Method for preparing a polymer film layer for a biosensor

Abstract

The present disclosure relates to a method for producing a polymer film layer for a biosensor, including: preparing a base layer reagent including a first type of polymer, a bonding layer reagent including a second type of polymer, and an outer layer reagent including a third type of polymer, the second type of polymer being a copolymer formed from a first monomer identical to a monomer of the first type of polymer and a second monomer identical to a monomer of the third type of polymer; and sequentially forming the base layer, a bonding layer having a bonding effect on the base layer, and an outer layer having biocompatibility on the bonding layer, wherein the bonding layer has diffusion control properties in the polymer film layer, and the bonding layer is bonded to the base layer and the outer layer by at least one of a conjugation effect, a like dissolves like effect, a hydrogen bond interaction, and cross-linking. Thus, a method for producing a polymer film layer for a biosensor having biocompatibility and capable of expanding a response linear range can be provided.

Description

[0001] This application was filed on [date]. September 1, 2020 The application number is 202010906747.9, and the invention title is... biology Sensors, their fabrication methods, and polymer films for biosensors A divisional application of the patent application. Technical Field

[0002] This disclosure relates to the field of biosensors, and more specifically to a method for preparing a polymer membrane for a biosensor. Background Technology

[0003] Biosensors are analytical devices that tightly integrate biological materials, bio-derived materials, or biomimetic materials with physicochemical sensors or sensing microsystems that are optical, electrochemical, temperature, piezoelectric, magnetic, or micromechanical. They are typically used to rapidly detect certain specific chemical substances in the human body, such as glucose, urea, uric acid, and a series of amino acid compounds.

[0004] Taking an implantable current sensor as an example, the working electrode typically includes a sensing layer that is in direct contact with the electrode conductive layer. When a chemical substance reaches the surface of the sensing layer and is consumed, there is a certain linear relationship between the detected current value and the concentration of the chemical substance. However, when the chemical substance consumption capacity is limited by the electrochemical kinetics of the sensing layer, the output current value is no longer linearly related to the concentration of the chemical substance reaching the surface of the sensing layer. Therefore, it is necessary to control the concentration of the chemical substance reaching the sensing layer to expand the linear range of the implantable current sensor's response to the chemical substance, thereby enabling the implantable current sensor to detect higher glucose concentrations. Moreover, since the implantable current sensor needs to be partially implanted in the body and in direct contact with the tissue, the part in contact with the tissue is required to have very good biocompatibility. Summary of the Invention

[0005] This disclosure is made in view of the above circumstances, and its purpose is to provide a biosensor capable of expanding the linear range of response and having biocompatibility, a method for preparing the same, and a polymer film layer for the biosensor.

[0006] Therefore, a first aspect of this disclosure provides a biosensor for detecting a analyte in vivo, comprising: a substrate, an electrode disposed on the substrate, a sensing enzyme layer disposed on the electrode, and a polymer film layer disposed on the sensing enzyme layer, the polymer film layer being used to control the diffusion of the analyte, the polymer film layer comprising a base layer, an adhesive layer formed on the base layer, and a biocompatible outer surface layer formed on the adhesive layer, the base layer being formed of a first type of polymer, the adhesive layer being formed of a second type of polymer, and the outer surface layer being formed of a third type of polymer, wherein the first type of polymer is a homopolymer having a benzene ring or heterocyclic ring, the second type of polymer is a copolymer formed of a first monomer that is the same as or similar to the monomer of the first type of polymer and a second monomer that is the same as or similar to the monomer of the third type of polymer, wherein the mass ratio of the first monomer to the second monomer is 3:7 to 7:3, and the thickness of the adhesive layer in the polymer film layer accounts for 40% to 50% of the thickness of the polymer film layer.

[0007] In the biosensor disclosed in the first aspect, by providing a polymer film layer for controlling the diffusion of the analyte on the sensing enzyme layer of the biosensor, the linear range of the biosensor response can be expanded, and the biosensor is made biocompatible by having a biocompatible outer layer of the polymer film layer.

[0008] Furthermore, in the biosensor disclosed in the first aspect, optionally, the first type of polymer is a water-swellable homopolymer, the second type of polymer is a water-swellable copolymer, and the third type of polymer is a water-soluble polymer. This can improve the diffusion control performance and biocompatibility of the polymer film, thereby helping to expand the linear response range of the biosensor and improve its biocompatibility.

[0009] Furthermore, in the biosensor disclosed in the first aspect of this invention, optionally, the water-swellable homopolymer is selected from polystyrene, polyurethane, ethoxyethyl polyacrylate, ethoxypropyl polyacrylate, poly-2-vinylpyridine, poly-4-vinylpyridine, polyhydroxyethyl methacrylate, and polyhydroxyethyl polyacrylate; and the water-soluble polymer is selected from polyvinylpyrrolidone, polyvinyl alcohol, chitosan, carboxymethyl chitosan, chitosan salt, alginate, alginate, hyaluronic acid, hyaluronic acid salt, cellulose ethers, cellulose esters, polyvinylpyrrolidone, polyacrylamide, polyacrylic acid, polyacryl alcohol, sodium polystyrene sulfonate, polyethylene glycol, and polyethylene glycol-polypropylene glycol copolymer. Thus, a polymer film layer with diffusion control properties and biocompatibility can be formed, thereby expanding the linear response range of the biosensor and improving its biocompatibility.

[0010] Additionally, in the biosensor disclosed in the first aspect of this invention, optionally, the water-swellable copolymer is selected from polyethylene glycol-block polystyrene, polyacrylic acid-block polystyrene, polyacrylic acid-copolymer-polystyrene, polyacrylamide-block polystyrene, polyacrylamide-copolymer-polystyrene, poly(2-vinylpyridine)-block polystyrene, poly(4-vinylpyridine)-copolymer-polystyrene, poly(4-vinylpyridine)-copolymer-polyvinylpyrrolidone, poly(2-vinylpyridine)-copolymer-polystyrene, poly(4-vinylpyridine)-block polystyrene, poly(4-vinylpyridine)-copolymer-polyacrylamide, poly(ethoxyethyl acrylate)-copolymer-hydroxyethyl acrylate, and poly(ethoxypropyl acrylate)-copolymer-polyvinyl alcohol. In this case, an adhesive layer with enhanced adhesion to the substrate layer and the outer surface layer can be formed, which facilitates the formation of the polymer film layer, thereby helping to expand the linear response range of the biosensor and improve the biocompatibility of the biosensor.

[0011] Furthermore, in the biosensor according to the first aspect of this disclosure, optionally, the molecular weight of the water-swellable homopolymer is 50,000 to 500,000 Da, the molecular weight of the water-swellable copolymer is 10,000 to 50,000 Da, and the molecular weight of the water-soluble polymer is 2,000 to 50,000 Da. This facilitates the formation of the polymer film layer, thereby helping to expand the linear response range of the biosensor.

[0012] Additionally, in the biosensor according to the first aspect of this disclosure, optionally, in the polymer film layer, the thickness of the base layer accounts for 30% to 40% of the thickness of the polymer film layer, and the thickness of the outer surface layer accounts for 20% to 30% of the thickness of the polymer film layer. In this case, the combination of the base layer, the adhesive layer, and the outer surface layer can further enhance the diffusion control performance of the polymer film layer.

[0013] Furthermore, in the biosensor according to the first aspect of this disclosure, optionally, the adhesive layer is bonded to the substrate layer and the outer surface layer through at least one of the following mechanisms: conjugation effect, similarity-compatibility, hydrogen bonding interaction, and crosslinking. This strengthens the bond between the adhesive layer and the substrate layer and the outer surface layer, thereby improving the diffusion control performance of the polymer film and, consequently, expanding the linear response range of the biosensor.

[0014] Furthermore, in the biosensor according to the first aspect of this disclosure, optionally, the substrate layer, the adhesive layer, and the outer surface layer are each cross-linked by the same cross-linking agent, wherein the cross-linking agent is at least one selected from active esters, epoxides, and sulfates. In this case, the substrate layer, adhesive layer, and outer surface layer can be bonded together through cross-linking, which can help improve the diffusion control performance of the polymer film layer, and thus help expand the linear response range of the biosensor.

[0015] A second aspect of this disclosure provides a method for fabricating a biosensor, comprising: preparing a substrate and disposing an electrode on the substrate; disposing a sensing enzyme layer on the electrode; and disposing a polymer film layer on the sensing enzyme layer, wherein the polymer film layer is prepared by the following steps: preparing a base layer reagent comprising a first type of polymer, an adhesive layer reagent comprising a second type of polymer, and an outer surface layer reagent comprising a third type of polymer, wherein the first type of polymer is a homopolymer having a benzene ring or heterocyclic structure, the second type of polymer is a copolymer formed from a first monomer that is the same as or similar to the monomer of the first type of polymer and a second monomer that is the same as or similar to the monomer of the third type of polymer, wherein the mass ratio of the first monomer to the second monomer is 3:7 to 7:3; and sequentially forming a base layer for controlling the diffusion of chemical substances, an adhesive layer on the base layer and having an adhesive effect, and an outer surface layer on the adhesive layer and having biocompatibility, wherein, in the polymer film layer, the thickness of the adhesive layer accounts for 40% to 50% of the thickness of the polymer film layer.

[0016] In a second aspect of this disclosure, a polymer film layer for controlling the diffusion of the analyte and having a biocompatible outer layer is formed on the sensing enzyme layer of the biosensor, thereby enabling the formation of a biosensor with an expanded linear response range and biocompatibility.

[0017] A third aspect of this disclosure provides a polymer membrane for a biosensor, comprising: a base layer, an adhesive layer formed on the base layer, and a biocompatible outer surface layer formed on the adhesive layer, wherein the base layer is formed of a first type of polymer, the adhesive layer is formed of a second type of polymer, and the outer surface layer is formed of a third type of polymer, wherein the first type of polymer is a homopolymer having benzene rings or heterocycles, the second type of polymer is a copolymer formed of a first monomer that is the same as or similar to the monomer of the first type of polymer and a second monomer that is the same as or similar to the monomer of the third type of polymer, and the mass ratio of the first monomer to the second monomer is 3:7 to 7:3, and the thickness of the adhesive layer accounts for 40% to 50% of the thickness of the polymer membrane.

[0018] In a third aspect of this disclosure, the adhesive layer is formed of a second type of polymer, which is the same as or similar to the monomers of the first type of polymer and the same as or similar to the monomers of the third type of polymer. This improves the adhesion of the adhesive layer to the base layer and the outer layer, enhances the stability of the polymer film, and thus helps to improve the diffusion control performance of the polymer film. This allows for a fixed concentration ratio of the analyte on both sides of the polymer film, and the biocompatible outer layer improves the biocompatibility of the polymer film.

[0019] According to this disclosure, a biosensor capable of expanding the linear range of response and having biocompatibility, a method for preparing the same, and a polymer film layer for the biosensor can be provided. Attached Figure Description

[0020] Figure 1 This is a diagram illustrating an application scenario of the biosensor involved in the examples of this disclosure.

[0021] Figure 2 This is a partial schematic diagram showing the probe of a biosensor involved in an example of this disclosure.

[0022] Figure 3 This is a schematic diagram illustrating the structure of the polymer film layer involved in the example of this disclosure.

[0023] Figure 4 This is a flowchart illustrating a method for fabricating a biosensor as described in the examples of this disclosure.

[0024] Figure 5 This is a flowchart illustrating a method for preparing a polymer film layer as described in the examples of this disclosure.

[0025] Figure 6 The diagram shows the current curve measured by the glucose biosensor of Embodiment 1 of this disclosure.

[0026] Figure 7 It shows Figure 6 The graph shows the linear relationship between current and glucose concentration.

[0027] Figure 8 This is a stained section of a polymer film layer from Example 1 of this disclosure. Detailed Implementation

[0028] Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same reference numerals are used for the same components, and repeated descriptions are omitted. Furthermore, the drawings are merely schematic diagrams, and the proportions of the components or the shapes of the components may differ from actual figures.

[0029] Figure 1This is a diagram illustrating an application scenario of the biosensor 1 involved in the examples of this disclosure.

[0030] The biosensor 1 disclosed herein can be used to detect small molecule chemical substances in tissues and physiological environments in vivo, such as for detecting blood glucose (e.g., glucose sensor), uric acid (e.g., uric acid sensor), and cholesterol (e.g., cholesterol sensor).

[0031] In this embodiment, the biosensor 1 can be used to detect a analyte within the body. In other words, the biosensor 1 can be used to detect a analyte in bodily fluids. Furthermore, the analyte can be a chemical substance in bodily fluids. For example, the analyte can be glucose, acetylcholine, amylase, bilirubin, cholesterol, human chorionic gonadotropin (hCG), creatine kinase, creatine, creatine anhydride, DNA, fructosamine, glucose, glutamine, growth hormone, hormones, ketone bodies, lactate, oxygen, peroxides, prostate-specific antigen (PSA), prothrombin, RNA, thyroid-stimulating hormone (TSH), or troponin. Additionally, the analyte can be a drug in bodily fluids, such as digoxin, theophylline, warfarin, or antibiotics (e.g., gentamicin, vancomycin, etc.).

[0032] In this embodiment, the biosensor 1 may include a probe P and an electronic system connected to the probe P. A portion of the probe P (particularly the sensing portion) may be implanted, for example, on the surface of a human body, in contact with tissue fluid within the body. Another portion of the probe P is connected to an electronic system located outside the body surface. When the biosensor 1 is operational, the probe P reacts with the tissue fluid or blood within the body to generate a sensing signal (e.g., an electrical signal), and transmits this sensing signal to the electronic system on the body surface. The electronic system processes the sensing signal to obtain the concentration of the analyte. Furthermore, although... Figure 1 The illustration shows the location where the biosensor 1 can be configured on the arm, but this embodiment is not limited to this. For example, the biosensor 1 can also be configured on the abdomen, waist, leg, etc.

[0033] Figure 2 This is a partial schematic diagram showing the probe P of the biosensor 1 involved in the example of this disclosure.

[0034] In some examples, probe P may include a substrate 10 and an electrode 20. In other words, biosensor 1 may include a substrate 10 and an electrode 20. The electrode 20 may be disposed on the substrate 10. In addition, the electrode 20 may serve as the sensing portion of probe P.

[0035] In some examples, substrate 10 may be a flexible substrate. The flexible substrate may be made generally of at least one of polyethylene (PE), polypropylene (PP), polyimide (PI), polystyrene (PS), polyethylene terephthalate (PET), and polyethylene terephthalate (PEN). Alternatively, in other examples, the flexible substrate may also be made generally of metal foil, ultrathin glass, a single-layer inorganic film, a multilayer organic film, or a multilayer inorganic film.

[0036] In some examples, substrate 10 may also be a non-flexible substrate. Non-flexible substrates may generally include materials with low conductivity, such as ceramics, alumina, or silicon dioxide. This allows for implantation into the body surface (e.g., superficial skin) without the need for auxiliary implantation devices (e.g., puncture needles).

[0037] In some examples, electrode 20 may include a working electrode. The working electrode can be used to acquire a current signal. Additionally, electrode 20 may include a counter electrode. In other examples, electrode 20 may also include a reference electrode, thereby enabling the formation of a three-electrode 20 sensor.

[0038] In some examples, electrode 20 may be made of platinum, silver, silver chloride, palladium, titanium, or iridium. This allows for good conductivity without affecting the electrochemical reaction of electrode 20. However, this embodiment is not limited to this; in other examples, electrode 20 may also be made of at least one selected from gold, glassy carbon, graphite, silver, silver chloride, palladium, titanium, or iridium. This reduces the impact on electrode 20 while maintaining good conductivity.

[0039] In some examples, the biosensor 1 may include a sensing enzyme layer. Additionally, the sensing enzyme layer may be disposed on the electrode 20 (which may be the working electrode). Furthermore, in the biosensor 1, the electrode 20 may be encapsulated or covered by the sensing enzyme layer. In some examples, the sensing enzyme layer may be formed on the electrode 20 (which may be the working electrode) by methods such as spin coating, dip-coating, drop coating, or spray coating.

[0040] In some examples, the sensing enzyme layer may contain a reactive enzyme. In some examples, the reactive enzyme in the sensing enzyme layer can be selected based on the analyte. For example, if the analyte is glucose, the reactive enzyme could be glucose oxidase or glucose dehydrogenase.

[0041] In some examples, the enzyme can serve as a detection substrate for the analyte. In other examples, the enzyme can react chemically with the analyte.

[0042] The following uses a glucose sensor as an example, with GO X (FAD) is used to describe the chemical reaction between glucose oxidase and glucose.

[0043] In the sensing enzyme layer, when GO X When (FAD) encounters glucose in tissues, the following reaction occurs:

[0044] Glucose + GOx(FAD) → Gluconolactone + GOx(FADH2) ... Reaction (I)

[0045] GOx(FADH2)+O2→GOx(FAD)+H2O2……Reaction (II).

[0046] In some examples, the electrode 20 is implanted into human skin, where the reactive enzyme can continuously react chemically with the test sample and convert it into a corresponding current signal, which is then transmitted to an external electronic system.

[0047] In some examples, the sensing enzyme layer may have a specific cross-linking agent. This allows the reactive enzyme to be immobilized on electrode 20 (working electrode). For example, the specific cross-linking agent may be pentylene glycol, bovine serum albumin-glutaraldehyde, polyepoxides, 1,4-butanediol diglycidyl ether, glutaraldehyde, active esters, acid anhydrides, azides, isocyanates, or acridines.

[0048] In some examples, the sensing enzyme layer may contain at least one of the following components: metal polymer, carbon nanotubes, graphene, porous titanium dioxide, and conductive organic salt.

[0049] In some examples, the thickness of the sensing enzyme layer can be from 0.1 μm to 100 μm. In other examples, preferably, the thickness of the sensing enzyme layer can be from 2 μm to 10 μm. In this case, controlling the thickness of the enzyme within a certain range avoids problems such as decreased adhesion due to excessive enzyme, causing material to detach in vivo, and also avoids problems such as insufficient reaction due to excessive enzyme, resulting in the inability to provide normal glucose concentration information.

[0050] In some examples, the thickness of the sensing enzyme layer can be 0.1 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 50 μm, 80 μm or 100 μm.

[0051] In some examples, the biosensor 1 may include a polymer membrane layer 30. Specifically, in the biosensor 1, a polymer membrane layer 30 may be disposed on the sensing enzyme layer. Additionally, the polymer membrane layer 30 may serve as a diffusion-limiting membrane. In other words, the polymer membrane layer 30 can be used to control the diffusion of the analyte.

[0052] In some examples, in biosensor 1, the sensing enzyme layer can be encapsulated or covered by the polymer membrane layer 30. This allows for control of the concentration of the analyte reaching the sensing enzyme layer using the polymer membrane layer 30.

[0053] In some examples, the polymer membrane layer 30 may include a base layer 31. The base layer 31 may be disposed on the sensing enzyme layer. Additionally, in some examples, the base layer 31 may encapsulate or cover the sensing enzyme layer.

[0054] In some examples, the base layer 31 may be formed from a first-type polymer. In other examples, the base layer 31 may be cross-linked from a first-type polymer. The base layer 31 may be formed by cross-linking a first-type polymer using a cross-linking agent.

[0055] In some examples, the first type of polymer can be a homopolymer having benzene rings or heterocycles. This allows the formation of a porous substrate layer 31, enabling the analyte to pass through it. Furthermore, the first type of polymer having benzene rings or heterocycles can facilitate the generation of conjugation effects (e.g., π-π conjugation).

[0056] In some examples, the substrate 31 may have diffusion control properties. In other examples, the substrate 31 may have water swelling properties. This improves the diffusion control properties of the substrate 31.

[0057] In some examples, the first type of polymer can be a water-swellable homopolymer. In this case, a substrate layer 31 with improved diffusion control performance can be formed, which can help improve the diffusion control performance of the polymer film layer 30, and thus help to expand the linear response range of the biosensor 1.

[0058] In some examples, the water-swellable homopolymer can be selected from polystyrene, polyurethane, polyethoxyethyl acrylate, polyethoxypropyl acrylate, poly-2-vinylpyridine, poly-4-vinylpyridine, polyhydroxyethyl methacrylate, and polyhydroxyethyl acrylate. This allows for the formation of a substrate layer 31 with good diffusion control performance, thereby improving the diffusion control performance of the polymer film layer 30 and contributing to expanding the linear response range of the biosensor 1.

[0059] In some examples, the molecular weight of the water-swellable homopolymer can be from 50,000 to 500,000 Da. This facilitates the formation of the substrate layer 31, which in turn facilitates the formation of the polymer film layer 30, thereby helping to expand the linear response range of the biosensor 1.

[0060] In some examples, the molecular weight of the water-swellable homopolymer can be 50,000 Da, 60,000 Da, 70,000 Da, 100,000 Da, 150,000 Da, 200,000 Da, 300,000 Da, 400,000 Da, or 500,000 Da.

[0061] In some examples, the thickness of the base layer 31 is not particularly limited. For example, the thickness of the base layer 31 can be from 5 μm to 20 μm. This facilitates cooperation with other components (such as the sensing enzyme layer and the adhesive layer 32).

[0062] In some examples, the thickness of the substrate 31 can be 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm or 20 μm.

[0063] In some examples, the polymer film layer 30 may include an adhesive layer 32. The adhesive layer 32 may be formed on the substrate layer 31. Additionally, the adhesive layer 32 may have an adhesive effect. The adhesive layer 32 may be bonded to the substrate layer 31. In other examples, the adhesive layer 32 may be bonded to the substrate layer 31 through at least one of the following mechanisms: conjugation, similarity-based solubility, hydrogen bonding, and crosslinking.

[0064] In some examples, adhesive layer 32 may be formed from a second type of polymer. Alternatively, adhesive layer 32 may be formed by crosslinking a second type of polymer. Furthermore, adhesive layer 32 may be formed by crosslinking a second type of polymer using a crosslinking agent.

[0065] In some examples, the second type of polymer may have a similar structure to the first type of polymer and the third type of polymer (described later), respectively. In this case, the adhesion strength of the adhesive layer 32 to the base layer 31 and the outer surface layer 33 can be improved, thereby increasing the stability of the polymer film 30 and preventing the base layer 31 and the outer surface layer 33 from detaching from the adhesive layer 32. This can help improve the diffusion control performance of the polymer film 30.

[0066] Specifically, the second type of polymer can be a copolymer formed from a first monomer that is the same as or similar to the monomer of the first type of polymer and a second monomer that is the same as or similar to the monomer of the third type of polymer. In other words, the structure of the first monomer can be the same as or similar to the structure of the monomer of the first type of polymer, and the structure of the second monomer can be the same as or similar to the structure of the monomer of the third type of polymer.

[0067] In some examples, the mass ratio of the first monomer to the second monomer in the second type of polymer can be 3:7 to 7:3. In this case, the diffusion control performance of the adhesive layer 32 can be improved, which in turn can help improve the diffusion control performance of the polymer film layer 30. In other words, in the monomers forming the second type of polymer, the mass fraction of the first monomer can be 30% to 70%, and the mass fraction of the second monomer can be 30% to 70%.

[0068] In some examples, the mass ratio of the first monomer to the second monomer can be 3:7, 7:13, etc.

[0069] In some examples, adhesive layer 32 may have diffusion control properties. Additionally, in some examples, adhesive layer 32 may have a certain degree of water-swellability. This improves the diffusion control properties of adhesive layer 32. In other examples, the second type of polymer may be a water-swellable copolymer.

[0070] In some examples, the water-swellable copolymer can be selected from polyethylene glycol-block polystyrene, polyacrylic acid-block polystyrene, polyacrylic acid-copolymer-polystyrene, polyacrylamide-block polystyrene, polyacrylamide-copolymer-polystyrene, poly(2-vinylpyridine)-block polystyrene, poly(4-vinylpyridine)-copolymer-polystyrene, poly(4-vinylpyridine)-copolymer-polyvinylpyrrolidone, poly(2-vinylpyridine)-copolymer-polystyrene, poly(4-vinylpyridine)-block polystyrene, poly(4-vinylpyridine)-copolymer-polyacrylamide, poly(ethoxyethyl acrylate)-copolymer-hydroxyethyl acrylate, and poly(ethoxypropyl acrylate)-copolymer-polyvinyl alcohol. In this case, an adhesive layer 32 with enhanced adhesion to the substrate layer 31 and the outer surface layer 33 can be formed, which can facilitate the formation of the polymer film layer 30 and thus help to expand the linear response range of the biosensor 1.

[0071] In some examples, the molecular weight of the water-swellable copolymer can be from 10,000 to 50,000 Da. This facilitates the film formation of the adhesive layer 32, which in turn facilitates the formation of the polymer film layer 30, thereby helping to expand the linear response range of the biosensor 1.

[0072] In some examples, the molecular weight of the water-swellable copolymer can be 10,000 Da, 12,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, or 50,000 Da.

[0073] In some examples, the thickness of the adhesive layer 32 is not particularly limited. For example, the thickness of the adhesive layer 32 can be from 5 μm to 20 μm. This facilitates the mating with other components (such as the base layer 31 and the outer layer 33).

[0074] In some examples, the thickness of the adhesive layer 32 can be 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm or 20 μm.

[0075] In some examples, the polymer film layer 30 may include an outer surface layer 33. The outer surface layer 33 may be formed on the adhesive layer 32. Additionally, the outer surface layer 33 may be biocompatible. In other examples, the outer surface layer 33 may be bonded to the adhesive layer 32 through at least one of the following mechanisms: conjugation, like dissolves like, hydrogen bonding, and crosslinking.

[0076] In some examples, the adhesive layer 32 can be bonded to the base layer 31 and the outer layer 33 through at least one of the following: conjugation effect (e.g., π-π conjugation), similarity solubility, hydrogen bonding interaction, and crosslinking. This strengthens the bond between the adhesive layer 32 and the base layer 31 and the outer layer 33, thereby improving the diffusion control performance of the polymer film layer 30 and consequently expanding the linear response range of the biosensor 1.

[0077] In some examples, the outer layer 33 may be formed from a third type of polymer. Alternatively, the outer layer 33 may be cross-linked from a third type of polymer. Furthermore, the outer layer 33 may be formed by cross-linking a third type of polymer using a cross-linking agent.

[0078] In some examples, the adhesive layer 32 and the outer layer 33 may be cross-linked with the same cross-linking agent. In other examples, the adhesive layer 32 and the base layer 31 may be cross-linked with the same cross-linking agent.

[0079] In some examples, the substrate layer 31, adhesive layer 32, and outer surface layer 33 can be cross-linked with the same cross-linking agent. In this case, the substrate layer 31, adhesive layer 32, and outer surface layer 33 can be combined through cross-linking, which can help improve the diffusion control performance of the polymer film layer 30 and thus help expand the linear response range of the biosensor 1.

[0080] In some examples, the crosslinking agent can be at least one of reactive esters, epoxides, and sulfates. For example, the crosslinking agent can be a polyisocyanate, a polyethylene glycol reactive ester, a glycidyl ester, a maleimide PEG reactive ester, polyethylene glycol ethylene oxide, 1,4-butanediol diglycidyl ether, glutaric anhydride, 1,4-diazid, bisacridone, or sodium sulfate.

[0081] In some examples, the third type of polymer can be biocompatible. Additionally, the third type of polymer can be a water-soluble polymer. This can help improve the biocompatibility of the outer surface layer 33, thereby improving the biocompatibility of the polymer film layer 30 and the biocompatibility of the biosensor 1.

[0082] In some examples, the water-soluble polymer may be selected from polyvinylpyrrolidone, polyvinyl alcohol, chitosan, carboxymethyl chitosan, chitosan salt, alginate, alginate, hyaluronic acid, hyaluronic acid salt, cellulose ethers, cellulose esters, polyvinylpyrrolidone, polyacrylamide, polyacrylic acid, polyacryl alcohol, sodium polystyrene sulfonate, polyethylene glycol, and polyethylene glycol-polypropylene glycol copolymers. This facilitates the formation of a biocompatible outer layer 33, thereby enabling the formation of a biocompatible polymer film layer 30, and further improving the biocompatibility of the biosensor 1.

[0083] In some examples, the molecular weight of the water-soluble polymer can be from 2,000 to 50,000 Da. This facilitates the formation of the outer layer 33, thereby promoting the formation of the polymer film 30 and improving the biocompatibility of the biosensor 1.

[0084] In some examples, the molecular weight of the water-soluble polymer can be 2000 Da, 5000 Da, 10000 Da, 15000 Da, 20000 Da, 25000 Da, 30000 Da, 35000 Da, 40000 Da, or 50000 Da.

[0085] In some examples, the thickness of the outer layer 33 is not particularly limited. For example, the thickness of the outer layer 33 can be from 5 μm to 20 μm. This facilitates mating with other components (e.g., adhesive layer 32). In some examples, the thickness of the outer layer 33 can be 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm, or 20 μm.

[0086] In some examples, the thickness of the polymer membrane 30 is not particularly limited. Additionally, in some examples, the thickness of the polymer membrane 30 may not exceed 100 μm. This facilitates the integration of the polymer membrane 30 with the sensing enzyme layer. For example, the thickness of the polymer membrane 30 can be 10 μm, 15 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm.

[0087] In some examples, the first, second, and third polymers can be matched with each other. For example, if the first polymer is poly(4-vinylpyridine) and the third polymer is polyvinylpyrrolidone, then the third polymer can be poly(4-vinylpyridine)-copolymer-polyvinylpyrrolidone.

[0088] In some examples, in the polymer film 30, the adhesive layer 32 may wrap around or cover the base layer 31. Additionally, in some examples, in the polymer film 30, the outer layer 33 may wrap around or cover the adhesive layer 32.

[0089] In this embodiment, the diffusion control performance of the polymer film 30 can be adjusted by changing the thickness ratio of the base layer 31, the adhesive layer 32, and the outer surface layer 33. In some examples, the thickness of the adhesive layer 32 in the polymer film 30 may be no less than the thickness of the base layer 31, and the thickness of the adhesive layer 32 may be greater than the thickness of the outer surface layer 33.

[0090] In some examples, the thickness of the adhesive layer 32 in the polymer film 30 can account for 40% to 50% of the thickness of the polymer film 30. This can help improve the diffusion control performance of the polymer film 30. For example, the thickness of the adhesive layer 32 can account for 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of the thickness of the polymer film 30.

[0091] In some examples, the thickness of the substrate layer 31 in the polymer film 30 can be 30% to 40% of the total thickness of the polymer film 30. In this case, the combination of the substrate layer 31, the adhesive layer 32, and the outer surface layer 33 can further enhance the diffusion control performance of the polymer film 30. For example, the thickness of the substrate layer 31 can be 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% of the total thickness of the polymer film 30.

[0092] In some examples, the thickness of the outer surface layer 32 in the polymer film 30 can be 20% to 30% of the total thickness of the polymer film 30. In this case, the combination of the base layer 31, the adhesive layer 32, and the outer surface layer 33 can further enhance the diffusion control performance of the polymer film 30. For example, the thickness of the outer surface layer 32 can be 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% of the total thickness of the polymer film 30.

[0093] In this embodiment, the base layer 31, adhesive layer 32, and outer surface layer 33 can all have diffusion control properties. That is, the base layer 31, adhesive layer 32, and outer surface layer 33 can all control the pass rate of the tested object.

[0094] In some examples, the diffusion control properties of the adhesive layer 32 in the polymer film layer 30 can be matched with the diffusion control properties of the base layer 31 and the outer layer 33.

[0095] In some examples, the diffusion control performance of the adhesive layer 32 can be between that of the substrate layer 31 and the outer surface layer 33. In other words, the throughput of the test sample through the adhesive layer 32 can be no less than that through the substrate layer 31 and no greater than that through the outer surface layer 33. In this case, the adhesive layer 32 can act as a transition layer, reducing the difference in diffusion control performance between the substrate layer 31 and the outer surface layer 33, and improving the diffusion control performance of the polymer film layer 30.

[0096] In the polymer film 30 involved in this embodiment, the adhesive layer 32 is formed of a second type of polymer, which is the same as or similar to the monomer of the first type of polymer and the same as or similar to the monomer of the third type of polymer. This can improve the adhesion of the adhesive layer 32 to the base layer 31 and the outer surface layer 33, thereby improving the stability of the polymer film 30. This can help improve the diffusion control performance of the polymer film 30, so that the concentration of the analyte on both sides of the polymer film 30 has a fixed concentration ratio. The biocompatible outer surface layer 33 can improve the biocompatibility of the polymer film 30.

[0097] In this embodiment, by providing a polymer film layer 30 for controlling the diffusion of the analyte on the sensing enzyme layer of the biosensor 1, the linear response range of the biosensor 1 can be expanded, and the biosensor 1 is made biocompatible by having a biocompatible outer surface layer 33 on the polymer film layer 30.

[0098] Furthermore, in this embodiment, by using the polymer film layer 30 to expand the linear range of the response of the biosensor 1, the upper limit of the detectable concentration of the biosensor 1 can be increased. In some examples, the upper limit of the detectable concentration can be increased to 40 mM.

[0099] The preparation method of biosensor 1 is described in detail below with reference to the accompanying drawings. Figure 4 This is a flowchart illustrating a method for preparing the biosensor 1 as described in the examples of this disclosure. Figure 5 This is a flowchart illustrating a method for preparing the polymer film 30 as described in the examples of this disclosure.

[0100] In this embodiment, such as Figure 4 As shown, the fabrication method of biosensor 1 may include preparing a substrate 10 and setting an electrode 20 on the substrate 10 (step S100). Furthermore, the substrate 10 and the electrode 20 can be referred to the description above.

[0101] In some examples, a working electrode may be formed on the substrate 10 in step S100. Additionally, a counter electrode may be formed on the substrate 10 in step S100. Furthermore, a reference electrode may be formed on the substrate 10 in step S100.

[0102] In some examples, in step S100, electrodes 20 can be formed on the substrate 10 by means of electroplating, evaporation, printing, extrusion, etc.

[0103] In this embodiment, such as Figure 4 As shown, the fabrication method of biosensor 1 may include depositing a sensing enzyme layer on electrode 20 (step S200). Specifically, in step S200, a sensing enzyme layer may be deposited on the working electrode. Furthermore, the sensing enzyme layer can be referred to the description above.

[0104] In this embodiment, such as Figure 4 As shown, the preparation method of biosensor 1 may include depositing a polymer membrane layer 30 on the sensing enzyme layer (step S300). The polymer membrane layer 30 can be referred to the description above.

[0105] In some examples, step S300 may include the preparation of the polymer film 30 (step S310). Additionally, in some examples, such as... Figure 5 As shown, step S310 may include preparing the base layer reagent, the adhesive layer reagent, and the outer surface layer reagent (step S311).

[0106] In some examples, the base layer reagent may include a first type of polymer, the adhesive layer reagent may include a second type of polymer, and the outer surface layer reagent may include a third type of polymer.

[0107] In some examples, the base layer reagent may contain a crosslinking agent. In other examples, the adhesive layer reagent may contain a crosslinking agent. Additionally, in some examples, the outer layer reagent may contain a crosslinking agent.

[0108] In some examples, in step S311, a base layer reagent can be formed by dissolving a first type of polymer in a first solvent. Specifically, in step S311, the first type of polymer can be mixed with the first solvent and subjected to treatments such as sonication, vibration, and stirring to obtain the base layer reagent.

[0109] In some examples, the first solvent in step S311 may be volatile. For example, the first solvent may be an aqueous solution of ethanol, tetrahydrofuran, isopropanol, methanol, dimethylformamide, dimethyl sulfoxide, or sulfolane.

[0110] In some examples, in step S311, the concentration of the first type of polymer in the base layer reagent can be from 30 to 120 mg / ml. For example, the concentration of the first type of polymer can be 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, or 120 mg / ml.

[0111] In some examples, in step S311, the base layer reagent can be formed by dissolving the first type of polymer and the crosslinking agent in a first solvent. In other examples, in step S311, the mass ratio of the first type of polymer and the crosslinking agent can be 8:1, 10:1, 15:1, 20:1, 30:1, 40:1, 60:1, 80:1, 100:1, 120:1, or 128:1.

[0112] In some examples, in step S311, the adhesive layer reagent can be formed by dissolving the second type of polymer in the second solvent. Specifically, in step S311, the second type of polymer can be mixed with the second solvent and subjected to treatments such as ultrasonication, vibration, and stirring to obtain the adhesive layer reagent.

[0113] In some examples, the second solvent in step S311 may be volatile. For example, the second solvent may be an aqueous solution of ethanol, tetrahydrofuran, isopropanol, methanol, dimethylformamide, dimethyl sulfoxide, or sulfolane. Alternatively, the second solvent may be the same as the first solvent.

[0114] In some examples, in step S311, the concentration of the second type of polymer in the adhesive layer agent can be from 30 to 120 mg / ml. For example, the concentration of the second type of polymer can be 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, or 120 mg / ml.

[0115] In some examples, in step S311, the base layer reagent can be formed by dissolving the second type of polymer and the crosslinking agent in a second solvent. In other examples, in step S311, the mass ratio of the second type of polymer and the crosslinking agent can be 8:1, 10:1, 15:1, 20:1, 30:1, 40:1, 60:1, 80:1, 100:1, 120:1, or 128:1.

[0116] In some examples, in step S311, the outer surface layer reagent can be formed by dissolving the third type of polymer in a third solvent. Specifically, in step S311, the third type of polymer can be mixed with the third solvent and subjected to treatments such as sonication, vibration, and stirring to obtain the outer surface layer reagent.

[0117] In some examples, the third solvent in step S311 may be volatile. For example, the third solvent may be an aqueous solution of ethanol, tetrahydrofuran, isopropanol, methanol, dimethylformamide, dimethyl sulfoxide, or sulfolane. Additionally, in some examples, the third solvent may be the same as the first solvent. In other examples, the third solvent may be the same as the second solvent. Furthermore, in some examples, the first solvent, the second solvent, and the third solvent may be the same.

[0118] In some examples, in step S311, the concentration of the third type of polymer in the outer surface layer reagent can be from 30 to 120 mg / ml. For example, the concentration of the third type of polymer can be 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, or 120 mg / ml.

[0119] In some examples, in step S311, the outer surface layer reagent can be formed by dissolving the third type of polymer and the crosslinking agent in a third solvent. In other examples, in step S311, the mass ratio of the third type of polymer and the crosslinking agent can be 8:1, 10:1, 15:1, 20:1, 30:1, 40:1, 60:1, 80:1, 100:1, 120:1, or 128:1.

[0120] In some examples, such as Figure 5 As shown, step S310 may include sequentially forming a base layer 31, an adhesive layer 32, and an outer surface layer 33 (step S312).

[0121] In some examples, in step S312, the substrate layer 31, the adhesive layer 32, and the outer layer 33 can be formed sequentially by spin coating, dip coating, drop coating, spray coating, etc., to form a polymer film layer 30. Alternatively, the polymer film layer 30 can be formed by drying in a nitrogen atmosphere.

[0122] In some examples, in step S300, the electrode 20 (which may refer to the working electrode) may be sequentially dipped in a base layer reagent, an adhesive layer reagent, and an outer layer reagent to form a polymer film 30 on the electrode 20.

[0123] In this embodiment, a polymer film layer 30 for controlling the diffusion of the analyte and having a biocompatible outer surface layer 33 is formed on the sensing enzyme layer of the biosensor 1, thereby enabling the formation of a biosensor 1 with an expanded linear response range and biocompatibility.

[0124] According to this disclosure, a biosensor 1 capable of expanding the linear range of response and having biocompatibility, a method for preparing the same, and a polymer film layer 30 for the biosensor 1 can be provided.

[0125] The embodiments of the present invention will be further described in detail below with reference to specific examples. Figure 6 The diagram shows the current curve measured by the glucose biosensor of Embodiment 1 of this disclosure. Figure 7 It shows Figure 6 The graph shows the linear relationship between current and glucose concentration. Figure 8 This is a stained section of a polymer film layer from Example 1 of this disclosure.

[0126]

Example

[0127] In this embodiment, a glucose biosensor with a three-electrode system incorporating glucose oxidase is used as the biosensor. The raw materials include poly(4-vinylpyridine) with a molecular weight of 50,000 Da, polyvinylpyrrolidone with a molecular weight of 5,000 Da, poly(4-vinylpyridine)-copolymer-polyvinylpyrrolidone with a molecular weight of 10,000 Da, poly(ethoxyethyl acrylate) with a molecular weight of 15,000 Da, poly(ethoxyethyl acrylate)-copolymer-hydroxyethyl acrylate with a molecular weight of 10,000 Da, hydroxyethyl acrylate with a molecular weight of 10,000 Da, polyacrylamide with a molecular weight of 8,000 Da, and poly(4-vinylpyridine)-copolymer-polyacrylamide with a molecular weight of 12,000 Da.

[0128] First, according to Table 1, prepare the base layer reagent raw materials for each embodiment (Example 1 to Example 3), and dissolve the first type of polymer in the first solvent, sonicate and vibrate until completely dissolved to obtain the base layer reagent; next, according to Table 1, prepare the adhesive layer reagent raw materials for each embodiment (Example 1 to Example 3), and dissolve the second type of polymer in the second solvent, sonicate and vibrate until completely dissolved to obtain the adhesive layer reagent; then, according to Table 1, prepare the outer surface layer reagent raw materials for each embodiment (Example 1 to Example 3), and dissolve the third type of polymer in the third solvent, sonicate and vibrate until completely dissolved to obtain the outer surface layer reagent.

[0129] Then, the working electrodes of the glucose biosensors of each embodiment were sequentially dipped into the base layer reagent, the adhesive layer reagent, and the outer surface reagent for 5 seconds, with a 10-minute interval between each dip before drying. Each reagent was dipped 12 times to form a polymer film layer 30, which was then dried in a nitrogen environment for 48 hours to complete the preparation.

[0130] Finally, the following two experiments were conducted: performance testing of the glucose biosensors obtained in each embodiment; and adhesion testing of the bonding strength between the base layer, adhesive layer, and outer surface layer in the polymer film in each embodiment. The specific steps are as follows:

[0131] (1) The glucose biosensor was immersed in standard PBS buffer (pH 7.4, 150 mM NaCl), followed by an initial pulse of 1.1 volts for 360 s. Each glucose biosensor was then subjected to residual measurements at 0.05 V. After 30 minutes to allow the glucose biosensor to reach a constant background, glucose concentrations of 0 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, and 40 mM were added to the solution to measure the linearity of the response. After each addition of glucose, the solution was allowed to equilibrate for 3 minutes, and the solution was continuously stirred during the measurement to ensure uniform concentration. The glucose biosensors of each embodiment exhibited good linearity in their response to glucose, specifically, for example, from... Figure 6 and Figure 7 As can be seen from the data, the glucose sensor in Example 1 exhibits a good linear correlation between glucose concentration and response current in the range of 0 mM to 40 mM.

[0132] (2) The working electrode of the glucose biosensor was fixed in an epoxy resin solution with a clamp and cured. After 24 hours, it was ground in a grinding mill to a depth of half the total width of the working electrode. It was then rinsed with deionized water and baked in a 45°C oven for 60 minutes. The dried working electrode was then immersed in a 0.1% Nile Red ethanol solution for 5 seconds, rinsed with ethanol, and air-dried at room temperature. Finally, the cut surface was observed under a 500x optical microscope. In each embodiment, the base layer, adhesive layer, and outer surface layer of the polymer film are firmly bonded. Specifically, for example, from... Figure 8 As can be seen, the polymer film in Example 1 has different adsorption degrees of dye in each layer, which can be clearly distinguished by color. After being ground by a grinding machine, there are no obvious cracks between the base layer, adhesive layer and outer layer, and the layers are firmly bonded together.

[0133] Table 1

[0134]

[0135]

[0136] While the present disclosure has been specifically described above in conjunction with the accompanying drawings and embodiments, it is to be understood that the above description does not limit the present disclosure in any way. Those skilled in the art can make modifications and variations to the present disclosure as needed without departing from its essential spirit and scope, and all such modifications and variations fall within the scope of the present disclosure.

Claims

1. A method for preparing a polymer film layer for a biosensor, characterized in that, The biosensor has a sensing enzyme layer with a biocompatible polymer film layer on its outer surface for controlling the diffusion of the analyte, and the preparation method includes: Prepare a base layer reagent comprising a first type of polymer, an adhesive layer reagent comprising a second type of polymer, and an outer layer reagent comprising a third type of polymer, wherein the first type of polymer is a homopolymer having a benzene ring or heterocyclic structure, the second type of polymer is a copolymer formed from a first monomer identical to the monomer of the first type of polymer and a second monomer identical to the monomer of the third type of polymer, the mass ratio of the first monomer to the second monomer being 3:7 to 7:3, and the second type of polymer is a water-swellable copolymer; and A base layer is sequentially formed, an adhesive layer with adhesive properties is formed on the base layer, and a biocompatible outer surface layer is formed on the adhesive layer. In the polymer film layer, the adhesive layer has diffusion control properties, and the adhesive layer is bonded to the base layer and the outer surface layer through at least one of the following: conjugation effect, similarity solubility, hydrogen bonding interaction, and crosslinking.

2. The preparation method according to claim 1, characterized in that, In the polymer film layer, the thickness of the base layer accounts for 30% to 40% of the thickness of the polymer film layer, the thickness of the adhesive layer accounts for 40% to 50% of the thickness of the polymer film layer, and the thickness of the outer surface layer accounts for 20% to 30% of the thickness of the polymer film layer.

3. The preparation method according to claim 1, characterized in that, The base layer, the adhesive layer, and the outer layer are each cross-linked by the same cross-linking agent.

4. The preparation method according to claim 3, characterized in that, The crosslinking agent is a polyisocyanate, polyethylene glycol active ester, glycidyl ester, maleimide PEG active ester, polyethylene glycol ethylene oxide, 1,4-butanediol diglycidyl ether, glutaric anhydride, 1,4-diazid, bisacridone, or sodium sulfate.

5. The preparation method according to claim 1, characterized in that, The base layer reagent is formed by dissolving the first type of polymer in a first solvent, wherein the first solvent is volatile and is an aqueous solution of ethanol, tetrahydrofuran, isopropanol, methanol, dimethylformamide, dimethyl sulfoxide, or sulfolane.

6. The preparation method according to claim 1, characterized in that, The adhesive layer is formed by dissolving the second type of polymer in a second solvent, wherein the second solvent is volatile and is an aqueous solution of ethanol, tetrahydrofuran, isopropanol, methanol, dimethylformamide, dimethyl sulfoxide, or sulfolane.

7. The preparation method according to claim 1, characterized in that, The outer surface layer reagent is formed by dissolving the third type of polymer in a third solvent, wherein the third solvent is volatile and is an aqueous solution of ethanol, tetrahydrofuran, isopropanol, methanol, dimethylformamide, dimethyl sulfoxide, or sulfolane.

8. The preparation method according to claim 1, characterized in that, In the base layer reagent, the concentration of the first type of polymer is 30 to 120 mg / ml; In the adhesive layer reagent, the concentration of the second type of polymer is 30 to 120 mg / ml; In the outer surface layer reagent, the concentration of the third type of polymer is 30 to 120 mg / ml.

9. The preparation method according to claim 1, characterized in that, The base layer reagent, the adhesive layer reagent, and the outer surface layer reagent are obtained by ultrasonication, vibration, and stirring, respectively.

10. The preparation method according to claim 1, characterized in that, The base layer, the adhesive layer, and the outer layer are formed sequentially by at least one method, such as spin coating, dip coating, drop coating, and spray coating, to form the polymer film layer.

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