Outer membrane of biosensor as well as preparation method and application of outer membrane

Abstract

The invention relates to the technical field of biosensors, in particular to an outer membrane of a biosensor and a preparation method and application of the outer membrane. According to the technical scheme, the outer membrane of the biosensor is provided, the provided outer membrane of the biosensor uses a three-dimensional complexing microcrystalline structure formed by the poly-L-lactic acid and the poly-D-lactic acid as a cross-linking structure, the formed cross-linking structure can increase the cross-linking density between membrane layers and improve the stability of a membrane material, and meanwhile, the polylactic acid has good biocompatibility, so that the biosensor has good biocompatibility. Compared with the prior art, the biosensor outer membrane has the advantages that the overall biocompatibility of the outer membrane material can be improved, and the formed cross-linked structure effectively avoids the use of a cross-linking agent harmful to a human body in a chemical cross-linked outer membrane formula, so that the obtained biosensor outer membrane is high in stability and safety, can accurately and continuously perform signal detection, and is beneficial to wide application.

Description

Technical Field

[0001] This application belongs to the technical field of biosensors, and particularly relates to an outer membrane of a biosensor, a preparation method thereof, and an application thereof. Background Art

[0002] A biosensor is an instrument that is sensitive to biological substances and converts their concentrations into electrical signals for detection. It is an analytical tool or system composed of an immobilized biological sensitive material as a recognition element (including biological active substances such as enzymes, antibodies, antigens, microorganisms, cells, tissues, nucleic acids, etc.), an appropriate physical and chemical transducer (such as an oxygen electrode, a photosensitive tube, a field effect transistor, a piezoelectric crystal, etc.), and a signal amplification device. A biosensor has the functions of a receiver and a converter.

[0003] One of the core components of a biosensor system based on the electrochemical method is an implanted / semi-implanted sensor, which usually consists of a conductive electrode, a sensing matrix layer participating in a specific reaction, and a polymer outer membrane that restricts the diffusion of the concentration of a specific substance. The polymer outer membrane is crucial for the detection range of the target substance concentration. However, in a complex physiological environment, there are risks such as damage and shedding of the outer membrane layer, which will affect the stability and accuracy of the continuous detection signal. Therefore, the preparation of the biosensor outer membrane needs to take into account both the linear detection range of the sensor and its own stability.

[0004] In order to provide the stability of the outer membrane, some solutions will choose to introduce a cross-linked structure into the polymer outer membrane. Although a structurally stable outer membrane with diffusion-limiting performance can be obtained after introducing the cross-linked structure, materials such as glutaraldehyde, which are commonly used cross-linking agents, are highly toxic and seriously affect the safety of the outer membrane. Moreover, most of the formed cross-linked structures are mainly chemical cross-links, and different cross-linking temperatures, cross-linking times, and the cross-linking ability of the cross-linking agent will all affect the density of the cross-linking points in the entire membrane layer; if the cross-linking density is too low, it will cause uneven coating of the sensor during manufacturing and affect the consistency of the sensor; if the cross-linking density is too high, the entire membrane solution will be in an insoluble and infusible gel-like structure, which will affect the modification of the polymer outer membrane layer and cause waste of materials. Therefore, there is an urgent need to provide an organic cross-linked outer membrane that can accurately and continuously detect signals, has high stability, and high safety. Summary of the Invention

[0005] The purpose of this application is to provide an outer membrane of a biosensor, a preparation method thereof, and an application thereof, aiming to solve the problem in the prior art that there is no organic cross-linked outer membrane that can accurately and continuously detect signals, has high stability, and high safety.

[0006] To achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] In a first aspect, the present application provides an outer membrane of a biosensor. The outer membrane includes a membrane skeleton constructed from a hydrophobic polymer, a flow-through channel constructed from a hydrophilic polymer, and a crosslinked structure. Among them, the crosslinked structure includes a stereocomplex microcrystalline structure formed by intermolecular hydrogen bonds between poly(L-lactic acid) and poly(D-lactic acid); the hydrophilic polymer is connected to the crosslinked structure by a covalent bond.

[0008] Furthermore, the covalent bond includes a carbon-carbon bond or a carbon-oxygen bond.

[0009] Furthermore, the stereocomplex microcrystalline structure includes stereocomplex microcrystalline crosslinking points and / or star-shaped crosslinking points.

[0010] Furthermore, the stereocomplex microcrystalline structure includes unit cells with different parameters.

[0011] In a second aspect, the present application provides a method for preparing a membrane material for preparing the outer membrane of a biosensor, including the following steps:

[0012] React a hydrophobic polymer and a hydrophilic polymer with poly(L-lactic acid) and poly(D-lactic acid) respectively through covalent bonds to obtain a poly(L-lactic acid) copolymer-containing product and a poly(D-lactic acid) copolymer-containing product respectively;

[0013] Blend the poly(L-lactic acid) copolymer-containing product and the poly(D-lactic acid) copolymer-containing product, and remove the solvent to obtain the membrane material.

[0014] Furthermore, the preparation method further includes:

[0015] Provide poly(L-lactic acid) monomers and poly(D-lactic acid) monomers;

[0016] Carry out a polymerization reaction on the poly(L-lactic acid) monomers to obtain a poly(L-lactic acid) homopolymer;

[0017] Carry out a polymerization reaction on the poly(D-lactic acid) monomers to obtain a poly(D-lactic acid) homopolymer;

[0018] Blend the solution of the poly(L-lactic acid) homopolymer and the solution of the poly(D-lactic acid) copolymer-containing product, and remove the solvent to obtain the membrane material.

[0019] Furthermore, in the step of reacting through covalent bonds, it includes: providing methacryloyl chloride, reacting methacryloyl chloride with poly(L-lactic acid) and poly(D-lactic acid) respectively to obtain methacrylated poly(L-lactic acid) and methacrylated poly(D-lactic acid);

[0020] Mix the hydrophobic polymer and the hydrophilic polymer with methacrylated poly(L-lactic acid) and methacrylated poly(D-lactic acid) respectively in an organic solvent, carry out deoxygenation treatment, and polymerize to obtain a terpolymer.

[0021] Further, in the step of reacting through covalent bonds, it includes: providing a compound containing hydroxyl groups, polymerizing with a hydrophilic compound and a hydrophobic compound to obtain a hydroxyl-containing binary copolymer;

[0022] Mix the hydroxyl-containing binary copolymer with the monomers of L-polylactic acid and D-polylactic acid respectively, and carry out ring-opening polymerization to obtain a ternary graft copolymer.

[0023] Further, in the step of reacting through covalent bonds, it includes: polymerizing an atom transfer radical polymerization / reversible addition-fragmentation chain transfer polymerization bifunctional initiator or chain transfer agent, a hydrophobic compound, and a hydrophilic compound to obtain a hydroxyl-terminated binary copolymer;

[0024] Mix the hydroxyl-terminated binary copolymer with the monomers of L-polylactic acid and D-polylactic acid respectively to obtain a ternary diblock copolymer.

[0025] In a third aspect, the present application provides a biosensor, including the outer membrane of the biosensor, or including the outer membrane of the biosensor prepared by the method for preparing a membrane.

[0026] Further, the biosensor includes any one of a glucose biosensor, a lactate biosensor, a uric acid biosensor, and a blood ketone biosensor.

[0027] The outer membrane of the biosensor provided in the first aspect of the present application utilizes the stereocomplex microcrystalline structure of the polylactic acid formed by L-polylactic acid and D-polylactic acid as a cross-linked structure. The formed cross-linked structure can increase the cross-linking density between the membrane layers and improve the stability of the membrane material. At the same time, polylactic acid has good biocompatibility, which can help improve the overall biocompatibility of the outer membrane material. And the formed cross-linked structure effectively avoids the use of cross-linking agents harmful to the human body in the chemical cross-linked outer membrane formulation. Therefore, the obtained biosensor outer membrane has high stability and high safety, can accurately and continuously detect signals, and is conducive to wide application.

[0028] The method for preparing the membrane material provided in the second aspect of the present application reacts a hydrophobic polymer, a hydrophilic polymer with L-polylactic acid and D-polylactic acid through covalent bonds respectively, and then blends the copolymer containing L-polylactic acid and the copolymer containing D-polylactic acid to make L-polylactic acid and D-polylactic acid carry out a complexation reaction to form a stereocomplex microcrystalline structure, so as to obtain the outer membrane of the biosensor. This preparation method is simple and efficient and is conducive to wide use.

[0029] The biosensor provided in the third aspect of the present application includes the outer membrane of the provided biosensor. Since the outer membrane of this biosensor has a large cross-linking density and high stability of the membrane material, the obtained biosensor has stable performance, can continuously detect signals, and has a wide range of uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0031] Figure 1 It is a three-dimensional cross-linking network mechanism diagram of the outer membrane of the biosensor provided by the embodiment of the present application.

[0032] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum ( Figure 2 (a)) and gel permeation chromatography ( Figure 2 (b)) analysis results of the material provided in Embodiment 1 of the present application.

[0033] Figure 3 It is the effect diagram provided in Embodiment 1 of the present application, Figure 3 (b) is the powder X-ray diffraction analysis results (XRD) of the material before and after film formation in Embodiment 2.

[0034] Figure 4 It is the cyclic voltammetry scanning test result provided in Embodiment 1 of the present application.

[0035] Figure 5 It is the graph of the change trend of current with glucose concentration (a) provided in Embodiment 1 of the present application and the statistical values of the current of the sensor after stabilization at different concentrations and the linear / nonlinear fitting results (b). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] In order to make the technical problems to be solved, technical solutions and beneficial effects of the present application more clear and understandable, the following further details the present application in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0037] The first aspect of the embodiment of the present application provides an outer membrane of a biosensor. As Figure 1 shown, the outer membrane includes a membrane skeleton constructed by a hydrophobic polymer, a flow channel constructed by a hydrophilic polymer, and a cross-linking structure. Among them, the cross-linking structure includes a stereocomplex microcrystalline structure formed by intermolecular hydrogen bonds between poly(L-lactic acid) and poly(D-lactic acid); the hydrophilic polymer is connected to the cross-linking structure by a covalent bond.

[0038] The outer membrane of the biosensor provided in the first aspect of the embodiments of the present application utilizes the stereocomplex microcrystalline structure of polylactic acid formed by L-polylactic acid and D-polylactic acid as a crosslinked structure. Since the formed crosslinked structure can further connect the membrane materials, the formed crosslinked structure can increase the crosslinking density between the membrane layers and improve the stability of the membrane materials. At the same time, polylactic acid has good biocompatibility, which is beneficial to improving the overall biocompatibility of the outer membrane materials. Moreover, the formed crosslinked structure effectively avoids the use of crosslinking agents harmful to the human body in the chemical crosslinked outer membrane formulation. Therefore, the obtained biosensor outer membrane has high stability and high safety, can accurately and continuously detect signals, and is conducive to wide application.

[0039] In some embodiments, the hydrophobic polymer includes but is not limited to one or more of polystyrene, polymethacrylate, and polyvinylpyridine. Providing a hydrophobic polymer as the membrane skeleton can help isolate water vapor and ensure high stability of the outer membrane materials.

[0040] In some embodiments, the hydrophilic polymer includes but is not limited to one or more of polyethylene glycol and its derivatives, poly(dimethylaminomethyl acrylate), poly(2-hydroxyethyl methacrylate), and polybetaine. The purpose of providing a hydrophilic polymer is to construct a hydrophilic flow channel, and the constructed hydrophilic flow channel is beneficial to the transport of hydrophilic substances and enables the hydrophilic substances to flow.

[0041] Furthermore, the membrane skeleton constructed by the hydrophobic polymer and the flow channel constructed by the hydrophilic polymer are mainly obtained by copolymerization in the presence of a catalyst or an initiator. In some embodiments, the provided initiator includes but is not limited to azobisisobutyronitrile; under the action of the initiator azobisisobutyronitrile, the hydrophobic polymer and the hydrophilic polymer can undergo a radical polymerization reaction.

[0042] Furthermore, the outer membrane also includes a stereocomplex microcrystalline structure formed by hydrogen bonding between L-polylactic acid and D-polylactic acid, and the formed stereocomplex microcrystalline structure can be three-dimensionally folded to improve the three-dimensional performance of the outer membrane materials.

[0043] In some embodiments, the hydrophilic polymer is connected to the crosslinked structure by a covalent bond, and the covalent bond includes a carbon-carbon bond or a carbon-oxygen bond.

[0044] In some embodiments, the formed stereocomplex microcrystalline structure includes stereocomplex microcrystalline crosslinking points and / or star-shaped crosslinking points. Among them, if the formed stereocomplex microcrystalline structure includes stereocomplex microcrystalline crosslinking points and star-shaped crosslinking points, it can further improve the crosslinking degree of the material and make the connection between the membrane layers closer.

[0045] In some embodiments, the formed stereocomplex microcrystalline structure includes unit cells with different parameters. In some specific embodiments, the 2θ angles for forming the stereocomplex microcrystalline structure include 11.9° or 20.7°; through analysis, it can be determined that the formed 2θ angles include 11.9° or 20.7°, and the resulting structure is a stereocomplex microcrystalline structure.

[0046] In some embodiments, the left-handed polylactic acid or right-handed polylactic acid includes, but is not limited to, copolymers or homopolymers. Among them, the formed homopolymers include, but are not limited to, linear homopolymers or star-shaped homopolymers. Further, the formed star-shaped homopolymers include, but are not limited to, three-arm, four-arm, six-arm, eight-arm, and hyperbranched star polymers. The density of the physical cross-linking regions of the formed star-shaped homopolymers is large, and they have good mechanical strength. As the cross-linking structure of the outer membrane, they can further improve the overall cross-linking strength of the membrane material.

[0047] The second aspect of the embodiments of the present application provides a method for preparing a membrane material for preparing the outer membrane of a biosensor, including the following steps:

[0048] S01. React a hydrophobic polymer and a hydrophilic polymer with left-handed polylactic acid and right-handed polylactic acid respectively through covalent bonds to obtain a copolymer containing left-handed polylactic acid and a copolymer containing right-handed polylactic acid respectively;

[0049] S02. Blend the copolymer containing left-handed polylactic acid and the copolymer containing right-handed polylactic acid, and remove the solvent to obtain the membrane material.

[0050] The method for preparing the membrane material provided by the second aspect of the embodiments of the present application reacts a hydrophobic polymer, a hydrophilic polymer with left-handed polylactic acid and right-handed polylactic acid respectively through covalent bonds, and then blends the copolymer containing left-handed polylactic acid and the copolymer containing right-handed polylactic acid to cause the left-handed polylactic acid and the right-handed polylactic acid to undergo a complexation reaction to form a stereocomplex microcrystalline structure, so as to obtain the outer membrane of the biosensor. This preparation method is simple and efficient, and is conducive to wide use.

[0051] In step S01, a hydrophobic polymer and a hydrophilic polymer are reacted with left-handed polylactic acid and right-handed polylactic acid respectively through covalent bonds to obtain a copolymer containing left-handed polylactic acid and a copolymer containing right-handed polylactic acid respectively.

[0052] In some embodiments, the hydrophobic polymer includes, but is not limited to, one or more of polystyrene, polymethacrylate, and polyvinylpyridine. Providing a hydrophobic polymer as the membrane skeleton can help isolate water vapor and ensure high stability of the outer membrane material.

[0053] In some embodiments, the hydrophilic polymer includes, but is not limited to, one or more of polyethylene glycol and its derivatives, poly(dimethylaminoethyl methacrylate), poly(2-hydroxyethyl methacrylate), and polybetaine. The purpose of providing the hydrophilic polymer is to construct a hydrophilic flow channel, and the constructed hydrophilic flow channel is beneficial to the transport of hydrophilic substances and enables the circulation of hydrophilic substances.

[0054] In some embodiments, the mass ratio of the hydrophobic polymer to the hydrophilic polymer is 90-99:1-10. By controlling the addition amounts of the hydrophobic polymer and the hydrophilic polymer, it is possible to effectively control the film skeleton constructed by the hydrophobic polymer and the flow channel constructed by the hydrophilic polymer in the prepared outer membrane, ensuring the stability of the formed outer membrane structure and excellent overall material properties.

[0055] In some embodiments, in the step of reacting through a covalent bond, it includes:

[0056] G01. Provide methacryloyl chloride, react methacryloyl chloride with L-polylactic acid and D-polylactic acid respectively to obtain L-methacryloylated polylactic acid and D-methacryloylated polylactic acid;

[0057] G02. Mix the hydrophobic polymer and the hydrophilic polymer with L-methacryloylated polylactic acid and D-methacryloylated polylactic acid in an organic solvent respectively, and perform deoxygenation treatment, and polymerize to obtain a terpolymer.

[0058] In step G01, the reaction formula of methacryloyl chloride and L-polylactic acid is as follows:

[0059]

[0060] In some embodiments, the mass ratio of methacryloyl chloride to L-polylactic acid is 1:1, ensuring that the provided methacryloyl chloride can be connected to L-polylactic acid one by one to avoid material waste.

[0061] The reaction formula of methacryloyl chloride and D-polylactic acid is as follows:

[0062]

[0063] In some embodiments, the mass ratio of methacryloyl chloride to D-polylactic acid is 1:1, ensuring that the provided methacryloyl chloride can be connected to D-polylactic acid one by one to avoid material waste.

[0064] In some embodiments, the molecular weight of the provided L-polylactic acid or D-polylactic acid is 500-5000 Da.

[0065] In step G02, a hydrophobic polymer and a hydrophilic polymer are respectively mixed with methacrylated L-polylactic acid and methacrylated D-polylactic acid in an organic solvent, and deoxygenation treatment is carried out, followed by polymerization to obtain a terpolymer.

[0066] In some embodiments, the weight ratio of the hydrophobic polymer, the hydrophilic polymer, and methacrylated L-polylactic acid is (10 - 60):(5 - 60):(5 - 20). Controlling the weight ratio of the three is beneficial for the three materials to react under appropriate proportion conditions, improving the reaction efficiency, and making the proportion of the mixture formed by the reaction of the obtained substances appropriate and the reaction efficiency high.

[0067] In some embodiments, the weight ratio of the hydrophobic polymer, the hydrophilic polymer, and methacrylated D-polylactic acid is (10 - 60):(5 - 60):(5 - 20).

[0068] In some embodiments, the organic solvent includes but is not limited to organic solvents such as dichloromethane, chloroform, and DMSO.

[0069] In some embodiments, the purpose of the deoxygenation treatment is to remove oxygen and impurities in the entire reaction system to ensure a relatively high purity of the obtained product. Among them, the time of the deoxygenation treatment includes but is not limited to 1 hour and can be controlled according to the specific reaction.

[0070] In some embodiments, an initiator is added during the polymerization process, and the heating time is determined according to the type of the initiator for heating reaction to obtain a terpolymer.

[0071] In some embodiments, based on the total mass of the hydrophobic polymer, the hydrophilic polymer, and methacrylated L-polylactic acid or methacrylated D-polylactic acid being 100%, the weight percentage content of the initiator is 0.001% - 0.1%. The weight of the added initiator is mainly added according to the overall reaction process. Among them, the initiator is selected from but not limited to azo initiators and nitroxide initiators.

[0072] In some embodiments, in the step of reacting through covalent bonds, it includes:

[0073] I01. Providing a compound containing a hydroxyl group, and polymerizing it with a hydrophilic compound and a hydrophobic compound to obtain a hydroxyl-containing binary copolymer;

[0074] I02. Mixing the hydroxyl-containing binary copolymer with monomers of L-polylactic acid and D-polylactic acid respectively, and carrying out ring-opening polymerization to obtain a ternary graft copolymer.

[0075] In Step I01, a compound containing a hydroxyl group is provided. The compound containing a hydroxyl group and the hydrophilic polymer are first mixed in an organic solvent. After deoxygenation, a reaction is carried out to obtain a hydrophilic polymer with a hydroxyl group at one end; then it is polymerized with a hydrophobic polymer to obtain a hydroxyl-containing diblock copolymer.

[0076] In some embodiments, the organic solvent includes but is not limited to at least one of dichloromethane, chloroform, DMF, THF, and DMSO.

[0077] In some embodiments, deoxygenation is carried out for 1 hour, which is beneficial to removing impurities in the reaction system and ensuring that oxidation does not occur during the reaction.

[0078] In some embodiments, the compound containing a hydroxyl group includes but is not limited to methanol, ethanol, etc. The provided compound containing a hydroxyl group can undergo a substitution reaction with the hydrophilic polymer to obtain a hydrophilic polymer with a hydroxyl group at one end.

[0079] In some embodiments, the ratio of the compound containing a hydroxyl group to the hydrophilic polymer is 1:1; by controlling the mass ratio of the two, it is ensured that the resulting material can react completely.

[0080] In some embodiments, the mass ratio of the hydrophobic polymer to the hydroxyl-containing hydrophilic polymer is 90 - 99:1 - 10. By controlling the addition amounts of the hydrophobic polymer and the hydrophilic polymer, it can effectively control the outer membrane prepared to be a membrane skeleton constructed by the hydrophobic polymer and a flow channel constructed by the hydrophilic polymer, ensuring that the formed outer membrane structure is stable and the overall performance of the material is excellent.

[0081] In some embodiments, the polymerization reaction further includes adding an initiator. The added initiator includes but is not limited to azo initiators or nitroxide initiators. And, based on the weight of the raw materials of the diblock copolymer being 100%, the addition amount of the initiator is 0.001% - 0.1%. Just adding the initiator can initiate the reaction.

[0082] In some embodiments, for the obtained hydroxyl-containing diblock copolymer, the hydroxyl content is 0.1 - 1 mmol. The added hydroxyl groups are mainly connected to the hydrophilic polymer and are mainly used to react with polylactic acid.

[0083] In some embodiments, the molar amount of added L-polylactic acid or D-polylactic acid is 50 - 5000 times the molar amount of hydroxyl groups in the diblock copolymer. Since L-polylactic acid or D-polylactic acid undergoes a covalent reaction with the hydrophilic polymer through hydroxyl groups, limiting the addition amount of L-polylactic acid or D-polylactic acid to 50 - 5000 times the molar content of hydroxyl groups in the hydroxyl-containing diblock copolymer can achieve the copolymerization reaction of the two reactants to a large extent.

[0084] In Step I02, the binary copolymer is respectively mixed with the monomers of L-polylactic acid and D-polylactic acid, and ring-opening polymerization is carried out to obtain a ternary graft copolymer.

[0085] In some embodiments, during the process of ring-opening polymerization, a catalyst is further included, and the weight percentage content of the catalyst is 0.1% - 0.2%. The provided catalyst includes but is not limited to tin-based catalysts, and the purpose is to catalyze the ring-opening polymerization of polylactic acid in the heating reaction to obtain a ternary graft copolymer.

[0086] In some embodiments, in the step of reacting through covalent bonds, it includes:

[0087] K01. Polymerize an atom transfer radical polymerization / reversible addition-fragmentation chain transfer polymerization bifunctional initiator or chain transfer agent, a hydrophobic compound, and a hydrophilic compound to obtain a binary copolymer containing terminal hydroxyl groups;

[0088] K02. Respectively mix the binary copolymer containing terminal hydroxyl groups with the monomers of L-polylactic acid and D-polylactic acid to obtain a ternary diblock copolymer.

[0089] In Step K01, in some embodiments, in the atom transfer radical polymerization / reversible addition-fragmentation chain transfer polymerization bifunctional initiator or chain transfer agent, it contains hydroxyl groups, and the content of the hydroxyl groups is 0.1 - 1 mmol / L. In some specific embodiments, the atom transfer radical polymerization / reversible addition-fragmentation chain transfer polymerization bifunctional initiator or chain transfer agent is selected from 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol.

[0090] Among them, the content of the hydroxyl groups is 0.1 - 1 mmol / L. Further, the molar amount of the hydrophobic polymer or hydrophilic polymer is 50 - 5000 times the molar amount of the hydroxyl groups in the ATRP / RAFT polymerization bifunctional initiator / chain transfer agent. Since L-polylactic acid or D-polylactic acid undergoes a covalent reaction with the hydrophilic polymer through hydroxyl groups, the addition amount of L-polylactic acid or D-polylactic acid is limited to 50 - 5000 times the molar content of the hydroxyl groups in the hydroxyl-containing diblock copolymer, and thus the copolymerization reaction of the two reactants can be achieved to a large extent.

[0091] Furthermore, an initiator is added to initiate the reaction, and the polymerization reaction is carried out under heating conditions to obtain a binary copolymer containing terminal hydroxyl groups.

[0092] In some embodiments, the weight of the binary copolymer containing terminal hydroxyl groups is 100%, and among them, the weight percentage content of the initiator is 0.001% - 0.1%.

[0093] In step K02, a hydroxyl-terminated diblock copolymer is mixed with monomers of L-polylactic acid and D-polylactic acid respectively to obtain a triblock copolymer.

[0094] Taking the total mass of the hydroxyl-terminated diblock copolymer and the L-polylactic acid monomer or the total mass of the hydroxyl-terminated diblock copolymer and the D-polylactic acid monomer as 100%, wherein a catalyst is added during the reaction, and the weight percentage of the catalyst is 0.1% - 0.2%. The provided catalyst includes but is not limited to tin-based catalysts, and the purpose is to catalyze the ring-opening polymerization of polylactic acid during the heating reaction to obtain a graft random copolymer.

[0095] In step S02, the L-polylactic acid copolymer and the D-polylactic acid copolymer are blended, and the solvent is removed to obtain a film material.

[0096] Among them, L-polylactic acid and D-polylactic acid undergo a complexation reaction to form a stereocomplex microcrystalline structure. The complexation reaction is mainly between the L-polylactic acid groups and the D-polylactic acid groups to form hydrogen bonds to form a stereocomplex microcrystalline structure.

[0097] In some embodiments, the preparation method further includes:

[0098] D01. Providing L-polylactic acid monomer and D-polylactic acid monomer;

[0099] D02. Polymerizing the L-polylactic acid monomer to obtain an L-polylactic acid homopolymer;

[0100] D03. Polymerizing the D-polylactic acid monomer to obtain a D-polylactic acid homopolymer;

[0101] D04. Blending the solution of the L-polylactic acid homopolymer and the solution containing the D-polylactic acid copolymer, and removing the solvent to obtain a film material.

[0102] In the specific reaction process, the obtained L-polylactic acid copolymer and the D-polylactic acid copolymer can be blended to obtain a film material; or the L-polylactic acid copolymer and the D-polylactic acid homopolymer can be reacted to obtain a film material.

[0103] In some embodiments, the formed outer film material can be coated on the surface of the sensor to form an outer film of the sensor. Among them, the coating methods include but are not limited to one or more of spraying, dip coating, and spin coating, and the number of coating times includes but is not limited to 1 - 100 times.

[0104] The third aspect of the embodiments of the present application provides a biosensor, including the outer film of the biosensor described above, or including the outer film of the biosensor prepared by using the above film preparation method.

[0105] The biosensor provided in the third aspect of the embodiments of the present application includes the outer membrane of the provided biosensor. Since the outer membrane of the biosensor has a high crosslinking density and high stability of the membrane material, the obtained biosensor has stable performance, can continuously detect signals, and has a wide range of applications.

[0106] In some embodiments, the biosensor includes any one of a glucose biosensor, a lactate biosensor, a uric acid biosensor, and a blood ketone biosensor.

[0107] The following is an illustration with specific embodiments.

[0108] Embodiment 1

[0109] An outer membrane of a biosensor and a preparation method thereof

[0110] The preparation method of the outer membrane of the biosensor is specifically as follows:

[0111] 1. Synthesis of high molecular weight linear PLLA and star-shaped PLLA

[0112] Preparation of high molecular weight linear PLLA homopolymer: Weigh a certain amount of n-hexadecanol, L-lactide, and stannous octoate in a clean and dry glass tube in a dry environment. After evacuating for 5 minutes, heat-seal the tube opening, polymerize at a certain temperature for 4 h, then dissolve the polymer with dichloromethane and precipitate it in methanol, and then filter out the precipitate. This process is repeated 5 times. After drying in a vacuum oven at 60 °C for 24 h, a methyl-capped monohydroxy high molecular weight linear PLLA homopolymer is obtained;

[0113] Preparation of star-shaped high molecular weight PLLA homopolymer: Weigh a certain amount of n-hexadecanol, L-lactide, and polyol in a clean and dry glass tube in a dry environment. After evacuating for 5 minutes, heat-seal the tube opening, polymerize at a certain temperature for 4 h, then dissolve the polymer with dichloromethane and precipitate it in methanol, and then filter out the precipitate. This process is repeated 5 times. After drying in a vacuum oven at 60 °C for 24 h, a methyl-capped monohydroxy star-shaped high molecular weight PLLA homopolymer is obtained;

[0114] 2. Synthesis of methacrylated PDLA prepolymer

[0115] Weigh 2.5 mmol of the above PDLA homopolymer and dissolve it in 60 mL of anhydrous dichloromethane. Add 420 μL of triethylamine under an argon protection atmosphere, stir evenly for 10 minutes, then dropwise add 10 mL of anhydrous dichloromethane dissolved with 275 μL of methacryloyl chloride at 0 °C. React at 0 °C for 5 h and then react at room temperature for 36 h;

[0116] The above reaction solution was filtered and then washed repeatedly 3 times with 100 mL of deionized water. The washed reaction solution was further dehydrated with anhydrous sodium sulfate, and after filtering the precipitate, rotary evaporation was carried out. The concentrated solution was further precipitated in n-hexane, and after the solvent volatilization was completed, it was dried in a vacuum oven at 60 °C for 24 h to obtain methacrylated PDLA prepolymer (PDLAMA).

[0117] 3. Synthesis of PMMA-co-PDMAEMA-co-PDLA terpolymer

[0118] MMA, DMAEMA, and PDLAMA were respectively added to a 250 mL two-necked round-bottom flask according to the molar ratio of monomers [MMA]:[DMAEMA]:[PDLAMA] = 100:80:1. At the same time, 15 mL of chloroform was added, and the mixture was continuously stirred for 1 h under an argon protection atmosphere until the solution became homogeneous. Subsequently, 0.1 wt% of azobisisobutyronitrile was added under an argon protection atmosphere, and after continuous stirring for 15 min, the flask was sealed and placed in an oil bath at 60 °C for continuous reaction for 24 h. The product after the reaction was washed by dissolving in dichloromethane and precipitating with ice-cold n-hexane. After repeating 3 times, the pure random copolymer PMMA-co-PDMAEMA-co-PDLA was obtained, and then it was dried in a vacuum oven at 60 °C for 24 h for standby.

[0119] Weighed 0.9 g of PMMA-co-PDMAEMA-co-PDLA copolymer and 0.1 g of high molecular weight PLLA homopolymer into an ampoule, and at the same time added 20 mL of dichloromethane to make a mixed sample solution of 0.05 g / mL. After stirring at room temperature for 24 h, the outer membrane material was obtained.

[0120] Biosensor, the specific preparation method includes:

[0121] The flexible electrode modified with a conductive layer and a sensing matrix was dip-coated and lifted in the above sample solution 4 times, with an interval of 5 min for each lift. After all the film layers were coated, it was left to dry naturally for 2 h for standby;

[0122] Pour the liquid of the outer membrane material of the biosensor obtained in Example 1 into a clean evaporation dish, and after the solvent volatilizes, natural film formation is completed.

[0123] Example 2

[0124] An outer membrane of a biosensor and its preparation method

[0125] 1. Synthesis of high molecular weight linear PLLA and star-shaped PLLA

[0126] Preparation of high molecular weight linear PLLA homopolymer: Weigh a certain amount of cetyl alcohol, L-lactide, and stannous octoate in a clean and dry glass tube in a dry environment. After evacuating for 5 minutes, heat-seal the tube mouth. Polymerize at a certain temperature for 4 hours, then dissolve the polymer in dichloromethane and precipitate it in methanol. Then filter out the precipitate, and repeat this process 5 times. Dry it in a vacuum oven at 60°C for 24 hours to obtain a methyl-capped monohydroxy linear high molecular weight PLLA homopolymer;

[0127] Preparation of star-shaped high molecular weight PLLA homopolymer: Weigh a certain amount of cetyl alcohol, L-lactide, and polyol in a clean and dry glass tube in a dry environment. After evacuating for 5 minutes, heat-seal the tube mouth. Polymerize at a certain temperature for 4 hours, then dissolve the polymer in dichloromethane and precipitate it in methanol. Then filter out the precipitate, and repeat this process 5 times. Dry it in a vacuum oven at 60°C for 24 hours to obtain a methyl-capped monohydroxy star-shaped high molecular weight PLLA homopolymer;

[0128] 2. Mix and react a compound containing a hydroxyl group and a hydrophilic polymer to obtain a hydrophilic polymer with a hydroxyl end; Mix 10 - 90% by weight of a hydrophobic polymer and 90 - 10% of the hydrophilic polymer with a hydroxyl end in an organic solvent dichloromethane, and then deoxygenate for 1 hour to obtain a mixture;

[0129] 3. Then add a nitroxide initiator with a weight fraction of 0.1%, heat for free radical polymerization, and then repeat the dissolution - precipitation - washing process. After drying, a diblock copolymer is obtained;

[0130] 4. Weigh the above diblock copolymer containing 0.1 - 1 mmol of hydroxyl groups and 50 - 5000 equivalents of L-lactide / D-lactide (PLA monomer) in an organic solvent (dichloromethane / chloroform / DMSO). After deoxygenation, add a tin catalyst with a weight fraction of 0.1%, heat for ring-opening polymerization, and then repeat the dissolution - precipitation - washing process. After drying, a ternary PLA grafted random copolymer is obtained;

[0131] 5. Weigh the ternary PDLA grafted random copolymer and 0.1 g of high molecular weight PLLA homopolymer in an ampoule, and simultaneously add 20 mL of dichloromethane to make a mixed sample solution with a concentration of 0.05 g / mL. Stir at room temperature for 24 hours to obtain an outer membrane material.

[0132] Biosensor, the specific preparation method includes:

[0133] Dip and lift the flexible electrode modified with a conductive layer and a sensing matrix in the above sample solution 4 times for coating, with a 5-minute interval for each lift. After all the film layers are coated, let it dry naturally for 2 hours for standby;

[0134] Pour the liquid of the outer membrane material of the biosensor obtained in Example 2 into a clean evaporating dish. After the solvent evaporates, natural film formation is completed.

[0135] Example 3

[0136] An outer membrane of a biosensor and a preparation method thereof

[0137] A preparation method of an outer membrane of a biosensor, the specific steps are as follows:

[0138] 1. Synthesis of high molecular weight linear PLLA and star-shaped PLLA

[0139] Preparation of high molecular weight linear PLLA homopolymer: Weigh a certain amount of cetyl alcohol, L-lactide and stannous octoate in a clean and dry glass tube in a dry environment. After evacuating for 5 minutes, heat-seal the tube mouth. After polymerization at a certain temperature for 4 h, dissolve the polymer in dichloromethane and precipitate it in methanol, then filter out the precipitate. This process is repeated 5 times. After drying in a vacuum oven at 60 °C for 24 h, a methyl-capped monohydroxy linear high molecular weight PLLA homopolymer is obtained;

[0140] Preparation of star-shaped high molecular weight PLLA homopolymer: Weigh a certain amount of cetyl alcohol, L-lactide and polyol in a clean and dry glass tube in a dry environment. After evacuating for 5 minutes, heat-seal the tube mouth. After polymerization at a certain temperature for 4 h, dissolve the polymer in dichloromethane and precipitate it in methanol, then filter out the precipitate. This process is repeated 5 times. After drying in a vacuum oven at 60 °C for 24 h, a methyl-capped monohydroxy star-shaped high molecular weight PLLA homopolymer is obtained;

[0141] 2. Weigh 0.1 - 1 mmol of 4-cyano-4-[(dodecylsulfonylthiocarbonyl)sulfamoyl]pentanol, 50 - 5000 equivalents of hydrophobic polymer monomers, and 50 - 500 equivalents of hydrophilic polymer monomers in dichloromethane, and deoxygenate for 1 h;

[0142] 3. Add 0.1% by weight of an azo initiator to the above solution, heat for radical polymerization, and then repeat the dissolution - precipitation - washing process. After drying, a diblock copolymer is obtained;

[0143] 4. Weigh the above diblock copolymer containing 0.1 - 1 mmol of hydroxyl groups, 50 - 5000 equivalents of L-lactide / D-lactide (PLA monomers) in dichloromethane. After deoxygenation, add 0.1% by weight of a tin catalyst, heat for ring-opening polymerization, and then repeat the dissolution - precipitation - washing process. After drying, a triblock copolymer formed by PLA and the above diblock copolymer is obtained;

[0144] 5: Weigh 0.9 g of the triblock copolymer containing PDLA and 0.1 g of the high molecular weight PLLA homopolymer into an ampoule. At the same time, add 20 mL of dichloromethane to make a mixed sample solution with a concentration of 0.05 g / mL. After stirring at room temperature for 24 h, an outer membrane slurry is obtained.

[0145] A biosensor, and the specific preparation method includes:

[0146] Dip and lift the flexible electrode modified with a conductive layer and a sensing matrix in the above sample solution 4 times for coating. The interval time for each lifting is 5 min. After all the film layers are coated, leave it to dry naturally for 2 h for standby;

[0147] Pour the liquid of the outer membrane material of the biosensor obtained in Example 3 into a clean evaporating dish. After the solvent evaporates, natural film formation is completed.

[0148] Property testing and result analysis

[0149] Perform property testing and analysis on the material obtained in Example 1

[0150] (1) Perform nuclear magnetic resonance hydrogen spectrum (1H NMR) and PLA-related polymer gel permeation chromatography (GPC) analysis on the terpolymer obtained in Example 1. The results are shown in Figure 2 (a) and 2(b). 1H NMR shows the characteristics of each polymer segment, and the GPC results show the homogeneity of the synthesized polymer. The statistical results of its molecular weight and molecular weight distribution are shown in Table 1.

[0151] Table 1

[0152]

[0153] (2) The natural film formation effect of the material on the surface of the biosensor is as shown in Figure 3 (a). After bending, the film layer still maintains good stability. Further, powder XRD tests are carried out on the material before and after film formation. The test results are shown in Figure 3 (b). The characteristic peaks at 2θ = 11.9°, 20.7°, and 24° respectively correspond to different crystal planes ((110), (300), (220)) of the PLA stereocomplex microcrystals, further indicating the accuracy of the designed structure as shown in Figure 1 (a).

[0154] (3) Electrochemical testing

[0155] Use cyclic voltammetry and chronoamperometry to test the uncoated and coated electrodes respectively, Figure 4The cyclic voltammetry results show that the sensor still maintains good conductivity after the outer membrane coating. The almost unchanged oxidation peak potential and reduction peak potential indicate that the outer membrane coating under this condition has little effect on the redox reaction of the sensing layer matrix, and the sensor has excellent reaction activity.

[0156] Furthermore, Figure 5 The results of chronoamperometry prove that the sensor coated with the above-mentioned polymer cross-linked outer membrane has an obvious response to glucose. The response current value of the uncoated sensor to glucose gradually tends to saturation as the glucose concentration increases. The coated sensor has a rapid response to different concentrations of glucose, especially high concentrations, and the linear detection range of glucose concentration can be broadened to 25 mM, which fully meets the detection of blood glucose concentration in human tissue fluid by the implantable sensor.

[0157] In summary, the outer membrane of the biosensor provided in this application utilizes the stereocomplex microcrystalline structure of polylactic acid formed by L-polylactic acid and D-polylactic acid as the cross-linking structure. The formed cross-linking structure can increase the cross-linking density between the membrane layers and improve the stability of the membrane material. At the same time, polylactic acid has good biocompatibility, which is conducive to improving the overall biocompatibility of the outer membrane material. Moreover, the formed cross-linking structure effectively avoids the use of cross-linking agents harmful to the human body in the chemical cross-linked outer membrane formulation. Therefore, the obtained biosensor outer membrane has high stability and high safety, can accurately and continuously detect signals, and is conducive to wide application.

[0158] The above are only the preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of this application shall be included in the protection scope of this application.

Claims

1. An outer membrane of a biosensor, Characterized in that, The outer membrane includes a membrane skeleton constructed by a hydrophobic polymer, a flow channel constructed by a hydrophilic polymer, and a cross-linked structure. Among them, the cross-linked structure includes a stereocomplex microcrystalline structure formed by intermolecular hydrogen bonds between poly(L-lactic acid) and poly(D-lactic acid); the hydrophilic polymer is connected to the cross-linked structure by a covalent bond.

2. The outer membrane of the biosensor according to claim 1, Characterized in that, The covalent bond includes a carbon-carbon bond or a carbon-oxygen bond.

3. The outer membrane of the biosensor according to claim 1, Characterized in that, The stereocomplex microcrystalline structure includes stereocomplex microcrystalline cross-linking points and / or star-shaped cross-linking points.

4. The outer membrane of the biosensor according to claim 1, Characterized in that, The stereocomplex microcrystalline structure includes unit cells with different parameters.

5. A preparation method of a membrane material, Characterized in that, For preparing the outer membrane of a biosensor, it includes the following steps: React a hydrophobic polymer and a hydrophilic polymer with poly(L-lactic acid) and poly(D-lactic acid) respectively through covalent bonds to obtain a poly(L-lactic acid) copolymer-containing product and a poly(D-lactic acid) copolymer-containing product respectively; Blend the poly(L-lactic acid) copolymer-containing product and the poly(D-lactic acid) copolymer-containing product, and remove the solvent to obtain the membrane material.

6. The preparation method of the membrane material according to claim 5, Characterized in that, The preparation method further includes: Providing poly(L-lactic acid) monomer and poly(D-lactic acid) monomer; Performing a polymerization reaction on the poly(L-lactic acid) monomer to obtain a poly(L-lactic acid) homopolymer; Performing a polymerization reaction on the poly(D-lactic acid) monomer to obtain a poly(D-lactic acid) homopolymer; Blend the solution of the poly(L-lactic acid) homopolymer and the solution of the poly(D-lactic acid) copolymer-containing product, and remove the solvent to obtain the membrane material.

7. The preparation method of the membrane material according to claim 5, Characterized in that, In the step of reacting through covalent bonds, it includes: providing methacryloyl chloride, reacting the methacryloyl chloride with the poly(L-lactic acid) and the poly(D-lactic acid) respectively to obtain methacrylated poly(L-lactic acid) and methacrylated poly(D-lactic acid); Mix the hydrophobic polymer and the hydrophilic polymer with the methacrylated poly(L-lactic acid) and the methacrylated poly(D-lactic acid) respectively in an organic solvent, and perform deoxygenation treatment, and polymerize to obtain a terpolymer.

8. The preparation method of the membrane material according to claim 5, Characterized in that, In the step of reacting through covalent bond connection, it includes: providing a compound containing a hydroxyl group, polymerizing with a hydrophilic compound and a hydrophobic compound to obtain a hydroxyl group-containing binary copolymer; Mix the hydroxyl group-containing binary copolymer with the monomers of poly(L-lactic acid) and poly(D-lactic acid) respectively, and perform ring-opening polymerization to obtain a ternary graft copolymer.

9. The preparation method of the membrane material according to claim 5, Characterized in that, In the step of reacting through covalent bond connection, it includes: polymerizing an atom transfer radical polymerization / reversible addition-fragmentation chain transfer polymerization bifunctional initiator or chain transfer agent, a hydrophobic compound, and a hydrophilic compound to obtain a hydroxyl group-containing binary copolymer; The binary copolymer containing terminal hydroxyl groups is respectively mixed with the monomers of L-polylactic acid and D-polylactic acid to obtain a ternary diblock copolymer.

10. A biosensor, characterized in that it includes the outer membrane of the biosensor according to any one of claims 1 to 4, or includes the outer membrane of the biosensor prepared by the preparation method of the membrane material according to any one of claims 5 to 9.

11. The biosensor according to claim 10, characterized in that the biosensor includes any one of a glucose biosensor, a lactic acid biosensor, a uric acid biosensor, and a blood ketone biosensor.

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