Composite Filter Membrane of a Biosensor and Its Preparation Method

Abstract

The present invention discloses a composite filtration membrane for a biosensor, which is loaded on the surface of the electrode of the sensor. The structure of the composite filtration membrane sequentially includes, from the outside to the inside: a biocompatible hydrophilic layer, a porous support layer, an analyte filtration layer, a bioactive functional layer, and a graphene derivative layer. The graphene derivative layer loaded on the electrode can improve the enzyme loading rate and reduce the interference of toxic substances exuded from the electrode to the bioactive functional layer; the analyte filtration layer improves the linear range of sensor detection and the service life of the bioactive functional layer; the porous support layer increases the mechanical strength of the membrane and prevents the bioactive functional layer from being squeezed; the biocompatible hydrophilic layer improves biocompatibility; the multi-layer composite structure of the composite filtration membrane of the present invention, as well as the selection of surface functional groups and segments between layers, improves the affinity between composite layers, improves the service life of the biosensor, reduces the cytotoxicity of the membrane, improves biocompatibility, and improves the service life.

Description

Technical Field

[0001] The present invention relates to the field of biosensors, specifically to the outer membrane of a sensor for testing biological indicators of body fluids or blood by an electrochemical method. Background Art

[0002] A biosensor is used to detect corresponding substances and biomolecules. The biosensor selectively recognizes biomolecules, converts chemical signals into corresponding physical signals such as light, electricity, sound, magnetism, and heat, and then quantitatively tests the signal molecules through a theoretical mathematical model. A biosensor is to convert the test substance into a signal that can be recognized by other electronic components through a layer of molecular recognition material.

[0003] Currently, the most widely used clinical biosensor is the blood glucose biosensor. Various sensors for in vitro and in vivo detection have been developed. Since blood glucose can cause various diseases, the principles of biosensors for testing blood glucose include enzyme-free and enzyme-based sensors. For enzyme-free sensors, an artificial synthetic material is used to replace the enzyme in the bioactive functional layer. Because enzyme-free sensors have poor stability and high manufacturing difficulty, currently, the main biosensors on the market are enzyme-based blood glucose sensors.

[0004] The basic structure of an enzyme-based sensor is to immobilize glucose oxidase on the sensor. The selectivity and specificity of the enzyme for glucose molecules are used to quantitatively test the concentration of glucose. Enzyme is a protein complex structure. After glucose contacts glucose oxidase, an oxidation-reduction reaction occurs, accompanied by an electron transfer process. The active center of glucose oxidase is generally in the center of the protein and does not directly contact the conductive material. The transfer of electrons requires a mediator to transmit.

[0005] According to the different processes of transferring electrons on glucose oxidase to the electrode surface, the sensors are divided into three generations. The first generation, the second generation, and the third generation. Their main differences lie in the different ways of transferring electrons on the electrode surface after the reaction between glucose oxidase and glucose. The first generation transfers electrons through oxygen, and the second generation uses other electron mediators, such as Fe2+ / Fe3+ , quinone derivatives and other redox pairs as electron transfer mediators. The third generation makes the redox electron pair into a polymer to form a conductive polymer, and transfers the electrons at the active center of the enzyme to the electrode, which is a more efficient transfer method.

[0006] Now, an implantable continuous monitoring blood glucose meter has been developed. In order to improve the biocompatibility of the sensor, the accuracy of detection, and expand the linear range of testing, and at the same time to protect the enzyme layer, an outer membrane must be coated on the surface of the sensor. The function of the outer membrane is to prevent interfering substances from entering, control the permeability of glucose and oxygen, extend the service life of the enzyme, the linear range, and the accuracy of testing.

[0007] Most of the current sensors on the market are first-generation sensors, and the test mechanism is as follows:

[0008]

[0009] According to the test principle, the outer membrane of the sensor needs to control the permeability of glucose and oxygen. Therefore, an outer membrane needs to be coated on the sensor surface to control the diffusion performance of substances. As the harm of hyperglycemia is increasing, patients need to monitor blood glucose 24 hours a day. Therefore, the demand for implantable continuous blood glucose meters is increasing. For the existing sensor outer membranes, the stability and filtration performance of the membranes are unstable. The solution is to mix more than 3 kinds of polyurethane materials, among which 2 kinds of polyurethanes contain PEG segments and siloxane segments respectively. The compatibility of these two materials is poor, and a third kind of polyurethane is needed as the continuous phase, with PEG polyurethane and siloxane polyurethane as the dispersed phases. After the three materials are blended, there is no chemical cross-linking or physical cross-linking. Due to the compatibility problem of the materials, over time, under the action of environmental stress, defects will appear in the outer membrane, cracking will occur, the integrity of the membrane will be damaged, and the function of the membrane will be lost.

[0010] In addition, for the polyurethane material containing PEG segments, because the PEG segments are prone to hydrolysis and not antioxidant, after a long implantation time, the outer membrane will be damaged due to the hydrolysis or oxidation of PEG, and the function of the outer membrane will be lost. To solve the problems of hydrolysis resistance and antioxidant property, Patent US20030042137A1 uses a nitrogen-containing water-absorbing polymer polyvinylpyridine. Although the stability of the outer membrane is improved, the compatibility of this material with oxygen-permeable materials is poor, and it cannot be blended with siloxane to make a complete outer membrane, and the oxygen permeability is too poor.

[0011] Patent WO2017089380A1 provides a method of adding PTFE particles or enzyme particles to a continuous-structured material and dispersing them in a polymer material to form a porous outer membrane structure. Since PTFE and enzymes are prone to aggregation and not easy to disperse, the pore size of the outer membrane made is very large, and it is difficult to control the pore size in the process, and the spatial distribution is uneven, making it difficult to achieve the effect of controlling the glucose permeability, and the mechanical properties of the membrane are poor.

[0012] Moreover, the existing outer membranes are mostly single-layer or double-layer structures with single functions, and toxic substances between the electrodes can also permeate through the outer membrane. In particular, the toxic substances in the printed electrode ink will permeate out, thus affecting the biocompatibility of the sensor.

[0013] The outer membrane of the sensor electrode needs to control the permeability of glucose and oxygen, improve the service life, linear range and test accuracy of the bioactive functional layer, and at the same time ensure a certain mechanical strength so that the outer membrane will not break. The single-layer or double-layer structure of the existing outer membranes is difficult to maintain the contradictions among the filtration performance, biocompatibility, mechanical properties of the outer membrane, antioxidant property and hydrolysis resistance.

[0014] Therefore, the contradiction between the outer membrane filtration performance and biocompatibility of the sensor and the mechanical properties, antioxidant and hydrolysis resistance of the outer membrane needs to be solved urgently. Summary of the Invention

[0015] The present invention provides a composite filtration membrane and its application in a biosensor, which can solve the above-mentioned defects in the prior art. The technical solution of the present invention is as follows:

[0016] A composite filtration membrane for a biosensor, the composite filtration membrane is loaded on the surface of the electrode of the biosensor, and the structure of the composite filtration membrane from the outside to the inside includes: a biocompatible hydrophilic layer, a porous support layer, an analyte filtration layer, a bioactive functional layer and a graphene derivative layer. The bioactive functional layer refers to the catalytic functional layer of the enzyme.

[0017] The structure of the composite filtration membrane of the present invention is applied to an implantable continuous monitoring sensor. The biocompatible hydrophilic layer improves the biocompatibility of the biosensor; the porous support layer is loaded on the analyte filtration layer, plays a mechanical support role, increases the mechanical strength of the membrane, and prevents the bioactive functional layer from being squeezed and affecting the performance of the bioactive functional layer; the analyte filtration layer adjusts the transmittance of the analyte, improves the linear range of the biosensor detection and the service life of the bioactive functional layer; the bioactive functional layer improves the selectivity and specificity for the test target; the graphene derivative layer can increase the specific surface area of the conductive substance, thereby increasing the enzyme loading rate, reducing the penetration of toxic substances in the electrode material, reducing interference, and reducing the energy consumption of the biosensor.

[0018] The multi-layer composite structure of the composite filtration membrane of the present invention, through the selection of materials for each layer and the cooperation of the structures, can reduce the penetration of toxic substances in the electrode out of the composite filtration membrane to generate interference signals, reduce cytotoxicity, improve the biocompatibility of the biosensor, and thus improve the service life.

[0019] Preferably, the material of the biocompatible hydrophilic layer is selected from one or more of collagen, sodium hyaluronate, and elastin. The biocompatible hydrophilic layer is made of biological materials, has good hydrophilicity, can improve the biocompatibility of the outer membrane, and does not affect the transmittance of oxygen or glucose. Specifically, polyethylene glycol and polyvinylpyrrolidone can also be added during preparation. These are widely used in medicine and human implant materials and are very safe biological materials.

[0020] Preferably, the material of the porous support layer is selected from one or more of polysulfone or its derivatives; the pore size of the porous support layer is 10 nanometers to 1000 nanometers, which increases the mechanical strength of the membrane.

[0021] Preferably, the analyte filtration layer comprises a hydrophilic material and an oxygen-permeable polymer material, and the hydrophilic material and the oxygen-permeable polymer material are blended or copolymerized to form the analyte filtration layer. In the present invention, the test object is glucose, and the analyte filtration layer is a glucose-permeable and oxygen-permeable layer, which resists hydrolysis and oxidation.

[0022] Preferably, the hydrophilic material is a polyurethane block copolymer selected from one or two of polyurethane block copolymers containing PVP segments and cyclodextrin segments; the oxygen-permeable polymer material is a polyurethane block copolymer containing silicone rubber segments. The oxygen-permeable polymer material can be natural rubber, silica gel or a mixture of both.

[0023] The analyte filtration layer comprises a continuous phase and a dispersed phase. The dispersed phase is a hydrophilic dispersed phase, and the continuous phase comprises a soft segment composed of siloxane. The compatibility of the continuous phase and the dispersed phase is increased by the hard segment component in the polyurethane block copolymer to prevent phase separation between the hydrophilic soft segment and the hydrophobic soft segment. The polyurethane block copolymer containing PVP segments has better hydrolysis resistance and antioxidant properties and good stability compared with the PEG segment polyurethane, and is a good physically cross-linked hydrophilic polyurethane material; the polyurethane containing siloxane is an elastomeric polyurethane with good hydrophobicity and oxygen permeability. Both PVP and silica gel are materials with good biocompatibility.

[0024] Preferably, since the glucose concentration in the human body is much higher than the oxygen concentration, the content ratio of silicone in the formula is higher than the content ratio of the PVP segment. The content ratio of the PVP segment to the silicone soft segment is 0.1-4:5-12, preferably 0.5-3:6-10. The polyurethane block copolymer of the silicone rubber segment is used as the continuous phase, and PU-b-PVP is used as the dispersed phase, so that the oxygen permeability can be controlled to be greater than the glucose permeability, hyperglycemia can be measured, and the linear range of the sensor is expanded to 35 mM / L.

[0025] By selecting the structures of the hard and soft segments of the polyurethane, the PU-b-PVP and the silicone rubber polyurethane have the same segments, and there is no need to use a third polyurethane to adjust their compatibility, thus improving the stability of the membrane.

[0026] Preferably, the material of the bioactive functional layer is a bioenzyme or a synthetic catalytic material.

[0027] The bioenzyme material can be glucose oxidase or lactate oxidase, and the corresponding enzyme is selected according to the test object; the synthetic catalytic materials are such as nano-gold and nano-silver.

[0028] Preferably, the graphene derivative layer is surface-modified graphene, and carboxyl, amino, hydroxyl or aldehyde groups are grafted on the surface of the graphene and coupled with a redox conductive polymer. Using a graphene derivative as a filter layer between the bioactive functional layer and the electrode can reduce the penetration of interfering substances to the electrode surface to generate interference signals. After the surface of the graphene film is derivatized and charged, it has good conductivity and specific surface area, which is beneficial to the adsorption and loading of enzymes; at the same time, the electrocatalytic activity of graphene can reduce polarization and lower the working voltage of the biosensor.

[0029] Graphene belongs to two-dimensional nanomaterials and is a good conductor. In the present invention, a graphene derivative is used as a filter layer between the bioactive functional layer and the electrode. After the surface of the graphene film is derivatized and charged, it has both catalytic properties and good conductivity and specific surface area, which is beneficial to the adsorption and loading of enzymes. The electrocatalytic activity of graphene can reduce polarization and lower the working voltage of the sensor.

[0030] A preparation method of a composite filter membrane for a biosensor includes the steps:

[0031] (1) Preparation method of the graphene derivative layer

[0032] Prepare a graphene oxide solution. For the surface-treated sensor electrode, deposit a layer of graphene on the platinum electrode by chemical vapor deposition (CVD) or electrochemical method, with a thickness between 5 nm and 100 nm, preferably 20 - 50 nm. After preparing a certain thickness of graphene oxide, modify carboxyl groups on the graphene oxide (GO) with succinyl peroxide or treat it with PLASMA plasma, or use CO2 Or N2 , NH3 Atmosphere to modify carboxyl or amino groups.

[0033] (2) Prepare a solution of the bioactive functional layer, coat the solution of the bioactive functional layer on the surface of the graphene derivative layer obtained in step (1), and crosslink; the material of the bioactive functional layer is a bioenzyme or a synthetic catalytic material.

[0034] (3) Preparation of the analyte filter layer

[0035] Mix a polyurethane block copolymer containing a PVP segment and a polyurethane block copolymer containing a siloxane in a certain proportion to form a homogeneous clear solution of the filter layer;

[0036] Adopt the dip-coating method to dip-coat the analyte filter layer on the surface of the bioactive functional layer in step (2), and dry it naturally or dry and crosslink it under vacuum. The temperature should not be higher than 37 °C to prevent the enzyme from being inactivated at high temperature. The coating method can also be spraying.

[0037] (4) Preparation of the porous support layer

[0038] Prepare a solution of polysulfone or its derivative material, mix and dissolve it with PVP to form a dipping solution. Dip the analytical substance filter layer in step (3) into the dipping solution, take it out and air-dry it naturally for 30 s - 2 min, then immerse it in water for 3 - 10 min, take it out and air-dry it naturally to obtain a porous support layer.

[0039] (5) Fabrication of biocompatible hydrophilic layer

[0040] Dissolve a biomaterial and a crosslinking agent in water to form a mixed solution. Dip the surface of the porous support layer in step (4) into the mixed solution, take it out and air-dry it naturally for 5 - 20 h, then soak it in water for 30 min - 3 h, and dry it to obtain a biosensor containing a composite filter membrane.

[0041] Preferably, the graphene layer is surface-modified with carboxyl, amino, hydroxyl or aldehyde groups by a chemical method or a plasma treatment method, and then a redox conductive polymer is modified by a coupling reaction, such as Poly(Aniline Quinone / Hydroquinone), polyaniline or a conductive polymer containing osmium or iron.

[0042] Further, in step (1), the concentration of the graphene oxide solution is 0.5 - 5 g / L, preferably 0.5 - 3 g / L; the thickness of the graphene layer is 5 nm - 100 nm, preferably 20 - 50 nm; in step (2), the bioactive functional layer solution includes BSA and a bio-enzyme, and the mass ratio of BSA to the bio-enzyme is 0.1~5:0.1~5, preferably 0.5~3:0.5~3; the mass concentration of the bioactive functional layer solution is 5 - 15%, preferably 7 - 14%; the crosslinking temperature is 25 - 37 °C, and the crosslinking time is 20 - 50 minutes, preferably 25 - 45 minutes; in step (3), the mass ratio of the polyurethane block copolymer containing a PVP segment to the polyurethane block copolymer containing a silicone rubber segment is 0.1~5:0.1~5, preferably 0.5~3:0.5~3; the mass concentration of the filter layer solution is 0.1 - 8%, preferably 1 - 6%; in step (4), the mass concentration of the polysulfone or its derivative material solution is 10 - 30%, preferably 15 - 25%; the content of PVP is 0.1 - 4%, preferably 1 - 3%; the dissolution time is 15 - 25 hours, preferably 18 - 23 hours; the time for volatilizing the solvent is 30 s - 2 min, and the immersion time in water is 3 - 10 min.

[0043] Preferably, in step (5), the biomaterial is selected from one, two or more of collagen, hyaluronic acid, sodium hyaluronate and elastin; the crosslinking agent is selected from one or more of glutaraldehyde, ADH, waterborne polycarbodiimide and waterborne isocyanate.

[0044] Further, the biomaterial at least comprises sodium hyaluronate and collagen, and the mass ratio of the sodium hyaluronate to the collagen is 0.1-10:0.1-10, preferably 0.1-5:1-10; the concentration of the mixed solution in the step (5) is 0.1-20%, preferably 5%-15%; the concentration of the crosslinking agent is 0.01%-1%, the time for volatilizing the solvent is 5-20 h, preferably 8-16 h; the soaking time in water is 30 min-3 h.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] First, for the biosensor of the present invention, the graphene derivative layer loaded on the electrode reduces the leakage of toxic substances from the electrode to the membrane surface, reduces interference, and reduces the cytotoxicity of the outer membrane; the analyte filtration layer improves the linear range of sensor detection and the service life of the bioactive functional layer; the porous support layer increases the mechanical strength of the membrane and prevents the bioactive functional layer from being extruded; the biocompatible hydrophilic layer improves biocompatibility; through the structural cooperation of the biocompatible hydrophilic layer, porous support layer, analyte filtration layer, bioactive functional layer and graphene derivative layer of the composite filtration membrane of the present invention, and the selection of materials for each layer, the leakage of toxic substances from the electrode material outside the composite filtration membrane is prevented, the cytotoxicity is reduced, the rejection reaction of the organism is reduced, the biocompatibility of the biosensor is improved, and thus the service life is improved; it well solves the contradiction between the filtration performance and biocompatibility of the outer membrane of the traditional sensor and the mechanical properties, antioxidant property and hydrolysis resistance of the outer membrane.

[0047] Second, the materials of the analyte filtration layer, porous support layer and biocompatible hydrophilic layer of the present invention all contain the same hard segment structure, which improves the composite force between the layers; at the same time, the graphene derivative layer is grafted with hydrophilic functional groups, which improves the binding force with the bioactive functional layer; the porous structure of the porous support layer improves the binding force between two adjacent layers of the analyte filtration layer and the biocompatible hydrophilic layer; the layers of the composite filtration layer of the present invention are tightly combined, preventing the composite filtration membrane from cracking, and improving the stability and service life of the membrane.

[0048] Third, through the selection of the continuous phase and dispersed phase materials of the analyte filtration layer of the present invention, and the selection of the structures of the hard and soft segments of the polyurethane block copolymer, the PU-b-PVP and the silicone rubber polyurethane structures have the same chain segments, which improves the material compatibility between the continuous phase and the dispersed phase, and does not require the use of a third polyurethane to adjust their compatibility, improves the stability of the membrane, and thus improves the service life of the composite filtration membrane.

[0049] Fourth, in the analyte filtration layer of the present invention, the content ratio of the silicone rubber segment is higher than that of the PVP segment. The polyurethane block copolymer of the silicone rubber segment serves as the continuous phase, and PU-b-PVP serves as the dispersed phase. In this way, the oxygen permeability can be controlled to be greater than the glucose permeability, enabling the measurement of high blood sugar, and the linear range of the sensor is extended to 35 mM / L.

[0050] Fifth, the biosensor with the composite filtration membrane of the present invention can operate for 20 days at a glucose concentration of 30 mM; moreover, the composite filtration membrane of the present invention has strong process operability.

[0051] Of course, it is not necessary for any product implementing the present invention to simultaneously achieve all the above-mentioned advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 is a schematic diagram of the composite filtration membrane of Example 1 of the present invention;

[0053] Figure 2 is an infrared spectrum test chart of the graphene derivative layer of Example 1 of the present invention;

[0054] Figure 3 is an HNMR chart of PU-b-PVP of Example 1 of the present invention;

[0055] Figure 4 is a SEM cross-sectional view of the porous support layer of Example 1 of the present invention;

[0056] Figure 5 is a SEM surface view of the porous support layer of Example 1 of the present invention;

[0057] Figure 6 is a linear test chart of the biosensor of Example 2 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0058] Regarding the biosensor, the relevant prior art research is as follows:

[0059] Patents CN1219676, CN108931568A, and CN2372689 disclose a sensor outer membrane structure in the form of a test strip. The stability and filtration performance of this outer membrane are unstable and cannot meet the requirements of the outer membrane of a continuous blood glucose meter implanted under the subcutaneous tissue. The solutions in US20020123087A1, US8050731, CN104761697A, and CN101018599 are to mix more than 3 polyurethane materials, among which 2 polyurethanes contain PEG segments and siloxane segments respectively. Because the above 2 materials have poor compatibility, a third polyurethane is required as the continuous phase, and PEG polyurethane and siloxane polyurethane are used as the 2 dispersed phases. After the 3 materials are blended, there is no chemical cross-linking or physical cross-linking. Due to the compatibility problem of the materials, the outer membrane will have defects. As time goes by, under the action of environmental stress, the outer membrane will have defects, cracks will appear, the integrity of the membrane will be damaged, the function of the membrane will be lost, and the service life of the sensor will be affected.

[0060] In addition, the existing sensor outer membranes are all single-layer structures, with a single function of the outer membrane. Toxic substances between the outer membrane and the electrode will also penetrate through the outer membrane. In particular, the toxic substances in the printed electrode ink will penetrate out, affecting the biocompatibility of the sensor. In the existing outer membrane polyurethane, the PEG and siloxane soft segment components are more, and the outer membrane material is very soft. When implanted under the subcutaneous tissue, due to the extrusion of the tissue, the outer membrane cannot play a supporting role, and the spatial structure of the enzyme layer is easily damaged by external forces, affecting the activity and efficiency of the enzyme and reducing the service life.

[0061] For the polyurethane material containing PEG segments, because the PEG segments are easy to hydrolyze and not antioxidant, the outer membrane will be damaged due to the hydrolysis or oxidation of PEG after a long implantation time, losing the function of the outer membrane. To solve this hydrolysis and antioxidant problem, patent US20030042137A1 uses a nitrogen-containing water-absorbing polymer polyvinylpyridine. Although the stability of the outer membrane in the body fluid environment is improved, the compatibility of this material with oxygen-permeable materials is poor, and it cannot be blended with siloxane to make a complete outer membrane. The oxygen permeability is too poor. For the first-generation sensors that require oxygen to participate in electron transfer, the reduction of oxygen content will affect the accuracy of the test results and the test linear range, especially when the blood sugar is high, the test is inaccurate.

[0062] Patent WO2017089380A1 provides a method of adding PTFE particles or enzyme particles to a continuous structure material and dispersing them in a polymer material to form a porous outer membrane structure. Since PTFE and enzymes are prone to aggregation and difficult to disperse, the pore size of the outer membrane made is very large. It is difficult to control the pore size in the process, and the spatial distribution is uneven. It is difficult to achieve the effect of controlling the glucose permeability. Moreover, the compatibility between the continuous material and PTFE and enzymes is very poor and it is difficult to disperse evenly. The formed outer membrane will be incomplete due to phase separation, and the mechanical properties are poor and it is easy to break, losing the function of the outer membrane.

[0063] Currently, in sensors, the bioactive functional layer is directly fixed on the electrode material. However, this has a drawback that the combination between the bioactive functional layer and the electrode is not firm, the loading rate is not high, and interfering substances can easily contact and react with the electrode through the outer membrane to generate interference signals. Small molecule substances released from the electrode material can affect the activity of enzymes. Therefore, a filter membrane needs to be added between the bioactive functional layer and the electrode. Patent CN103462615B uses cellulose acetate (CTA) as this filter membrane layer. Since CTA is not resistant to H2O2 oxidation, its service life is not long, about 7 days.

[0064] The working environment where the outer membrane is located requires a balance between hydrophilicity and hydrolysis resistance, between transmittance and mechanical properties, and between filtration function and biocompatibility for the outer membrane. To solve the contradictions among material compatibility, filtration, mechanical properties, antioxidant property, and hydrolysis resistance, the present invention provides a composite filter membrane for a biosensor. The composite filter membrane is loaded on the surface of the electrode of the biosensor. The structure of the composite filter membrane from the outside to the inside sequentially includes: a biocompatible hydrophilic layer, a porous support layer, an analyte filtration layer, a bioactive functional layer, and a graphene derivative layer.

[0065] Among them, the continuous phase and the dispersed phase of the analyte filtration layer increase the material compatibility through the hard segment component in the polyurethane block copolymer, preventing phase separation between the hydrophilic soft segment and the hydrophobic soft segment. The polyurethane block copolymer containing a PVP segment has better hydrolysis resistance and antioxidant properties, good stability, and is a good physically cross-linked hydrophilic polyurethane material; the polyurethane containing siloxane is an elastomeric polyurethane with good hydrophobic oxygen permeability, which solves the contradictions among material compatibility, antioxidant property, and hydrolysis resistance. By providing a porous support layer on the analyte filtration layer, the mechanical properties of the membrane are improved. At the same time, by providing a graphene derivative layer between the electrode and the bioactive functional layer, and a biocompatible hydrophilic layer on the surface of the porous support layer, the toxic substances exuded from the electrode are prevented from passing through the outer membrane, the cytotoxicity is reduced, and the biocompatibility is improved.

[0066] The multi-layer composite structure of the composite filter membrane of the present invention improves the compatibility of each layer structure through material selection, and at the same time improves the compatibility between the functional groups on the graphene derivative layer and the hydrophilic material of the analyte filtration layer; and the porous structure of the porous support layer improves the bonding force with the analyte filtration layer and the biocompatible hydrophilic layer, prevents the composite filter membrane from cracking, and improves the service life of the sensor.

[0067] In this text, a range expressed as "from one numerical value to another numerical value" is a summary representation method to avoid listing all the numerical values within this range one by one in the specification. Therefore, the description of a specific numerical range encompasses any numerical value within this range and the smaller numerical ranges defined by any numerical values within this range, as if such arbitrary numerical values and smaller numerical ranges were clearly written in the specification.

[0068] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the protection scope of the present invention. Improvements and adjustments made by those skilled in the art according to the present invention in actual applications still fall within the protection scope of the present invention.

[0069] Example 1 of the bioactive functional layer

[0070] The biosensor of this embodiment uses a printed flexible electrode with a three - electrode system. The insulating material is PI. The working electrode and the counter electrode are printed with gold paste, and the silver electrode is used as the reference electrode. A layer of platinum black is electroplated on the working electrode and the counter electrode. A composite filter membrane is prepared outside the working electrode, the counter electrode and the reference electrode. As Figure 1 shown, the composite filter membrane is prepared on the surface of electrode 1. The structure of the composite filter membrane from the outside to the inside successively includes: a biocompatible hydrophilic layer 1 - 5, a porous support layer 1 - 4, an analyte filtration layer 1 - 3, a bioactive functional layer 1 - 2, and a graphene derivative layer 1 - 1. The composite filter membrane undergoes the following manufacturing process:

[0071] The first step. Preparation of the graphene derivative layer:

[0072] The graphene oxide used is prepared by the improved Hummers method:

[0073] Take an appropriate amount of graphene oxide, add it to a certain amount of deionized water, and perform ultrasonic dispersion to prepare a 1 g / L graphene oxide solution for later use. Using the cleaned working electrode of the sensor as the positive electrode and the platinum electrode as the negative electrode, deposition and film - forming are carried out in the above - prepared solution for 300 seconds. After taking it out and drying it, and then curing it, a graphene oxide film layer is deposited on the surface of the sensor.

[0074] The thickness of the graphene oxide film layer is between 5 nm and 100 nm, preferably 20 - 50 nm.

[0075] After preparing a certain thickness of graphene oxide, carboxyl groups are modified on the graphene oxide (GO) with succinyl peroxide.

[0076] Specifically, the sensor coated with the graphene oxide film layer is immersed in a DMF solution of succinyl peroxide at 5 g / L and reacted at 85 °C for 72 h. After the reaction, it is ultrasonically treated with pure water for 5 min. Or it is treated with PLASMA plasma and modified with carboxyl or amino groups using CO2 Or N2 , NH3 atmosphere.

[0077] The prepared biosensor sample is tested by ATR of FT-IR, using a. succinic acid (SA), b. succinyl peroxide, c. GO (graphene oxide), d. GO-COOH (grafted carboxyl graphene oxide). The test results are shown in Figure 2 as follows.

[0078] In the infrared spectrum test, for the four curves of a, b, c, and d, the d curve has characteristic peaks of graphene oxide ( 1735cm-1 and 1125cm-1 ), indicating that there is graphene on the electrode surface; and the peak at 3443cm-1 indicates that the derivative of graphene on the electrode surface has a carboxyl group.

[0079] The graphene derivative layer is a surface modification of graphene. The graphene derivative layer can increase the specific surface area of the conductive substance, thereby increasing the enzyme loading rate. Graphene has a high conductivity, which can reduce the working voltage and reduce the interference signal generated by the electrochemical reaction of interfering substances, reducing the energy consumption of the sensor. At the same time, the surface of graphene is modified to be charged, and the pore size is in the nanometer range. Without affecting the penetration of the test substance, it can reduce the penetration of charged interfering substances through graphene and contact with the platinum electrode to generate an electrochemical reaction, thereby reducing the interference signal and improving the test accuracy. Because of the filtering function of graphene, the toxic substances of the electrode material are reduced from permeating out of the graphene layer and affecting the bioactive functional layer and biocompatibility.

[0080] Step 2. Preparation of the glucose oxidase layer:

[0081] Using a phosphate buffer solution of BSA and glucose oxidase, the mass ratio of BSA to glucose oxidase is 1:1, and the total concentration is 10% (m / v). Add 0.01% glutaraldehyde and coat it on the graphene derivative layer prepared in the first step. Print it 3 times with an inkjet printer and then crosslink it at 37 °C for 30 min.

[0082] Step 3. Preparation of the analyte filtration layer:

[0083] Synthesize PU-b-PVP. Dissolve a mixture of the synthesized PU-b-PVP and TPSE345 (thermoplastic silicone elastomer, Wacker Chemie AG, Germany) with a mass ratio of 1:1 in THF. The prepared solution has a mass concentration of 3%. Stir it at room temperature for 5 days and then filter it to obtain the filtration layer solution for standby.

[0084] The filtration layer solution was coated on the glucose oxidase layer prepared in the second step by dip coating. After dip coating, it was volatilized and dried in a THF atmosphere for 5 min, and dip coating was performed 5 times.

[0085] The synthesis route and process parameters of the synthesized PU-b-PVP are as follows:

[0086]

[0087] The HNMR spectrum of the synthesized PU-b-PVP is as Figure 3 shown. The CH2 nuclear magnetic peaks of the PVP segment are between 1.85 and 2.50 ppm, and the synthesis peaks of CH are between 4.35 and 4.50 ppm. According to the H spectrum, the characteristic peaks of PVP can be seen, proving the presence of the PVP segment.

[0088] A tetrahydrofuran solution containing 3% PU-b-PVP was prepared, and then cast into a 10-μm-thick film. It was cut into rectangular samples with a width of 2 cm and a length of 8 cm. The mass of the sample was weighed, and after soaking in water for 3 min, it was taken out and weighed again. The mass difference before and after soaking divided by the mass of the sample before soaking was the water absorption rate. The water absorption rate of the sample was 100%, and the water absorption also indicated the presence of the PVP segment structure.

[0089] The water-absorbed sample was stretched with a 100-g weight for 5 min without breaking, indicating that the water-absorbed sample still had mechanical strength and was a physical cross-linking.

[0090] In the synthesized PU-b-PVP, the content of PVP is 20%, and the silane content of TPSE is 90%. The mass ratio of PVP to silane in the analytical substance filtration layer is 2:9. PU-b-PVP and TPSE have the same segments, and there is no need to use a third polyurethane to adjust their compatibility, solving the contradiction between the stability of the membrane and its antioxidant and hydrolysis resistance, and improving the stability of the membrane.

[0091] Step 4. Fabrication of the porous support layer:

[0092] The psf particles (solvaypsf 1700) were configured into a 20% (m / v) dry DMF solution, 2% PVP was added, and it was stirred and dissolved at room temperature for 20 h and then filtered. The filtered solution was used as the coating solution for standby.

[0093] The coating solution was coated on the analytical substance filtration layer prepared in the third step by dip coating. After dip coating, it was air-dried naturally for 2 min, then immersed in water for 30 min, taken out and air-dried naturally, and the fabrication of the porous support layer was completed.

[0094] The SEM cross-sectional view (magnified 500X) of the porous support layer is asFigure 4 As shown in the SEM surface image (magnified 5000X) of the porous support layer, Figure 5 As shown in the SEM images of the surface and cross-section, the porous support layer PSF has pores on its surface, which can increase the contact area between the porous support layer and the adjacent layer, and improve the bonding force between the porous support layer and the analytical material filtration layer and the biocompatible hydrophilic layer. At the same time, the porous structure has good oxygen and glucose permeability, and can improve the mechanical strength of the porous support layer.

[0095] Step 5. Preparation of the biocompatible hydrophilic layer:

[0096] A phosphate buffer solution of sodium hyaluronate and collagen, with a mass ratio of sodium hyaluronate to collagen of 2:8 and a total concentration of 10%, is added with 0.01% glutaraldehyde and printed onto the porous support layer obtained in the fourth step using an inkjet printer three times, and then crosslinked at 37°C for 30 min.

[0097] The preparation of the composite filtration membrane of the biosensor is completed.

[0098] Example 2

[0099] The fabricated biosensor was tested after polarization in a 50 mM glucose solution for 24 h. During the test, the glucose concentrations were 0, 4, 10, 15, 20, 25, and 30 mM.

[0100] The test results are as Figure 6 shown, with a very good linearity of 0.998 and a high sensitivity of k = 11.29.

[0101] Example 3

[0102] Cytotoxicity test of the outer membrane, MTT method, at 37°C, extraction for 72 h, and the cytotoxicity was 89.7% (the standard requires more than 70% to be qualified).

[0103] Example 4

[0104] The sensor of Example 1 was used for in vitro life test, that is, using the I-T method of an electrochemical workstation, the sensor was placed in 30 mM glucose and tested at 0.55 V for 20 days. The linearity and K value of the sensor were tested on the 0th day, the 10th day, and the 20th day respectively. The test results are shown in the following table:

[0105] Table 1

[0106] The data in Table 1 show that after 20 days of operation, the linearity and K value of the sensor changed very little, indicating that the sensor can work for 20 days at a glucose concentration of 30 mM. The life of the sensor is mainly determined by the outer membrane. The life of similar sensors on the market is 15 days, and the sensor using the composite filtration outer membrane of the present invention has a longer life.

[0107] The above disclosure is only the preferred embodiment of the present invention. The preferred embodiment does not describe all the details in detail, nor does it limit the invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification to better explain the principles and practical applications of the present invention, so that those skilled in the art can make good use of the present invention. The present invention is only limited by the claims and their full scope and equivalents.

[0108] Under the teachings of the present invention and the above embodiments, those skilled in the art can easily foresee that the present invention can be implemented by each of the raw materials or their equivalent substitutes, each processing method or their equivalent substitutes listed or exemplified in the present invention, and the upper and lower limit values and interval values of the parameters of each raw material and processing method can also implement the present invention. Examples are not listed one by one here.

Claims

1. A composite filtration membrane for a biosensor, characterized in that, The composite filtration membrane is loaded on the surface of the electrode of the biosensor. The structure of the composite filtration membrane from the outside to the inside includes: a biocompatible hydrophilic layer, a porous support layer, an analyte filtration layer, a bioactive functional layer, and a graphene derivative layer; wherein, The materials of the analyte filtration layer, the porous support layer, and the biocompatible hydrophilic layer contain the same hard segment structure; The graphene derivative layer is grafted with hydrophilic functional groups; and, The analyte filtration layer contains a hydrophilic material and an oxygen-permeable polymer material. The hydrophilic material and the oxygen-permeable polymer material are blended or copolymerized to form the analyte filtration layer. The hydrophilic material is a polyurethane block copolymer containing a PVP segment, and the oxygen-permeable polymer material is a polyurethane block copolymer containing a siloxane soft segment. The polyurethane block copolymer containing a siloxane soft segment serves as the continuous phase, and the polyurethane block copolymer containing a PVP segment serves as the dispersed phase, and the content ratio of the siloxane soft segment is higher than the content ratio of the PVP segment.

2. The composite filtration membrane according to claim 1, wherein The material of the biocompatible hydrophilic layer is selected from one or a combination of collagen, sodium hyaluronate, and elastin.

3. The composite filtration membrane according to claim 1, characterized in that, The material of the porous support layer contains one or more of polysulfone or its derivatives; the pore size of the porous support layer is 10 nanometers to 1000 nanometers.

4. The composite filter membrane according to claim 1, wherein The content ratio of the PVP segment to the siloxane soft segment is 0.1~4:5~12.

5. The composite filtration membrane according to claim 1, wherein The material of the bioactive functional layer is a bioenzyme or a catalytic material; the bioenzyme is selected from one of glucose oxidase or lactate oxidase, and the catalytic material is selected from nano-gold or nano-silver.

6. The composite filter membrane according to claim 1, wherein The thickness of the graphene derivative layer is 5nm - 100nm; the graphene surface is grafted with carboxyl, amino, hydroxyl, or aldehyde groups, and is coupled with a redox conductive polymer.

7. A method for preparing a composite filtration membrane of the biosensor according to any one of claims 1-6, characterized in that, Including steps: (1) Prepare a graphene oxide solution and coat it on the surface of the electrode of the biosensor to obtain a graphene layer, and perform surface modification on the graphene layer to obtain a graphene derivative layer; (2) Prepare a solution of the bioactive functional layer and coat it on the surface of the graphene derivative layer obtained in step (1), and crosslink to obtain a bioactive functional layer; (3) Mix the polyurethane block copolymer containing a PVP segment and the polyurethane block copolymer containing a siloxane soft segment in a predetermined ratio to form a uniform filtration layer solution, coat it on the surface of the bioactive functional layer in step (2), and dry to obtain an analyte filtration layer; (4) Prepare a solution of polysulfone or its derivative material, mix and dissolve it with PVP to form a dipping solution, coat it on the surface of the analyte filtration layer prepared in step (3), volatilize the solvent, then immerse it in water, take it out and dry to obtain a porous support layer; (5) Dissolve the biological material and the crosslinking agent in water to form a mixed solution, coat it on the surface of the porous support layer prepared in step (4), volatilize the solvent, then soak it in water, and dry to obtain a biosensor containing a composite filtration membrane.

8. The preparation method according to claim 7, characterized in that, In step (1), the graphene layer is surface-modified with carboxyl, amino, hydroxyl, or aldehyde groups by a chemical method or a plasma treatment method, and then modified with a redox conductive polymer through a coupling reaction.

9. The preparation method according to claim 7, wherein In the step (1), the concentration of the graphene oxide solution is 0.5-5 g / L, and the thickness of the graphene layer is 5 nm-100 nm; in the step (2), the bioactive functional layer solution includes BSA and bioenzyme, and the mass ratio of BSA to the bioenzyme is 0.1-5:0.1-5. The mass concentration of the bioactive functional layer solution is 5-15%, the crosslinking temperature is 25-37 °C, and the crosslinking time is 20-50 minutes; in the step (3), the mass ratio of the polyurethane block copolymer containing PVP segments to the polyurethane block copolymer containing siloxane soft segments is 0.1-5:0.1-5; the mass concentration of the filter layer solution is 0.1-8%; in the step (4), the mass concentration of the polysulfone or its derivative material solution is 10-30%, the content of PVP is 0.1-4%, the dissolution time is 15-25 hours, the time for volatilizing the solvent is 30 s-2 min, and the soaking time in water is 3-10 min.

10. The preparation method according to claim 7, characterized in that, In the step (5), the biological material is selected from one or more of collagen, hyaluronic acid, sodium hyaluronate, and elastin; the crosslinking agent is selected from one or more of glutaraldehyde, ADH, aqueous polycarbodiimide, or aqueous isocyanate.

11. According to the preparation method described in claim 10, wherein The biological material at least includes sodium hyaluronate and collagen, and the mass ratio of sodium hyaluronate to collagen is 0.1-10:0.1-10. The concentration of the mixed solution in the step (5) is 0.1-20%, the concentration of the crosslinking agent is 0.01%-2%, the time for volatilizing the solvent is 5-20 h, and the soaking time in water is 30 min-3 h.

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