Biosensor polymer outer membrane and preparation method thereof

Abstract

The invention belongs to the technical field of medical instruments, and discloses a biosensor polymer outer membrane and a preparation method thereof, and a biosensor comprising the outer membrane. The outer film structure comprises a double-layer structure of an inner layer film and a surface layer film, the inner layer film and the surface layer film form an integral structure in a chemical cross-linking mode, the inner layer film is of a continuous compact structure or a microporous structure and is 10-100 microns thick, the surface layer film is 1-20 microns thick, and the outer layer film is of a hollow structure. The hydration performance, the anti-interference performance, the biocompatibility and the like of the existing biosensor can be effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of medical device technology, specifically relating to a biosensor outer membrane and its preparation method, as well as a biosensor containing the outer membrane. Background Technology

[0002] Biosensors are devices that convert the concentration of a analyte into a detectable signal by utilizing the specific recognition function of biomolecules, and they have wide applications in the medical and health field. Compared with in vitro monitoring, implantable flexible sensors provide real-time, accurate, and continuous vital sign information, and have broader application prospects in medical diagnosis, health management, and drug development. However, their widespread application still faces challenges related to accuracy and stability. Inflammatory reactions and fibrotic encapsulation after sensor implantation are among the important factors leading to a decrease in the accuracy and stability of implantable biosensors.

[0003] The outer membrane of a sensor, in direct contact with the analyte and the testing environment, is a crucial interface component for the long-term, accurate operation of implantable biosensors. Its core function is to regulate the mass transfer process of target molecules and to block the adsorption of interfering substances and non-specific biomolecules. For implantable biosensors, the structure and performance of the outer membrane directly affect the accuracy and stability of the sensor during long-term operation in vivo.

[0004] Currently, commercial and cutting-edge research primarily utilizes polymer materials based on polyurethane (TPU), Nafion, etc., to construct the outer membrane of sensors. The performance of these membranes is then modulated through mixing or composite methods. They have a certain application basis in regulating small molecule mass transfer and electrochemical stability. However, under long-term implantation conditions, they are still susceptible to protein adsorption, biocontamination, and changes in the local microenvironment, leading to a decline in sensing performance.

[0005] As an unavoidable challenge for implantable biosensors, the acute inflammatory response induced after implantation and the subsequent formation of fibrotic encapsulation alter the mass transfer conditions and local physicochemical environment (such as pH and oxygen partial pressure) around the outer membrane, leading to signal drift and measurement errors. Furthermore, existing outer membrane structures struggle to achieve an effective balance between permeability to target molecules such as glucose, interference resistance, rapid response performance, long-term stability, and biocompatibility, limiting further improvements in the overall performance of implantable biosensors.

[0006] Zwitterionic materials possess superhydrophilic and electrically neutral properties. The zwitterions on their surface can form strong hydrogen bonds with water molecules, creating a tight hydration layer that acts as a physical barrier. This effectively resists the adsorption and deposition of proteins on the sensor surface, reduces foreign body reactions, and minimizes the adhesion of macrophages and fibroblasts, ensuring the accuracy, stability, and lifespan of implantable biosensors. Multilayer membrane systems, with their functional decoupling and synergistic optimization characteristics, can overcome the performance limitations of single materials, further enhancing sensor performance.

[0007] Therefore, developing an outer membrane system that ensures effective delivery of target molecules while also taking into account resistance to biocontamination, structural stability, and biocompatibility is of great significance for improving the long-term reliability of implantable biosensors. Summary of the Invention

[0008] To address the problems existing in the prior art, the present invention aims to provide a biosensor outer membrane and its preparation method, as well as a biosensor comprising the outer membrane structure. This outer membrane structure can effectively improve the hydration performance, anti-interference performance, and biocompatibility of current biosensors.

[0009] According to one aspect of the present invention, an object of the present invention is to provide a biosensor outer membrane, said outer membrane being a bilayer structure comprising an inner membrane and a surface membrane, wherein the inner membrane and the surface membrane are formed into an integral structure by chemical cross-linking, the inner membrane being a continuous dense structure or a microporous structure with a thickness of 10 to 100 μm, and the surface membrane having a thickness of 1 to 20 μm. The inner membrane has a polymer structure represented by the following general formula 1: General Formula 1 Where the subscripts x, y, z, and m represent the molar content of each block, not their positional relationship within the polymer molecule. The ratio of x : y : m is 1-10 : 1-10 : 0-1, and z is less than x. When the structure... When it does not exist, z is zero; when the structure does not exist, z is zero. When m does not exist, m is zero.

[0010] Group A is selected from 5-12 membered heterocyclic groups containing 1-3 nitrogen atoms or substituted C2-C6 alkenyl groups. In substituted C2-C6 alkenyl groups, "substituted" means containing 1-3 substituents. The substituents are selected from amino, carboxyl, hydroxyl, ester, acid anhydride, mercapto, and isocyanate groups. When group A is located in the middle of the molecular chain, it is the corresponding subgroup structure. Group B is selected from C2-C6 alkenyl groups containing 1-3 substituents selected from carboxyl, sulfonic acid, phosphoric acid and sulfinic acid groups; Group C is selected from bromopolyacid alkane structures, cyclic sulfonyl lactone structures, or copolymers of small molecule compounds.

[0011] Preferably, group C is selected from 3-bromopropanephosphine, 3-bromopropionic acid, 2-bromoethylphosphine, 4-bromobutylphosphine, 1,3-propanesulfonyl lactone, 1,4-butanesulfonyl lactone, 2-bromoacetic acid sulfonyl lactone, or a copolymer of any one of polyethylene glycol glycidyl ether, 1,4-butanediol diglycidyl ether, triethylene glycol diglycidyl ether, and neopentyl glycol diglycidyl ether with any one of any one of acids selected from 1,4-amino-1,3-benzenedisulfonic acid, 2,2-amino-4,6-disulfonicobenzoic acid, 3,5-amino-2,4-disulfonicobenzenesulfonamide, and aniline disulfonic acid.

[0012] The weight-average molecular weight of the inner membrane polymer is 200,000 to 1,000,000, preferably 300,000 to 500,000; the number-average molecular weight of the inner membrane polymer is 100,000 to 500,000, preferably 200,000 to 300,000; the molecular weight distribution of the inner membrane polymer can be 1.5 to 2.5. The surface membrane has the polymer structure represented by the following structural formula 2: General Formula 2 in, The subscripts x, y, and z represent the molar content of each block, not their positional relationship within the polymer molecule. The x:y:z ratio is 1-10:1-10:0-1. When z does not exist, z is zero.

[0013] The group O is selected from C2-C6 alkenyl groups containing active groups; Group P is selected from groups containing a betaine structure; Group Q is selected from hydrophilic olefin structures.

[0014] In general formula 1: Preferably, the subscripts x : y : m are 4-8 : 1-9 : 0-1.

[0015] Preferably, group A is selected from 5-12 membered heterocyclic groups containing 1 or 2 nitrogen atoms or substituted C2-C4 alkenyl groups. In substituted C2-C4 alkenyl groups, "substituted" means containing 1 or 2 substituents, which are selected from amino, carboxyl, hydroxyl, ester, acid anhydride, mercapto, and isocyanate groups.

[0016] Preferably, group A is selected from, but not limited to, pyridine, bipyridine, imidazole, biimidazole, maleic anhydride, allylamine, butenamine, allyl alcohol, propenol, butenol, transbutenic acid, itaconic acid, allyl thiol, 3-mercaptoacrylic acid, vinyl isocyanate, and ethyl isocyanate acrylate.

[0017] Preferably, group B is selected from C2-C4 alkenyl groups containing one or two substituents selected from carboxyl, sulfonic acid, phosphoric acid and sulfinic acid groups; Preferably, group B is selected from C2-C3 alkenyl groups containing one or two substituents selected from carboxyl, sulfonic acid, phosphoric acid and sulfinic acid groups; Preferably, group B is selected from vinyl sulfonic acid, allyl sulfonic acid, acrylic acid, crotonic acid, maleic acid, vinyl phosphoric acid, and vinyl sulfinic acid.

[0018] Preferably, group C is selected from copolymers of 3-bromopropanephosphine, 1,3-propanesulfonic acid lactone, polyethylene glycol glycidyl ether, and aniline disulfonic acid.

[0019] Preferably, the inner membrane polymer structure is selected from the following structures: Structural Formula 1 The molar ratio of 4-vinylpyridine (x) to styrene (y) is 5:5 to 9:1, the molar ratio of polyethylene glycol glycidyl ether to aniline disulfonic acid is 1:1, and the amount of polyethylene glycol glycidyl ether / aniline disulfonic acid is 5% to 20% of the mass of poly(4-vinylpyridine-co-styrene). Structural Formula 2 The molar ratio of butenamine (x): styrene (y): allyl sulfonic acid (m) is 5:4:1 to 8:1:1; or Structural Formula 3 The molar ratio of 4-vinylpyridine (xz): styrene (y): maleic acid (m) is 5:4:1 to 8:1:1, and the amount of 3-bromopropanephosphoric acid is 1% to 10% of the mass of poly(4-vinylpyridine-co-styrene-co-maleic acid).

[0020] In general formula 2: Preferably, x : y : z is 4-8 : 1-9 : 1.

[0021] Preferably, the group O is selected from glycidyl methacrylate, glycidyl acrylate, allyl glycidyl ether, 2-glycidyloxyethyl methacrylate, 3,4-epoxycyclohexyl methyl methacrylate, glycidyloxyphenyl methacrylate, tetrahydrofurfuryl glycidyl methacrylate, bisphenol A diglycidyl methacrylate, isocyanate methacrylate, isocyanate acrylate, vinyl isocyanate, hydroxyethyl methacrylate, 2-carboxyethyl acrylate, N-hydroxymethylacrylamide, N,N-dimethylaminoethyl methacrylate, N,N-diethylaminoethyl methacrylate, 2-mercaptoethyl methacrylate, 3-mercaptopropyl methacrylate, and 4-mercapto-1-butene.

[0022] Preferably, the group P is selected from polycarboxybetaine, polysulfobetaine, phosphatidylcholine, etc., such as methacryloyloxyethyl phosphorylcholine, methacryloyloxyethyl sulfobetaine, methacryloyloxyethyl carboxybetaine, and allyl phosphorylcholine.

[0023] Preferably, the group Q is selected from polyethylene glycol methacrylate, polyethylene glycol acrylate, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, polyethylene glycol methyl ether acrylate, ethoxylated hydroxyethyl methacrylate, and ethoxylated bisphenol A diacrylate.

[0024] Preferably, the surface membrane polymer structure is selected from the following structures: Structural Formula 4 The molar ratio of glycidyl methacrylate (x) to sulfobetaine (y) is 5:5 to 1:9; or Structural Formula 5 The molar ratio of glycidyl methacrylate (x): phosphatidylcholine (y): polyethylene glycol methacrylate (z) is 4:5:1 to 1:8:1.

[0025] The weight-average molecular weight of the surface membrane polymer is 20,000 to 100,000, preferably 20,000 to 50,000; the number-average molecular weight of the surface membrane polymer is 10,000 to 50,000, preferably 10,000 to 20,000; and the molecular weight distribution of the surface membrane polymer can be 1.5 to 2.5.

[0026] Preferably, the inner membrane polymer structure and the outer membrane polymer structure can be prepared by conventional polymerization methods.

[0027] According to another aspect of the present invention, another object of the present invention is to provide a method for preparing the outer membrane of the biosensor, comprising the following steps: Step 1. Dissolve the polymer represented by structural formula 1 prepared above in a mixed solvent of ethanol and 10 mM HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) to form a solution with a mass concentration of 20-80 mg / ml, wherein the volume ratio of ethanol to 10 mM HEPES is 10:1 to 1:1, and label it as solution A; Step 2. Dissolve the crosslinking agent in a mixed solvent of ethanol and 10 mM HEPES to form a solution with a mass concentration of 20-100 mg / ml, wherein the volume ratio of ethanol to 10 mM HEPES is 10:1 to 1:1, and label it as solution B. Then mix solution A and solution B at a volume ratio of 10:1, coat the mixture onto the electrode, and cure for 15 to 60 minutes. Step 3. Dissolve the polymer represented by structural formula 2 prepared above in a mixed solvent of ethanol and 10 mM HEPES to form a solution with a mass concentration of 14-60 mg / ml, wherein the volume ratio of ethanol to 10 mM HEPES is 1:10 to 1:1, and label it as solution C. Coat solution C onto the electrode after curing in step 2. Step 4. Place the electrode in a constant temperature and humidity chamber for crosslinking at 20°C and 60% for 48 hours. After the polymer solution has crosslinked on the electrode, remove the electrode from the crosslinking chamber, immerse it in PBS solution for 2 hours, and then transfer it to an electrochemical testing device to test its linear response in glucose solution.

[0028] Preferably, in step 1), the volume ratio of ethanol to 10 mM HEPES is 5:1 to 1:1, more preferably 3:1.

[0029] Preferably, the mass concentration of the polymer represented by structural formula 1 in step 1) is 20-80 mg / ml, more preferably 60 mg / ml.

[0030] Preferably, in step 2), the volume ratio of ethanol to 10 mM HEPES is 1:5 to 1:1, more preferably 1:3.

[0031] Preferably, the mass concentration of the polymer represented by structural formula 1 in step 2) is 50-100 mg / ml, more preferably 80 mg / ml.

[0032] Preferably, the crosslinking agent in step 2) includes, but is not limited to, polyethylene glycol glycidyl ether, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, neopentyl glycol diglycidyl ether, glycerol triglycidyl ether, polypropylene glycol diglycidyl ether, and mannitol diglycidyl ether.

[0033] Preferably, in step 3), the volume ratio of ethanol to 10 mM HEPES is 1:3 to 1:1, more preferably 1:1.

[0034] Preferably, in step 3), the mass concentration of the polymer represented by structural formula 1 is 80-120 mg / ml, more preferably 100 mg / ml.

[0035] According to another aspect of the invention, another object of the invention is to provide a biosensor comprising the outer membrane.

[0036] Beneficial effects The biosensor membrane prepared in this invention employs a multilayer membrane structure, combining glucose flow limitation, interference resistance, and good biocompatibility. The surface layer of the outer membrane contains zwitterionic structures, endowing the sensor with excellent hydration, biocompatibility, and antifouling properties. The inner membrane introduces cross-linking sites, while the surface membrane provides reactive groups. Through covalent cross-linking, the surface membrane uniformly covers the inner membrane, forming a stable three-dimensional network structure and achieving stable construction of the multilayer membrane structure. Attached Figure Description

[0037] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 This is a schematic diagram of the outer membrane structure of the biosensor according to the present invention.

[0039] Figure 2 This is a graph showing the electrode's response to glucose concentration.

[0040] Figure 3 The study demonstrated that the protein adsorption density of the sensor was significantly reduced after coating the outer membrane.

[0041] Figure 4 The infrared spectra are used to characterize the chemical structure and characteristic functional groups of the polymers prepared in Examples 1 and 4.

[0042] Figure 5 for Figure 4 A magnified view of the rectangular marked area. Detailed Implementation

[0043] The present invention will now be described in detail. Before proceeding with the description, it should be understood that the terminology used in this specification and the appended claims should not be construed as limited to its general or dictionary meaning, but rather should be interpreted according to the meaning and concept corresponding to the technical aspects of the invention, based on the principle that the inventors are allowed to appropriately define the terms for the best interpretation. Therefore, the description presented herein is merely a preferred example for illustrative purposes and is not intended to limit the scope of the invention. It should be understood that other equivalents or modifications can be obtained from it without departing from the spirit and scope of the invention.

[0044] In this document, the terms “comprising,” “including,” “having,” “containing,” or any other similar terms are open-ended conjunctions intended to cover non-exclusive inclusions. For example, a composition or article containing a plurality of elements is not limited to those listed herein, but may also include other elements not explicitly listed but typically inherent to the composition or article. Furthermore, unless explicitly stated to the contrary, the term “or” is inclusive, not exclusive. For example, the condition “A or B” is satisfied in any of the following cases: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); A and B are both true (or exist). Moreover, in this document, the terms “comprising,” “including,” “having,” and “containing” should be interpreted as specifically disclosed and simultaneously cover closed or semi-closed conjunctions such as “composed of” and “substantially composed of.”

[0045] In this document, all features or conditions defined in the form of numerical ranges or percentage ranges are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible secondary ranges and individual values ​​within those ranges, particularly integer values. For example, a range description of "1 to 8" should be considered as specifically disclosing all secondary ranges such as 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8, etc., particularly secondary ranges defined by all integer values, and should be considered as specifically disclosing individual values ​​within those ranges such as 1, 2, 3, 4, 5, 6, 7, 8, etc. Unless otherwise specified, the foregoing interpretation applies to all content throughout this invention, regardless of its scope.

[0046] If a quantity or other numerical value or parameter is expressed as a range, a preferred range, or a series of upper and lower limits, it should be understood that this document has specifically disclosed all ranges consisting of any upper or preferred value of that range and the lower or preferred value of that range, regardless of whether such ranges are separately disclosed. Furthermore, when a range of numerical values ​​is mentioned herein, unless otherwise stated, the range shall include its endpoints and all integers and fractions within the range.

[0047] In this document, numerical values ​​are to be understood as having a precision with significant digits, provided that the purpose of the invention can be achieved. For example, the number 40.0 should be understood to cover a range from 39.50 to 40.49.

[0048] In this invention, the outer membrane of the biosensor employs a multilayer membrane structure system. The surface membrane structure is rich in zwitterionic ions, providing hydration performance, antifouling performance, and biocompatibility, while the inner membrane structure provides flow restriction and anti-interference functions. Through synergistic optimization and covalent bonding of the multilayer membrane functions, the anti-interference performance, biocompatibility, and stability of the existing biosensor outer membrane are improved, extending the sensor's lifespan.

[0049] The inner membrane is a polymer film layer located outside the sensor's functional layer. It has a continuous, dense structure or a microporous structure with a thickness of 10–100 μm. Active groups for cross-linking reactions are introduced onto the surface or interior of the inner membrane. These active groups include, but are not limited to, amino, carboxyl, hydroxyl, thiol, and epoxy groups. The inner membrane serves as a cross-linking site providing layer, offering stable and controllable reaction sites and a suitable chemical environment for subsequent chemical cross-linking reactions, thereby ensuring the stability and selectivity of interlayer bonding. Furthermore, the inner membrane also plays a fundamental role in regulating the mass transfer process of target molecules and acts as a barrier against some interfering substances.

[0050] The surface membrane, disposed outside the inner membrane, is a polymer membrane layer with cross-linking capabilities. The surface membrane itself contains active groups capable of participating in cross-linking reactions. These active groups can chemically cross-link with the active groups on the inner membrane, thereby forming a stable three-dimensional cross-linked network structure between the layers. The surface membrane primarily serves to construct the interface between the sensor and the biological environment. Its cross-linked structure is preferentially distributed on the surface region of the outer membrane to enhance the overall structural stability and interface durability of the outer membrane, and to impart good anti-interference performance and biocompatibility to the outer membrane.

[0051] The inner and outer membranes can be constructed into an integral outer membrane structure through sequential coating, interface-induced crosslinking, or in-situ crosslinking, so as to achieve synergistic optimization of the multilayer membrane in terms of structural stability, mass transfer regulation, and biocompatibility.

[0052] Polymer molecular weight and molecular weight distribution were analyzed using a Waters high-performance gel permeation chromatography system, with a calibration curve based on standard polystyrene. Elution was performed using the corresponding mobile phase system, and M was collected and calculated. n M w The polydispersity index (PDI) was used; the chemical structure and characteristic functional groups of the polymer were characterized using a Thermo Fisher Fourier transform infrared spectroscopy (FT-IR) instrument.

[0053] All reagents were purchased from Sigma or Aladdin.

[0054] The following embodiments are merely examples illustrating implementations of the present invention and do not constitute any limitation on the present invention. Those skilled in the art will understand that modifications made without departing from the spirit and concept of the present invention fall within the protection scope of the present invention. Unless otherwise specified, the reagents and instruments used in the following embodiments are commercially available products.

[0055] Example 1: Preparation of compounds of structural formula 1 Structural Formula 1 Step 1: Using azobisisobutyronitrile (AIBN) as an initiator, the free radical polymerization of 4-vinylpyridine and styrene was initiated to obtain a poly(4-vinylpyridine-co-styrene) compound. The molar ratio of 4-vinylpyridine to styrene was 9:1, the amount of AIBN was 2% of the total monomer mass, and purified water was used as the solvent. The reaction was carried out at 60°C for 24 h under nitrogen atmosphere. After the reaction, the compound was first dissolved in ethanol, then co-precipitated twice using an ethanol / purified water system, and finally dried under vacuum.

[0056] Step 2: Polyethylene glycol glycidyl ether and aniline disulfonic acid were reacted in an ethanol / 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid solution at 55°C for 72 h. The molar ratio of polyethylene glycol glycidyl ether to aniline disulfonic acid was 1:1, and the volume ratio of ethanol to 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid was 4:1.

[0057] Step 3: Dissolve the poly(4-vinylpyridine-co-styrene) obtained in Step 1 in ethanol and add it to the reaction solution from Step 2. Continue the reaction for 72 h. The amount of polyethylene glycol glycidyl ether and aniline disulfonic acid copolymer used is 10% of the molar amount of 4-vinylpyridine. After the reaction is complete, precipitate with purified water first, then co-precipitate and purify twice using an ethanol / purified water system, and finally perform vacuum drying.

[0058] In structural formula 1, the subscript (xz): z: y is approximately equal to 8.1: 0.9: 1. The subscript ratio here only indicates the molar ratio of each polymer unit and does not represent the specific degree of polymerization.

[0059] The obtained polymer had a weight-average molecular weight of 433,000, a number-average molecular weight of 219,000, and a molecular weight distribution of 2.0.

[0060] Example 2: Preparation of the compound of structural formula 2 Structural Formula 2 A poly(butenylamine-co-styrene-co-allylsulfonic acid) compound was obtained by free radical polymerization of butenylamine, styrene, and allylsulfonic acid using azobisisobutyronitrile (AIBN) as an initiator. The molar ratio of AIBN, styrene, and allylsulfonic acid was 8:1:1, and the amount of AIBN was 2% of the total mass of the monomers. Purified water was used as the solvent. The reaction was carried out at 60°C for 24 h under nitrogen atmosphere. After the reaction, the compound was first dissolved in ethanol, then co-precipitated twice using an ethanol / purified water system, and finally dried under vacuum.

[0061] In structural formula 2, the subscripts x : y : m are approximately equal to 8 : 1 : 1. The subscript ratio here only indicates the molar ratio of each polymer unit and does not represent the specific degree of polymerization.

[0062] The obtained polymer had a weight-average molecular weight of 405,000, a number-average molecular weight of 214,000, and a molecular weight distribution of 1.9.

[0063] Example 3: Preparation of compounds with structural formula 3 Structural Formula 3 Step 1: Using azobisisobutyronitrile (AIBN) as an initiator, free radical polymerization of 4-vinylpyridine, styrene, and maleic acid was initiated to obtain a poly(4-vinylpyridine-co-styrene-co-maleic acid) compound, wherein the molar ratio of 4-vinylpyridine, styrene, and maleic acid was 8:1:1, the amount of AIBN was 2% of the total monomer mass, purified water was used as the solvent, and the reaction was carried out at 60°C for 24 h under nitrogen atmosphere. After the reaction, the compound was first dissolved in ethanol, then co-precipitated twice using an ethanol / purified water system, and finally vacuum dried for later use. In the second step, poly(4-vinylpyridine-co-styrene-co-maleic acid) was dissolved in ethanol, and then 3-bromopropane phosphoric acid was added for copolymerization to obtain the target product. The amount of 3-bromopropane phosphoric acid used was 5% of the molar amount of 4-vinylpyridine, and the reaction was carried out at 80°C for 24 h under nitrogen atmosphere. After the reaction, the product was first precipitated with purified water, then co-precipitated twice using an ethanol / purified water system, and finally dried under vacuum.

[0064] In structural formula 3, the subscript (xz): z: y: m is approximately equal to 7.6: 0.4: 1: 1. The subscript ratio here only indicates the molar ratio of each polymer unit and does not represent the specific degree of polymerization.

[0065] The obtained polymer had a weight-average molecular weight of 490,000, a number-average molecular weight of 300,000, and a molecular weight distribution of 1.6.

[0066] Example 4: Preparation of the compound of structure 4 Structural Formula 4 The target product was obtained by free radical polymerization of glycidyl methacrylate and sulfobetaine using azobisisobutyronitrile (AIBN) as an initiator. The molar ratio of AIBN to sulfobetaine was 3:7, the amount of AIBN was 2% of the total monomer mass, and the solvent was purified water / isopropanol (volume ratio of purified water to isopropanol was 1:1). The reaction was carried out at 70°C for 24 h under nitrogen atmosphere. After the reaction, the product was first precipitated with tetrahydrofuran, then co-precipitated twice using a tetrahydrofuran / purified water system, and finally freeze-dried.

[0067] In structural formula 4, the subscript x : y is approximately equal to 3 : 7. The subscript ratio here only indicates the molar ratio of each polymer unit and does not represent the specific degree of polymerization.

[0068] The obtained polymer had a weight-average molecular weight of 22,000, a number-average molecular weight of 13,000, and a molecular weight distribution of 1.7.

[0069] Example 5: Preparation of the compound of structural formula 5 Structural Formula 5 The target product was obtained by free radical polymerization of glycidyl methacrylate, phosphatidylcholine, and polyethylene glycol methacrylate using azobisisobutyronitrile (AIBN) as an initiator. The molar ratio of AIBN to Glycidyl methacrylate, phosphatidylcholine, and polyethylene glycol methacrylate was 2:7:1, the amount of AIBN was 2% of the total monomer mass, and the solvent was purified water / isopropanol (volume ratio of purified water to isopropanol was 1:1). The reaction was carried out at 70°C for 24 h under nitrogen atmosphere. After the reaction, the product was first precipitated with tetrahydrofuran, then co-precipitated twice using a tetrahydrofuran / purified water system, and finally freeze-dried.

[0070] In structural formula 5, the subscripts x : y : z are approximately equal to 2 : 7 : 1. The subscript ratio here only indicates the molar ratio of each polymer unit and does not represent the specific degree of polymerization.

[0071] The obtained polymer had a weight-average molecular weight of 26,000, a number-average molecular weight of 15,000, and a molecular weight distribution of 1.7.

[0072] Figure 4 The infrared spectra characterizing the chemical structure and characteristic functional groups of the polymers prepared in Examples 1 and 4 are shown. The spectra of Example 1 show that the structure at 1596 cm⁻¹... -1 1554 cm -1 1412 cm -1 and 821 cm -1 A distinct characteristic absorption peak appears at 910 cm⁻¹, which can be attributed to the characteristic vibrations of the pyridine ring and its nitrogen atom. The structure in Example 4 shows an absorption peak at 910 cm⁻¹.-1 The characteristic stretching vibration absorption peak of the typical epoxy group appears at the location. The pyridine nitrogen in the structure of Example 1 provides crosslinking sites, and the epoxy group in the structure of Example 4 provides reactivity. Through covalent crosslinking, the surface film is uniformly covered on the inner film, thereby forming a stable three-dimensional network structure and realizing the stable construction of the multilayer film structure.

[0073] Preparation Example 1 Preparation and coating of the outer membrane solution: 1) Inner membrane: The polymer prepared in Example 1 was dissolved in ethanol and 10 mM HEPES solution at a mass concentration of 60 mg / ml (V 乙醇 V HEPES =3∶1), labeled as solution A; 2) Dissolve the crosslinking agent polyethylene glycol diglycidyl ether at a mass concentration of 80 mg / ml in ethanol and 10 mM HEPES solution (V 乙醇 V HEPES The ratio of A to B is 3:1, and this solution is labeled as solution B. Then, solutions A and B are mixed evenly at a volume ratio of 10:1 and coated onto the electrode. 3) Surface film: The polymer prepared in Example 4 above was dissolved in ethanol and 10mM HEPES solution at a mass concentration of 100 mg / ml (V 乙醇 ∶ V HEPES =1∶1), labeled as solution C, and coated onto the electrode containing the inner membrane; 4) After the polymer solution has cross-linked on the electrode, the sensor electrode is obtained, immersed in PBS solution, and continuous performance testing is performed.

[0074] Test Example 1: Response Test to Glucose Concentration The performance of the electrodes was tested using a Chenhua electrochemical workstation. 1) Select the it (current-time curve) test mode; 2) Set the test parameters: operating voltage 0.04V, sampling interval 0.1s, running time 10000s; 3) At 37℃, the steady-state current response of electrodes without outer membrane coating and with outer membrane coating was tested at glucose concentrations of 0, 2, 5, 10, 15, 20, and 25 mM, respectively. 4) After the test, record the response current at the corresponding concentration and plot it to compare its linear range. Performance test results: as follows Figure 2 As shown, the sensing electrode coated with the outer film exhibits good linear response over a wide range of glucose concentrations.

[0075] Test Example 2: Anti-protein adsorption capacity test The protein density adsorbed on the outer membrane surface was determined using the micro-BCA method: BSA was diluted to 500 μg / mL with sterile PBS, and then... 2 The BSA protein solution was uniformly coated on the electrode surface and allowed to freely adsorb for 2 hours at 37 °C. Then, the unadsorbed BSA protein solution was removed by pipetting.

[0076] 1) Prepare protein standard curve: Dilute the 2 mg / uL standard BSA to 0, 100 ug / mL, 200 ug / mL, 300 ug / mL, 400 ug / mL, and 500 ug / mL, and take 100 uL in a 96-well plate; 2) Take 100 μL of the unadsorbed BSA protein solution and put it into a 96-well plate; 3) Prepare micro-BCA working solution: Mix micro-BCA working solution evenly in the following ratio (A∶B∶C=25∶24∶1); 4) BSA protein solution reaction: Add 100 μL of the prepared micro-BCA working solution to each well of a 96-well plate containing diluted protein standard solution and unadsorbed BSA protein solution, and incubate at 37°C for 1 h. 5) Measure absorbance: After the temperature returns to room temperature, use an ELISA reader to record the absorbance value at a wavelength of 570 nm.

[0077] Protein adsorption density test results: as follows Figure 3 As shown, the protein adsorption density of the sensor is significantly reduced after coating with the outer membrane.

[0078] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A biosensor outer membrane, wherein the outer membrane is a bilayer structure comprising an inner membrane and a surface membrane, the inner membrane and the surface membrane being chemically cross-linked to form an integral structure, the inner membrane being a continuous dense structure or a microporous structure with a thickness of 10 to 100 μm, and the surface membrane having a thickness of 1 to 20 μm. in, The inner membrane has a polymer structure represented by the following general formula 1: General Formula 1 Where the subscripts x, y, z, and m represent the molar content of each block, not their positional relationship within the polymer molecule. The ratio of x : y : m is 1-10 : 1-10 : 0-1, and z is less than x. When the structure... When it does not exist, z is zero; when the structure does not exist, z is zero. When m does not exist, m is zero; Group A is selected from 5-12 membered heterocyclic groups containing 1-3 nitrogen atoms or substituted C2-C6 alkenyl groups. In substituted C2-C6 alkenyl groups, the substitution refers to the presence of 1-3 substituents. The substituents are selected from amino, carboxyl, hydroxyl, ester, acid anhydride, mercapto, and isocyanate groups. When group A is located in the middle of the molecular chain, it is the corresponding subgroup structure. Group B is selected from C2-C6 alkenyl groups containing 1-3 substituents selected from carboxyl, sulfonic acid, phosphoric acid and sulfinic acid groups; Group C is selected from bromopolyacid alkane structures, cyclic sulfonyl lactone structures, or copolymers of small molecule compounds; The surface film has a polymer structure represented by the following structural formula 2: General Formula 2 in, The subscripts x, y, and z represent the molar content of each block, not their positional relationship within the polymer molecule. The x:y:z ratio is 1-10:1-10:0-1. When z does not exist, z is zero; The group O is selected from C2-C6 alkenyl groups containing active groups; Group P is selected from groups containing a betaine structure; Group Q is selected from hydrophilic olefin structures.

2. The biosensor outer membrane according to claim 1, characterized in that, In general formula 1: The subscripts x : y : m are 4-8 : 1-9 : 0-1; Group A is selected from 5-12 membered heterocyclic groups containing 1 or 2 nitrogen atoms or substituted C2-C4 alkenyl groups. In substituted C2-C4 alkenyl groups, the substitution refers to the presence of 1 or 2 substituents, and the substituents are selected from amino groups, carboxyl groups, hydroxyl groups, ester groups, acid anhydrides, mercapto groups and isocyanate groups. Alternatively, group A is selected from pyridine, bipyridine, imidazole, biimidazole, maleic anhydride, allylamine, butenamine, allyl alcohol, propenol, butenol, trans-butenic acid, itaconic acid, allyl thiol, 3-mercaptoacrylic acid, vinyl isocyanate, ethyl isocyanate acrylate. Group B is selected from C2-C4 alkenyl groups containing one or two substituents selected from carboxyl, sulfonic acid, phosphoric acid and sulfinic acid groups; Alternatively, group B is selected from C2-C3 alkenyl groups containing one or two substituents selected from carboxyl, sulfonic acid, phosphoric acid and sulfinic acid groups; Alternatively, group B is selected from vinyl sulfonic acid, allyl sulfonic acid, acrylic acid, crotonic acid, maleic acid, vinyl phosphoric acid, and vinyl sulfinic acid; Group C is selected from 3-bromopropanephosphine, 3-bromopropionic acid, 2-bromoethylphosphine, 4-bromobutylphosphine, 1,3-propanesulfonyl lactone, 1,4-butanesulfonyl lactone, 2-bromoacetic acid sulfonyl lactone, or a copolymer of any ether selected from polyethylene glycol glycidyl ether, 1,4-butanediol diglycidyl ether, triethylene glycol diglycidyl ether, and neopentyl glycol diglycidyl ether with any acid selected from 1,4-amino-1,3-benzenedisulfonic acid, 2,2-amino-4,6-disulfonicobenzoic acid, 3,5-amino-2,4-disulfonicobenzenesulfonamide, and aniline disulfonic acid; Alternatively, group C is selected from copolymers of 3-bromopropanephosphine, 1,3-propanesulfonic acid lactone, polyethylene glycol glycidyl ether, and aniline disulfonic acid.

3. The biosensor outer membrane according to claim 1, characterized in that, The inner membrane polymer structure is selected from the following structures: Structural Formula 1 The molar ratio of 4-vinylpyridine (x) to styrene (y) is 5:5 to 9:1, the molar ratio of polyethylene glycol glycidyl ether to aniline disulfonic acid is 1:1, and the amount of polyethylene glycol glycidyl ether / aniline disulfonic acid is 5% to 20% of the mass of poly(4-vinylpyridine-co-styrene). Structural Formula 2 The molar ratio of butenamine (x): styrene (y): allyl sulfonic acid (m) is 5:4:1 to 8:1:1; or Structural Formula 3 The molar ratio of 4-vinylpyridine (xz): styrene (y): maleic acid (m) is 5:4:1 to 8:1:1, and the amount of 3-bromopropanephosphoric acid is 1% to 10% of the mass of poly(4-vinylpyridine-co-styrene-co-maleic acid).

4. The biosensor outer membrane according to claim 1, characterized in that, The weight-average molecular weight of the inner membrane polymer is 200,000 to 1,000,000, or 300,000 to 500,000; the number-average molecular weight of the inner membrane polymer is 100,000 to 500,000, or 200,000 to 300,000; and the molecular weight distribution of the inner membrane polymer is 1.5 to 2.

5.

5. The biosensor outer membrane according to claim 1, characterized in that, In general formula 2: The x : y : z ratio is 4-8 : 1-9 : 1; The group O is selected from glycidyl methacrylate, glycidyl acrylate, allyl glycidyl ether, 2-glycidyloxyethyl methacrylate, 3,4-epoxycyclohexyl methyl methacrylate, glycidyloxyphenyl methacrylate, tetrahydrofurfuryl glycidyl methacrylate, bisphenol A diglycidyl methacrylate, isocyanate methacrylate, isocyanate acrylate, vinyl isocyanate, hydroxyethyl methacrylate, 2-carboxyethyl acrylate, N-hydroxymethylacrylamide, N,N-dimethylaminoethyl methacrylate, N,N-diethylaminoethyl methacrylate, 2-mercaptoethyl methacrylate, 3-mercaptopropyl methacrylate, and 4-mercapto-1-butene. The group P is selected from polycarboxybetaine, polysulfobetaine, and phosphatidylcholine, such as methacryloyloxyethylphosphatidylcholine, methacryloyloxyethylsulfobetaine, methacryloyloxyethylcarboxybetaine, and allylphosphatidylcholine. Group Q is selected from polyethylene glycol methacrylate, polyethylene glycol acrylate, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, polyethylene glycol methyl ether acrylate, ethoxylated hydroxyethyl methacrylate, and ethoxylated bisphenol A diacrylate.

6. The biosensor outer membrane according to claim 1, characterized in that, The surface membrane polymer structure is selected from the following structures: Structural Formula 4 The molar ratio of glycidyl methacrylate (x) to sulfobetaine (y) is 5:5 to 1:9; or Structural Formula 5 The molar ratio of glycidyl methacrylate (x): phosphatidylcholine (y): polyethylene glycol methacrylate (z) is 4:5:1 to 1:8:

1.

7. The biosensor outer membrane according to claim 1, characterized in that, The weight-average molecular weight of the surface membrane polymer is 20,000 to 100,000, or 20,000 to 50,000; the number-average molecular weight of the surface membrane polymer is 10,000 to 50,000, or 10,000 to 20,000; and the molecular weight distribution of the surface membrane polymer is 1.5 to 2.

5.

8. A method for preparing the outer membrane of a biosensor according to any one of claims 1 to 7, comprising the following steps: Step 1. Dissolve the polymer represented by structural formula 1 in a mixed solvent of ethanol and 10 mM HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) to form a solution with a mass concentration of 20-80 mg / ml, wherein the volume ratio of ethanol to 10 mM HEPES is 10:1 to 1:1, and label it as solution A; Step 2. Dissolve the crosslinking agent in a mixed solvent of ethanol and 10 mM HEPES to form a solution with a mass concentration of 20-100 mg / ml, wherein the volume ratio of ethanol to 10 mM HEPES is 10:1 to 1:1, and label it as solution B. Then mix solution A and solution B at a volume ratio of 10:1, coat the mixture onto the electrode, and cure for 15 to 60 minutes. Step 3. Dissolve the polymer represented by structural formula 2 in a mixed solvent of ethanol and 10 mM HEPES to form a solution with a mass concentration of 14-60 mg / ml, wherein the volume ratio of ethanol to 10 mM HEPES is 1:10 to 1:1, and label it as solution C. Coat solution C onto the electrode after curing in step 2. Step 4. Place the electrode in a constant temperature and humidity chamber for crosslinking at 20°C and 60% for 48 hours. After the polymer solution has crosslinked on the electrode, remove the electrode from the crosslinking chamber, immerse it in PBS solution for 2 hours, and then transfer it to an electrochemical testing device to test its linear response in glucose solution.

9. The method for preparing the outer membrane of the biosensor according to claim 8, characterized in that, In step 1), the volume ratio of ethanol to 10 mM HEPES is 5:1 to 1:1, or 3:

1. In step 1), the mass concentration of the polymer represented by structural formula 1 is 20-80 mg / ml, or 60 mg / ml; In step 2), the volume ratio of ethanol to 10 mM HEPES is 1:5 to 1:1, or 1:

3. In step 2), the mass concentration of the polymer represented by structural formula 1 is 50-100 mg / ml, or 80 mg / ml; The crosslinking agent mentioned in step 2) includes polyethylene glycol glycidyl ether, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, neopentyl glycol diglycidyl ether, glycerol triglycidyl ether, polypropylene glycol diglycidyl ether, and mannitol diglycidyl ether. In step 3), the volume ratio of ethanol to 10 mM HEPES is 1:3 to 1:1, or 1:

1. In step 3), the mass concentration of the polymer represented by structural formula 1 is 80-120 mg / ml, more preferably 100 mg / ml.

10. A biosensor comprising a biosensor outer membrane according to any one of claims 1 to 7.

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