a mixed solution of HEPES aqueous solution and ethanol, and then mixing the resulting product with the polymer to prepare a porous polymer membrane material.

[0060] In this application, the content of the solvent is not particularly limited, as long as it is sufficient to dissolve the reactants.

[0061] According to the embodiments of this application, the method further includes a post-processing step, such as washing, filtering, and drying the prepared product.

[0062] As an exemplary embodiment of this application, the method for preparing the porous polymer membrane material is more specifically as follows:

[0063] (S1) At room temperature, the polymer is dissolved by stirring with N,N-dimethylformamide. After dissolution, 1,3-propanesulfonic acid lactone is added, and the temperature is raised to 60℃-120℃ for 12h. After the reaction is completed, ethanol is added for dilution. The resulting solution is added dropwise to purified water, and a solid is precipitated. After filtration, the solid is dried under vacuum to obtain the zwitterionic outer membrane material of the polymer, i.e., the porous polymer membrane material.

[0064] As an exemplary embodiment of this application, the method for preparing the porous polymer membrane material is specifically as follows:

[0065] (S1) At room temperature, the polymer is dissolved by stirring with N,N-dimethylformamide. After dissolution, 2-(2-methoxyethoxy) halogenated derivatives, sulfonates, halogenated alkanes, carbonates of halogenated derivatives, or phosphate esters of halogenated derivatives are added. The temperature is raised to 60℃-100℃ and reacted for 12 hours. After the reaction is completed, ethanol is added for dilution. The resulting solution is added dropwise to methyl tert-butyl ether, filtered, and vacuum dried to obtain the outer membrane material of the cationic product, i.e., the porous polymer membrane material.

[0066] As an exemplary embodiment of this application, the method for preparing the porous polymer membrane material is specifically as follows:

[0067] (S2) The low molecular weight polymer is added to a mixed solution of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) and ethanol (the concentration of the low molecular weight polymer in the mixed solution is 10-30 M / V%). The reaction is carried out at 50°C for 72 hours. After the reaction is completed, the temperature is lowered to room temperature. At room temperature, the resulting solution is added to the ethanol solution of the polymer. The reaction is carried out at 55°C for 48 hours. The temperature is then lowered to room temperature, diluted with ethanol, and added dropwise to water. A solid is precipitated. The solid is collected after filtration with a filter cloth and dried with a forced air for 48 hours. The resulting solid is then pulverized with a pulverizer and filtered through a 200-mesh sieve. The resulting powder is vacuum dried at 50°C for 48 hours and the product is collected, which is the porous polymer membrane material.

[0068] This application also provides a biosensor, which includes a conductive substrate and a sensing membrane coated on the surface of the conductive substrate, wherein the sensing membrane includes the aforementioned porous polymer membrane material and a responsive enzyme.

[0069] According to the embodiments of this application, the responding enzyme includes any one of glucose oxidase, lactate oxidase, L-glutamate oxidase or xanthine oxidase.

[0070] According to an embodiment of this application, the step of forming a sensing film on the surface of the conductive substrate includes:

[0071] A coating reagent is prepared, wherein the coating reagent comprises at least the responsive enzyme, the electron transport agent, the porous polymer membrane material, and the crosslinking agent; and...

[0072] The coating agent is applied to the surface of the conductive substrate and then cured.

[0073] Preferably, the curing temperature is 20-60℃.

[0074] According to embodiments of this application, the crosslinking agent includes, but is not limited to, polyethylene glycol diglycidyl ether (PEGDGE), glutaraldehyde, or carbodiimide.

[0075] According to the embodiments of this application, the electron transport agent can be a compound with redox properties, such as a transition metal complex or quinone, for example, selected from osmium metal complexes (e.g., a tridentate osmium metal complex with bimidazole ligand).

[0076] According to an embodiment of this application, the sensing membrane further includes an enzyme stabilizer, such as human serum albumin, catalase, or trehalose.

[0077] According to an embodiment of this application, the coating agent further includes a low molecular weight polymer.

[0078] According to an embodiment of this application, in the coating reagent, the mass of the low molecular weight polymer is 0.005-20 wt% of the mass of the porous polymer membrane material.

[0079] According to the embodiments of this application, the coating agent further includes small molecule additives, which are selected from plasticizers or anti-glycation agents;

[0080] The plasticizer is selected from triethyl citrate (with a weight-average molecular weight of 276) or acetyl tributyl citrate (with a weight-average molecular weight of 402).

[0081] The anti-glycolytic agent is selected from mannitol (with a weight-average molecular weight of 182) or sodium DL-glyceraldehyde fluoride.

[0082] According to the embodiments of this application, the mass of the small molecule additive is 0.1-20 wt% of the mass of the porous polymer membrane material.

[0083] According to the embodiments of this application, the shape of the coating agent when it is coated on the surface of the conductive substrate can be a straight line, a dashed line, a single point, multiple points, or a surface.

[0084] This application also provides the above-mentioned biosensors for applications including but not limited to the detection of glucose, lactic acid, blood ketones, uric acid, and cholesterol.

[0085] The beneficial effects of this application are:

[0086] The porous polymer membrane material of this application has both hydrophilic and hydrophobic structures. When applied to a biosensor, the polymer membrane formed by the combined action of the hydrophilic and hydrophobic structures has a complex network structure, which makes the polymer membrane have good adhesion and tensile properties on the surface of the biosensor. Moreover, the large-pore porous structure can control the diffusion of the analyte to the sensing layer of the biosensor at an appropriate concentration to meet the detection requirements.

[0087] The nucleophilicity of the N on the heterocyclic substituted vinyl monomer shown in Formula I of this application can react with halogenated compounds and active esters to obtain porous polymer membrane materials of amphoteric or cationic compounds.

[0088] The biosensor of this application can detect chemical substances in the physiological environment within the body. Its sensing membrane extends at least to the active region of the working electrode, effectively limiting analyte throughput, expanding the sensor's linear response range, and exhibiting good biocompatibility. This active region can be configured into different shapes through deposition, including but not limited to straight lines, dashed lines, squares, single-point, multi-point, or planar shapes, with a total area of ​​approximately 0.05-0.5 mm². 2 between. Attached Figure Description

[0089] Figure 1 is a cross-sectional SEM image of the coated material after molding in Application Example 1.

[0090] Figure 2 shows the time-current curve of the biosensor in Application Example 1.

[0091] Figure 3 is a graph showing the linear relationship between the current value and the glucose concentration in Application Example 1.

[0092] Figure 4 is a cross-sectional SEM image of the coated material after molding in Application Example 2.

[0093] Figure 5 shows the time-current curve of the biosensor in Application Example 2.

[0094] Figure 6 is a linear relationship between the current value of the biosensor and the glucose concentration in Application Example 2.

[0095] Figure 7 shows the current response values ​​of the biosensor in Application Example 3 at different lactic acid concentrations.

[0096] Figure 8 shows the current response values ​​of the biosensor in Application Example 4 at different lactic acid concentrations.

[0097] Figure 9 is a cross-sectional SEM image of the coated material after molding in Comparative Application Example 1.

[0098] Figure 10 shows the time-current curves of the biosensor in Comparative Application Example 1.

[0099] Figure 11 is a graph showing the linear relationship between current value and glucose concentration in Comparative Application Example 1. Detailed Implementation

[0100] The technical solutions of this application will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of this application, and should not be construed as limiting the scope of protection of this application. All technologies implemented based on the above content of this application are covered within the scope of protection intended by this application.

[0101] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0102] Example 1

[0103] Add 50 ml of N,N-dimethylformamide to a 500 ml three-necked flask, followed by 30 g of 4-vinylpyridine, 15 g of 1-vinylimidazole, 5 g of styrene, and 200 mg of azobisisobutyronitrile. After stirring and mixing thoroughly, purge the mixture three times with nitrogen to maintain a positive nitrogen pressure. Heat the mixture to 65 °C and stir mechanically for 20 h. After the reaction is complete, dissolve the solid in 200 ml of ethanol and add 200 mg of hydroquinone. After complete dissolution, add the reaction mixture dropwise to 5 L of methyl tert-butyl ether. The precipitated solid is filtered through a 200-mesh filter cloth, collected, and dried under forced air at 50 °C for 48 h. The collected dry material was dissolved again with ethanol (200 mL) and then added dropwise to 4 L of pure water. The solid precipitated, filtered, and then dried in a forced-air condition at 50 °C for 48 hours. After that, it was pulverized by a pulverizer and filtered through a 200-mesh sieve. The resulting powder was vacuum dried at 50 °C for 48 hours and the product was collected as PVP-PS-PVI (45 g, yield 90%).

[0104] MW: 370436; Mn: 211709; PDI: 1.75

[0105] Example 2

[0106] Add 60 ml of N,N-dimethylformamide to a 250 ml flask, add 10 g of PVP-PS-PVI from Example 1, stir to dissolve at room temperature, add 1.5 g of 1,3-propanesulfonic acid lactone, heat to 60 °C and react for 12 h, after the reaction is complete, add 35 ml of ethanol to dilute, add the resulting solution dropwise to 6 L of purified water, filter and vacuum dry to obtain the outer membrane material (10 g) of the zwitterionic product of white product PVP-PS-PVI, i.e., porous polymer membrane material.

[0107] Example 3

[0108] Add 60 ml of N,N-dimethylformamide to a 250 ml flask, then add 10 g of PVP-PS-PVI from Example 1. Stir at room temperature to dissolve. After dissolution, add 1 g of diethylene glycol mono-p-toluenesulfonate. Heat to 90 °C and react for 12 h. After the reaction is complete, add 150 ml of ethanol to dilute. Add the resulting solution dropwise to 3 L of methyl tert-butyl ether. After filtration, vacuum dry to obtain the outer membrane material (10 g) of the white product PVP-PS-PVI cationic product.

[0109] Example 4

[0110] Add 60 ml of N,N-dimethylformamide to a 250 ml flask, then add 5 g of PVP-PS-PVI from Example 2. Stir and dissolve at room temperature. After dissolution, add 0.6 g of 1-bromo-2-(2-methoxyethoxy)ethane. Heat to 90 °C and react for 12 h. After the reaction is complete, add 25 ml of ethanol for dilution. Add the resulting solution dropwise to 500 mL of methyl tert-butyl ether. After filtration, vacuum dry to obtain the outer membrane material (5.2 g) of the zwitterionic cationic product of white product PVP-PS-PVI.

[0111] Example 5

[0112] Add 50 ml of N,N-dimethylformamide to a 500 ml three-necked flask, followed by 70 g of 4-vinylpyridine, 10 g of 1-vinylimidazole, 10 g of styrene, and 10 g of methyl methacrylate. Add 100 mg of azobisisobutyronitrile (AIBN) and stir thoroughly until homogeneous. After mixing, purge the mixture three times with nitrogen to maintain a positive nitrogen pressure. Heat to 75 °C and stir mechanically for 20 h. After the reaction is complete, dissolve the solid in 400 ml of ethanol and add 200 mg of hydroquinone. After complete dissolution, add the reaction mixture dropwise to 7.5 L of methyl tert-butyl ether. The precipitated solid is filtered through a 200-mesh filter cloth, collected, and dried at 50 °C for 48 h. The collected dry material was dissolved again with ethanol (400 mL) and then added dropwise to 6 L of pure water. The solid precipitated, filtered, and then dried in a forced-air condition at 50 °C for 48 hours. After that, it was pulverized by a pulverizer and filtered through a 200-mesh sieve. The resulting powder was vacuum dried at 50 °C for 48 hours and the product was collected as PVP-PS-PVI-PMMA (80 g, yield 90%).

[0113] MW: 589779; Mn: 277586; PDI: 2.12

[0114] Example 6

[0115] Add 60 ml of N,N-dimethylformamide to a 250 ml flask, then add 30 g of PVP-PS-PVI-PMMA from Example 5. Stir at room temperature to dissolve, then add 3 g of 1,3-propanesulfonic acid lactone. Heat to 60 °C and react for 12 h. After the reaction is complete, add 270 ml of ethanol to dilute. Add the resulting solution dropwise to 10 L of purified water, filter, and vacuum dry to obtain 30 g of the white product PVP-PS-PVI-PMMA zwitterionic outer membrane material.

[0116] Example 7

[0117] Polyetheramine (4.4 g) and polyethylene glycol diglycidyl ether (1.1 g) were added to a mixed solution of 0.5% HEPES aqueous solution (10 ml) and ethanol (40 ml). The reaction was carried out at 50 °C for 72 hours. After the reaction was completed, the solution was cooled to room temperature. The resulting solution was added to an ethanol (60 ml) solution of PVP-PS-PVI-PMMA zwitterionic outer membrane material (30 g) in Example 6 at room temperature. The reaction was carried out at 55 °C for 48 hours. The solution was then cooled to room temperature, diluted with ethanol (200 ml), and added dropwise to 3 L of water. A solid precipitated out. The solid was collected after filtration with a filter cloth and dried by forced air for 48 hours. The resulting solid was then pulverized with a pulverizer and filtered through a 200-mesh sieve. The resulting powder was vacuum dried at 50 °C for 48 hours. The product was collected as PVP-PS-PVI-PMMA polymer outer membrane material (30 g).

[0118] Example 8

[0119] The difference between Example 8 and Example 2 is that 2.6g of methyl 1-bromo-2-(2-methoxyethoxy)acetate was used instead of 1.5g of 1,3-propanesulfonic acid lactone.

[0120] Example 9

[0121] The difference between Example 9 and Example 2 is that 2.5g of methyl 1-chloro-2-(2-methoxyethoxy)phosphate is used instead of 1.5g of 1,3-propanesulfonic acid lactone.

[0122] Example 10

[0123] In a 2L three-necked flask, 150g of polyethylene glycol (PEG400), 1000mL of dichloromethane, and 59.6g of p-toluenesulfonyl chloride were added sequentially. The mixture was cooled to 0°C in an ice bath, and then 31.6g of triethylamine was added dropwise. After the addition was complete, the reaction was allowed to proceed overnight. The mixture was diluted with 6L of ethyl acetate, then washed three times with 1M HCl and once with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated, and the concentrate was crystallized from petroleum ether and ethyl acetate to give 72g of a white solid product, which was polyethylene glycol mono-p-toluenesulfonate.

[0124] Biosensors

[0125] Application Example 1

[0126] A biosensor is used to detect glucose in the human body at a low potential. The biosensor contains a functional glucose oxidase.

[0127] A biosensor, the coating material of which is prepared using the materials and proportions shown in Tables 1, 2, and 3 below. The specific method is as follows:

[0128] At room temperature, the components in Table 2 were added to an aqueous solution of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) and an ethanol solution (volume ratio of 20:80), stirred and dissolved. After dissolution, the low molecular weight polymer PDMS and crosslinking agent PEGDGE2000 in Table 2 were added, and after ultrasonic dissolution, the coating reagent A of the outer membrane solution was obtained.

[0129] At room temperature, the components in Table 3 were added to an aqueous solution of 4-hydroxyethylpiperazine ethanesulfonic acid and an ethanol solution (volume ratio of 20:80), stirred and dissolved. After dissolution, the low molecular weight polymer PDMS and crosslinking agent PEGDGE2000 in Table 3 were added, and after ultrasonic dissolution, the coating reagent B of the outer membrane solution was obtained.

[0130] The responsive enzyme and mediator prepared according to the formulation in Table 1 were coated with the reagent to deposit 8 spots (approximately 0.02 mm each). 2 The coating is applied to the surface of the working electrode of the biosensor using the following method. After the active area is cured, the coating reagent of the above outer membrane solution is repeatedly applied to the active area of ​​the biosensor through immersion coating. The specific coating method is as follows: First, complete two immersion coatings using coating reagent A of the outer membrane solution in Table 2, and cure at 25°C for 30 min; Second, complete four immersion coatings using coating reagent B of the outer membrane solution in Table 3, and cure at 25°C for 24 h, and then cure at 50°C for 48 h.

[0131] Table 1 Enzyme Solution Formulation

[0132] In Table 1, “concentration” refers to the concentration of each component in the HEPES aqueous solution.

[0133] Table 2 shows the coating reagent A for the outer membrane solution in Application Example 1.

[0134] Table 3 shows the coating reagent B for the outer membrane solution in Application Example 1.

[0135] In Tables 2 and 3, “concentration” refers to the concentration of each component in a mixed solution of ethanol and HEPES, where the volume ratio of ethanol to HEPES is 80:20.

[0136] Figure 1 is a cross-sectional SEM image of the coating material after molding in Application Example 1. Its pore structure is created by the aging process, and its pores are an important channel for glucose in the tissue fluid to enter the functional area of ​​the biosensor.

[0137] Figure 2 shows the time-current curve of the biosensor in Application Example 1. As can be seen from Figure 2, the current value is stable and does not decay for different concentrations of glucose solution, thus demonstrating the potential for fabrication into a biosensor.

[0138] Figure 3 shows the linear relationship between the current value of the biosensor and the glucose concentration in Application Example 1. The specific testing procedure was as follows: In a 100Mm PBS buffer solution at pH 7.4, with the working voltage set to 40mV, glucose solutions of different concentrations (2mM, 3mM, 5mM, 10mM, 15mM, 20mM, and 25mM) were added to the PBS buffer to achieve the response current of the biosensor at different glucose concentrations. The test results, as shown in Figure 3, demonstrate a good linear relationship between different current values ​​and different glucose concentrations, indicating a positive correlation between glucose concentration and current value. Different glucose concentrations can be inferred from different current values, thus demonstrating that the polymer of this application can be applied to biosensors.

[0139] Application Example 2

[0140] A biosensor is used to detect glucose in the human body at a low potential. The biosensor contains a functional glucose oxidase.

[0141] A biosensor, the coating material of which is prepared using the materials and proportions specified in Tables 1, 4, and 5. The specific method is as follows:

[0142] At room temperature, the components in Table 4 were added to an aqueous solution of 4-hydroxyethylpiperazine ethanesulfonic acid and an ethanol solution, stirred and dissolved. After dissolution, the low molecular weight polymer PDMS and crosslinking agent PEGDGE2000 in Table 4 were added. After ultrasonic dissolution, the coating reagent A of the outer membrane solution was obtained.

[0143] Similarly, following the steps above, prepare coating reagent B for the outer membrane solution according to the raw materials and their proportions in Table 5;

[0144] The coating reagents for the responsive enzymes and mediators, prepared according to the formulations in Table 1, were deposited onto the surface of the working electrode of the biosensor in a straight line approximately 1.5 mm long. After curing the active area, the active area on the biosensor was coated multiple times using coating reagents A and B of the outer membrane solution in Tables 4 and 5. The specific coating method was as follows: First, 2-3 immersion coatings were performed using coating reagent A of the outer membrane solution in Table 4, and cured at 25°C for 30 min. Second, 4-6 immersion coatings were performed using coating reagent B of the outer membrane solution in Table 5, and cured at 25°C for 24 h, followed by curing at 50°C for 48 h.

[0145] Table 4 shows the coating reagent A for the outer membrane solution in Application Example 2.

[0146] Table 5. Coating reagent B for the outer membrane solution in Application Example 2.

[0147] Figure 4 is a cross-sectional SEM image of the coating material after molding in Application Example 2. Its pore structure is created by the aging process, and its pores are an important channel for glucose in the tissue fluid to enter the functional area of ​​the biosensor.

[0148] Figure 5 shows the time-current curves of the biosensor used in Example 2. As can be seen from Figure 5, the current value remains stable and does not decay for different concentrations of glucose solution.

[0149] Figure 6 shows the linear relationship between the current value of the biosensor and the glucose concentration in Application Example 2. The specific testing procedure was as follows: In a 100Mm PBS buffer solution at pH 7.4, with the operating voltage set to 40mV, glucose solutions of different concentrations were added to the PBS buffer solution to achieve the response current of the biosensor at different glucose concentrations (2mM, 3mM, 5mM, 10mM, 15mM, 20mM, and 25mM). The test results are shown in Figure 6. The linear relationship between different current values ​​and different glucose concentrations is good, indicating a positive correlation between glucose concentration and current value. Therefore, different glucose concentrations can be inferred from the detected different current values. Thus, the polymer of this application can be applied to biosensors.

[0150] Application Example 3

[0151] The preparation of biosensors for detecting lactate is similar to that of corresponding glucose-responsive analyte sensors, except that a lactate-responsive enzyme is used instead of glucose oxidase. The lactate-responsive enzyme can be lactate oxidase.

[0152] A biosensor whose coating material is formulated using the materials and proportions in Tables 6, 4 and 5.

[0153] The responsive enzyme and mediator coating reagent prepared according to the formulation in Table 6 were deposited on the surface of the working electrode to form an area of ​​approximately 0.15 mm². 2 The dotted linear regions, the coating reagent A and coating reagent B of the outer membrane solution, and the coating method are the same as in application example 2.

[0154] Table 6 Enzyme Solution Formulation

[0155] Figure 7 shows the current response values ​​of the biosensor in Application Example 3 at different lactic acid concentrations (2 mM, 3 mM, 5 mM, 7 mM, 10 mM, 15 mM, 20 mM, and 25 mM). The specific testing procedure was as follows: the operating voltage was set to 40 mV, and the test was conducted at 37°C in 100 mM PBS buffer. Different concentrations of lactic acid solution were added to the PBS buffer to obtain the sensor's response current at different concentrations, showing a good linear relationship. The test results, as shown in Figure 7, demonstrate a good linear relationship between different current values ​​and different lactic acid concentrations, indicating a positive correlation between lactic acid concentration and current value. Different lactic acid concentrations can be inferred from the detected different current values, thus demonstrating that the polymer of this application can be applied in biosensors.

[0156] Application Example 4

[0157] The preparation of biosensors for detecting lactate is similar to that of corresponding glucose-responsive analyte sensors, except that a lactate-responsive enzyme is used instead of glucose oxidase. The lactate-responsive enzyme is lactate dehydrogenase.

[0158] A biosensor whose coating material is formulated using the materials and proportions in Tables 7, 4 and 5.

[0159] The responsive enzyme and mediator coating reagent prepared according to the formulation in Table 7 were deposited on the surface of the working electrode to form an area of ​​approximately 0.15 mm². 2 The square area, the coating reagent A and coating reagent B of the outer membrane solution, and the coating method are the same as in application example 2.

[0160] Table 7 Enzyme Solution Formulation

[0161] Figure 8 shows the current response of the biosensor in Application Example 4 at different lactate concentrations (2 mM, 3 mM, 5 mM, 7 mM, 10 mM, 15 mM, 20 mM, and 25 mM). The operating voltage was set to 40 mV, and the test was conducted at 37°C in 100 mM PBS buffer. Different concentrations of lactate solution were added to the PBS buffer to obtain the sensor's response current at different concentrations, showing a good linear relationship.

[0162] Comparative application example 1:

[0163] A biosensor is used to detect glucose in the human body at a low potential. The biosensor contains a functional glucose oxidase. It is prepared using the formulation ratios specified in Table 1.

[0164] A biosensor, the coating material of which is prepared using the materials and proportions shown in Table 1 and Table 8 below. The specific method is as follows:

[0165] At room temperature, the components in Table 8 were added to an aqueous solution of 4-hydroxyethylpiperazine ethanesulfonic acid and an ethanol solution (volume ratio of 5:95), stirred and dissolved. After dissolution, a crosslinking agent was added, and the solution was dissolved by ultrasonication to prepare the coating reagent for the outer membrane solution.

[0166] The responsive enzyme and mediator coating reagent, prepared according to the formulation in Table 1, were deposited on the surface of the biosensing electrode to a depth of approximately 0.02 mm. 2 A small dot of the desired size was applied to the surface of the biosensor's working electrode. After curing the active area, the active area on the biosensor was coated multiple times using the coating reagents for the outer membrane solution in Table 8. After the coating process, the biosensor was cured at 25°C and then at 50°C for 48 hours.

[0167] Table 8 Coating reagents for outer membrane solution

[0168] Figure 9 shows a cross-sectional SEM image of the membrane fluid after it has been formed in Comparative Application Example 1. Its pore structure is created by the aging process, and its pores are an important channel for glucose in the tissue fluid to enter the functional area of ​​the biosensor.

[0169] Figure 10 shows the time-current curves of the biosensor in Comparative Application Example 1. The sensor is made of poly(tetravinylpyridine-co-styrene) cross-linked with a cross-linking agent and uniformly coated on the surface of the biosensor. In 100 μM PBS buffer at pH 7.4, with the operating voltage set to 40 mV, glucose solutions of different concentrations were added to the PBS buffer to realize the response current of the biosensor at different sugar concentrations.

[0170] Figure 11 is a linear relationship graph between current value and glucose concentration in Comparative Application Example 1. The dashed line represents the simulated linear relationship graph, and the solid line represents the linear relationship graph of the measured values. Comparing the linear relationship graphs between current value and glucose concentration in Application Example 1 (i.e., Figure 3) and Comparative Application Example 1, it can be found that the linear relationship graph in Application Example 1 (i.e., Figure 3) shows a better linear relationship. When such a material with a good linear relationship is used as the outer membrane of a biosensor, the measured data will be more accurate, thus demonstrating better product performance.

[0171] The embodiments of this application have been described above by way of example. However, the scope of protection of this application is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A polymer, characterized in that, The polymer comprises three or more of the following structural units: The polymer comprises at least one structural unit of a compound represented by the following general formula I. In formula I, R a R b and R c Whether they are the same or different, they are independently selected from H and C. 1-6 Alkyl, sulfonic acid, ether, or ester substituents; R d The ring is a substituted or unsubstituted imidazole or pyridine ring; the substitution or unsubstituent refers to the presence or absence of substituents on the carbon atoms of the imidazole or pyridine ring, wherein the substituents are selected from C14. 1-6 Alkyl, C 1-6 Alkoxy, sulfonic acid, sulfonic acid amino, amino, methylamino, dimethylamino, ethylamino, propylamino, or butylamino; The structural unit of styrene compounds; And, (meth)acrylic acid compounds; The polymer may optionally include the structure shown in Formula II: Where A is O, N, or C; and B is O, N, or C; R3 and R4 may be the same or different, and are independently selected from H, CH3, OMe, and SO3. 2- CO2 2- PO3 2- NR5R6; R5 and R6 may be the same or different, and are independently selected from H and C. 1-6 alkyl; x is a number between 0 and 10; y is a number between 0 and 10; This represents the bond connecting to the N atom on the pyridine or imidazole ring.

2. The polymer according to claim 1, characterized in that, The compound represented by Formula I is selected from at least one of 4-vinylpyridine, 2-vinylpyridine and 3-vinylpyridine, 1-vinylimidazole, 2-vinylimidazole and 4-vinylimidazole; The styrene compounds are selected from at least one of styrene, 2-methylstyrene, 3-methylstyrene, 4-methylstyrene, o-methoxystyrene, m-methoxystyrene, p-methoxystyrene, and m-sulfonated styrene; The (meth)acrylic acid compounds are selected from at least one of (meth)acrylates, (meth)acrylic acid, (meth)acrylamide, (meth)propenol, (meth)acrylate ethoxylate, 2-methacryloyloxyethyl phosphate choline, and (meth)acrylate hydroxyalkyl ester. Preferably, the (meth)acrylate is selected from (meth)acrylate C. 1-6 Alkyl esters. Preferably, the (meth)acrylate hydroxyalkyl ester is selected from (meth)acrylate C 1-6 Hydroxyalkyl esters, hydroxyethyl acrylate, hydroxyethyl methacrylate, hydroxypropyl acrylate, hydroxypropyl methacrylate.

3. The polymer according to claim 1 or 2, characterized in that, The sulfonic acid substituent is selected from one of methanesulfonic acid group, ethylsulfonic acid group, propylsulfonic acid group, isopropylsulfonic acid group or butylsulfonic acid group; The ether substituent is selected from one of methoxyether, ethoxyether, phenyl ether, benzyl ether, methyl thioether or ethyl thioether; The ester substituents are selected from C. 1-6 Alkyl ester group, such as methoxymethyl ester, ethoxymethyl ester, n-propoxymethyl ester, and isopropoxymethyl ester.

4. A method for preparing the polymer according to any one of claims 1-3, characterized in that, The method includes: Step (1): Mix the above-mentioned at least three monomers and initiator in a solvent to prepare the polymer.

5. The method according to claim 4, characterized in that, The method further includes step (2): reacting the polymer obtained in step (1) with the compound shown in formula III to prepare a polymer containing the group shown in formula II; Where A is O, N, or C; and B is O, N, or C; R3 and R4 may be the same or different, and are independently selected from H, CH3, OMe, and SO3. 2- CO2 2- PO3 2- NR5R6; R5 and R6 may be the same or different, and are independently selected from H and C1-C6 alkyl groups; x is a number between 0 and 10; y is an integer between 0 and 10; R is a leaving group.

6. The method according to claim 5, characterized in that, The compound with the structure shown in Formula III is selected from 2-(2-methoxyethoxy) halogens or sulfonates or haloalkanes or halogenated carbonates or halogenated phosphates, specifically from 1,3-propanesulfonate lactone, diethylene glycol monotoluenesulfonate or 1-bromo-2-(2-methoxyethoxy)ethane, methyl 1-bromo-2-(2-methoxyethoxy)acetate or trimethyl 1-chloro-2-(2-methoxyethoxy)phosphoate.

7. A porous polymer membrane material, characterized in that, The porous polymer membrane material contains the polymer according to any one of claims 1-3.

8. A method for preparing the porous polymer membrane material according to claim 7, characterized in that, The method is as follows: the polymer is mixed and reacted with a low molecular weight polymer in a solvent to obtain the porous polymer membrane material. The low molecular weight polymer is selected from polyoxypropylene-polyoxyethylene block copolymer, carbomer, polyetheramine, polydimethylsiloxane, polyethylene glycol diglycidyl ether, polyacrylate, hydroxypropyl methylcellulose, povidone, Dowsil 57 non-reactive ethylene glycol copolymer surfactant, or Dowsil 205SL 50% silicone polyether copolymer.

9. A biosensor, characterized in that, It includes a conductive substrate and a sensing membrane coated on the surface of the conductive substrate, wherein the sensing membrane includes the porous polymer membrane material as described in claim 7 and a responsive enzyme.

10. The biosensor of claim 9, wherein the step of coating the sensing membrane onto the surface of the conductive substrate comprises: A coating reagent is prepared, wherein the coating reagent includes at least the responsive enzyme, the electron transport agent, the porous polymer membrane material, and the crosslinking agent; and The coating agent is applied to the surface of the conductive substrate and then cured. Preferably, the coating agent can be coated on the surface of the conductive substrate in the shape of a straight line, a dashed line, a single point, multiple points, or a surface.

11. The application of the biosensor according to claims 9 and 10 in the detection of glucose, lactic acid, blood ketones, uric acid and cholesterol.

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