A biosensor and a method for preparing the same

Abstract

The present invention provides a biosensor and a preparation method thereof, wherein the biosensor comprises: an electrode structure, comprising a substrate, a first conductive layer, a first insulating layer, a second conductive layer, a third conductive layer and a second insulating layer; wherein the first conductive layer is formed on the substrate; the first insulating layer is formed on the first conductive layer, and a part of the first conductive layer is exposed to form a working electrode; the second conductive layer is formed on the first insulating layer; the third conductive layer is formed on a part of the second conductive layer; the second insulating layer is at least formed on the second conductive layer, and a part of the second conductive layer is exposed to form a counter electrode, and at least a part of the third conductive layer is exposed to form a reference electrode; and a reaction film layer is formed on the working electrode for electrochemical reaction with a target. The biosensor has a simple and reasonable structure and is conducive to mass production.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and in particular to a biosensor and a preparation method thereof. Background Art

[0002] A biosensor is defined as "a device that uses immobilized biomolecules combined with transducers to detect environmental chemicals inside or outside a living organism or to respond after specific interactions with them". Based on the differences in the biological substances used in the sensor, biosensors can be divided into tissue sensors, cell sensors, enzyme sensors, etc. Based on the physical quantity, chemical quantity or biological quantity monitored, biosensors can be divided into thermal sensors, optical sensors, insulin sensors, etc., and can also be divided into immune sensors, drug sensors, etc. based on their uses. So far, the most commercially successful biosensor is the glucose sensor, which is used to monitor glucose in human body fluids such as blood, interstitial fluid, and sweat. Due to the huge number of diabetes patients worldwide, glucose sensors have a huge market share.

[0003] Glucose monitoring requires the use of a special glucose detector, and the core component of the glucose detector is the glucose sensor. Glucose sensors can be divided into in vitro sensors, fully implanted sensors, and subcutaneous implanted sensors. Among them, in vitro sensors monitor blood glucose data at a single point by sampling blood from the fingertips. Fully implanted sensors have biocompatibility issues, and surgical implantation and removal are relatively complicated. Subcutaneous implanted sensors are quickly implanted or removed in a minimally invasive manner, and can also continuously monitor glucose. However, subcutaneous implantable glucose sensors in the prior art have problems such as complex preparation process, high cost, short life, poor anti-interference ability, and sensitivity attenuation. Summary of the invention

[0004] The object of the present invention is to provide a biosensor and a preparation method thereof. The biosensor has a simple structure and is easy to prepare.

[0005] To achieve the above object, the present invention provides a biosensor, comprising:

[0006] An electrode structure, comprising a substrate, a first conductive layer, a first insulating layer, a second conductive layer, a third conductive layer, and a second insulating layer; wherein the first conductive layer is formed on the substrate; the first insulating layer is formed on the first conductive layer, and a portion of the first conductive layer is exposed to form a working electrode; the second conductive layer is formed on the first insulating layer; the third conductive layer is formed on a portion of the second conductive layer; the second insulating layer is at least formed on the second conductive layer, and a portion of the second conductive layer is exposed to form a counter electrode, and at least a portion of the third conductive layer is exposed to form a reference electrode; and,

[0007] The reaction film layer is formed on the working electrode and is used for electrochemical reaction with the target object.

[0008] Optionally, the reaction film layer is formed by coating a reaction reagent on the working electrode and solidifying the reaction reagent; the reaction reagent includes a metal complex, a bioreaction enzyme, a polypeptide macromolecule and a first cross-linking agent.

[0009] Optionally, the metal complex comprises a transition metal complex.

[0010] Optionally, the bioreaction enzyme includes any one of glucose oxidase, lactate oxidase, L-glutamate oxidase or xanthine oxidase.

[0011] Optionally, the reaction reagent also includes a stabilizer. Taking the total amount of the metal complex, the bioreaction enzyme, the polypeptide macromolecule, the stabilizer and the first cross-linking agent as 100%, the mass percentage of the metal complex is 5% to 50%, the mass percentage of the stabilizer is 1% to 20%, the mass percentage of the polypeptide macromolecule is 1% to 20%, the mass percentage of the first cross-linking agent is 0.1% to 10%, and the remainder is the bioreaction enzyme.

[0012] Optionally, the stabilizer includes a polymer prepolymer solution.

[0013] Optionally, the biosensor further includes a functional membrane layer; the functional membrane layer includes an anti-interference membrane layer, the anti-interference layer is at least arranged on the reaction membrane layer, the counter electrode and the reference electrode, and is used to prevent interfering substances from passing through the functional membrane layer.

[0014] Optionally, the anti-interference film layer includes at least one of naphthol, cellulose acetate, polylysine, polyvinyl pyridine and modified copolymers thereof, and polyurethane.

[0015] Optionally, the functional film layer further includes a regulating film layer, which is disposed on the anti-interference film layer and is used to regulate the passing rate of the target object on the functional film layer.

[0016] Optionally, the adjustment membrane layer includes a hydrophilic polymer, a hydrophobic polymer and a second cross-linking agent.

[0017] Optionally, the adjustment film layer includes a first adjustment film layer and a second adjustment film layer, the first adjustment film layer is formed on the anti-interference film layer, and the second adjustment film layer is formed on the first adjustment film layer; the content of the hydrophobic polymer in the first adjustment film layer is greater than the content of the hydrophobic polymer in the second adjustment film layer.

[0018] Optionally, in the first adjustment film layer, the weight ratio of the hydrophilic polymer to the hydrophobic polymer is 1:9 to 1:1.1, and in the second adjustment film layer, the weight ratio of the hydrophilic polymer to the hydrophobic polymer is 9:1 to 1:1.

[0019] Optionally, the hydrophilic polymer includes at least one of polyethylene glycol, polyhydroxyethyl methacrylate, polyacrylic acid, polypropylene alcohol, chitosan, hydrophilic cellulose, hydrophilic modified silane condensation polymer, hydrophilic modified polyurethane, polyDMAEMA, polyNIPAM, polymethacrylamide, polydopamine, alginic acid, hyaluronic acid, sodium polystyrene sulfonate, polyethylene glycol-modified vinyl pyridine, polysulfonic acid-modified vinyl pyridine, and polycarboxyl-modified 4-vinyl pyrrolidone; the hydrophobic polymer includes at least one of polystyrene, polymethyl methacrylate, polyvinyl pyridine, polyvinyl pyrrolidone, polysilanes, polyurethane, and polycarbonate; and / or,

[0020] The molecular weight distribution of the hydrophilic polymer and the hydrophobic polymer is 10000Da to 1000000Da.

[0021] Optionally, the biosensor includes an implantation part, which is used to be implanted into a target object, and the working electrode, the counter electrode, the reference electrode, and the reaction film layer are all located on the implantation part; the biosensor also includes a biocompatible layer, which is located on the implantation part, and the biocompatible layer is used to cover the surface of the implantation part together with the functional film layer.

[0022] To achieve the above object, the present invention further provides a method for preparing a biosensor, which is used to prepare the biosensor as described in any of the preceding items, and the preparation method comprises the following steps:

[0023] providing the substrate;

[0024] forming the first conductive layer on the substrate;

[0025] forming the first insulating layer on the first conductive layer;

[0026] forming the second conductive layer on the first insulating layer;

[0027] forming the third conductive layer on the second conductive layer;

[0028] forming the second insulating layer at least on the second conductive layer;

[0029] The reaction film layer is formed on the working electrode.

[0030] Optionally, the reaction film layer is formed on the working electrode by dispensing or inkjet process.

[0031] Optionally, a functional film layer is formed on the reaction film layer, the counter electrode and the reference electrode.

[0032] Optionally, the biosensor includes an implant part, the working electrode, the counter electrode, the reference electrode, and the reaction film layer are all located on the implant part, and the preparation method also includes: the biosensor includes an implant part, the working electrode, the counter electrode, the reference electrode, and the reaction film layer are all located on the implant part, and the preparation method also includes: forming a biocompatible layer on the implant part, and the biocompatible layer is used to jointly cover the surface of the implant part with the functional film layer.

[0033] Compared with the prior art, the biosensor and the preparation method thereof of the present invention have the following advantages:

[0034] The aforementioned biosensor includes a substrate, an electrode structure and a reaction film layer; the electrode structure includes a first conductive layer, a first insulating layer, a second conductive layer, a third conductive layer and a second insulating layer, the first conductive layer is formed on the substrate, the first insulating layer is formed on the first conductive layer, and a portion of the first conductive layer is exposed to form a working electrode, the second conductive layer is formed on the first insulating layer, the third conductive layer is formed on a portion of the second conductive layer, the second insulating layer is at least formed on the second conductive layer, and a portion of the second conductive layer is exposed to form a counter electrode, and at least a portion of the third conductive layer is exposed to form a reference electrode; the reaction film layer is formed on the working electrode and is used to undergo an electrochemical reaction with a target, the target being, for example, any one of glucose, lactic acid, xanthine and L-glutamic acid in body fluids. The biosensor can be formed by sequentially coating with a coating and then curing, and the preparation method is simple and low in cost.

[0035] Furthermore, the reaction membrane layer includes a metal complex, a bioreaction enzyme, a polypeptide macromolecule and a first cross-linking agent, wherein the amino, carboxyl, hydroxyl and other groups on the polypeptide macromolecule can react with the metal complex and the bioreaction enzyme to form a covalent bond, and form a core-shell structure under the action of the stabilizer, thereby improving the stability and life of the biosensor.

[0036] Furthermore, the biosensor also includes a functional film layer, which may include an anti-interference film layer and an adjustment film layer. The anti-interference film layer is used to prevent interfering substances from passing through the functional film layer to reduce interference with the detection of the target object. The adjustment film layer is used to adjust the pass rate of the target object on the functional film layer, thereby adjusting the amount of the target object entering the reaction film layer and improving the sensitivity of the biosensor. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings are used to better understand the present invention and do not constitute an improper limitation of the present invention.

[0038] Figure 1 is a schematic diagram of the overall structure of a biosensor provided according to an embodiment of the present invention;

[0039] Figure 2 is a cross-sectional view of a biosensor provided according to one embodiment of the present invention;

[0040] Figure 3 is a schematic diagram of a core-shell structure in a reaction membrane layer of a biosensor provided according to an embodiment of the present invention;

[0041] Figure 4 is a flow chart of the preparation of a biosensor provided according to one embodiment of the present invention;

[0042] Figure 5 is a flow chart of the preparation of an electrode structure of a biosensor provided according to one embodiment of the present invention;

[0043] Figures 6a to 6f A schematic diagram showing the preparation process of the electrode structure of the biosensor is shown;

[0044] Figure 7 is a graph showing the response current of the biosensor provided by one embodiment of the present invention to different concentrations of glucose;

[0045] Figure 8 4 is a linear relationship diagram between the response current and glucose concentration of the biosensor provided in one embodiment of the present invention.

[0046] [Description of reference numerals is as follows]:

[0047] 1100-electrode structure, 1101-implantation part, 1110-substrate, 1111-joining area, 1112-electrode area, 1111a-first pin, 1111b-second pin, 1111c-third pin, 1120-first conductive layer, 1121-first part, 1121a-first end, 1121b-second end, 1122-second part, 1123-third part, 1130-first insulating layer, 1140-second conductive layer, 1141-fourth part, 1141a-third end, 1141b-fourth end, 1142-fifth part, 1142a-fifth end, 1142b-sixth end, 1143-sixth part, 1150-third conductive layer, 1160-second insulating layer, 1001-working electrode, 1002-counter electrode, 1003-reference electrode;

[0048] 1200-reaction film layer;

[0049] 1300-functional film layer, 1310-anti-interference film layer, 1320-adjustment film layer, 1321-first adjustment film layer, 1322-second adjustment film layer;

[0050] 1400-Biocompatible layer. DETAILED DESCRIPTION

[0051] The following is an explanation of the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the present embodiment only illustrate the basic concept of the present invention in a schematic manner, and only the components related to the present invention are shown in the figure instead of being drawn according to the number, shape and size of the components in the actual implementation. The type, quantity and proportion of each component in the actual implementation can be a random change, and the component layout type may also be more complicated.

[0052] In addition, each embodiment of the following description has one or more technical features, but this does not mean that the user of the present invention must implement all the technical features in any embodiment at the same time, or can only implement part or all of the technical features in different embodiments separately. In other words, under the premise that implementation is possible, those skilled in the art can selectively implement part or all of the technical features in any embodiment according to the disclosure of the present invention and according to the design specifications or implementation requirements, or selectively implement a combination of part or all of the technical features in multiple embodiments, thereby increasing the flexibility of the implementation of the present invention.

[0053] As used in this specification, the singular forms "a", "an", and "the" include plural objects, and the plural form "plurality" includes more than two objects, unless the content clearly indicates otherwise. As used in this specification, the term "or" is generally used to include the meaning of "and / or", unless the content clearly indicates otherwise, and the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection. It can be a mechanical connection or an electrical connection. It can be directly connected or indirectly connected through an intermediate medium, and it can be a connection between the internal parts of two elements or an interaction relationship between two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to the specific circumstances.

[0054] In order to make the purpose, advantages and features of the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings. It should be noted that the drawings are all in very simplified form and in non-precise proportions, and are only used to conveniently and clearly assist in explaining the purpose of the embodiments of the present invention. The same or similar reference numerals in the drawings represent the same or similar components.

[0055] Figure 1 The overall structure diagram of the biosensor provided by the embodiment of the present invention is shown. Figure 2 is a cross-sectional view of the biosensor.

[0056] Please refer to Figure 1 and Figure 2, the biosensor includes an electrode structure 1100 and a reaction membrane layer 1200. The electrode structure 1100 includes a substrate 1110, a first conductive layer 1120, a first insulating layer 1130, a second conductive layer 1140, a third conductive layer 1150, and a second insulating layer 1160. The first conductive layer 1120 is formed on the substrate 1100. The first insulating layer 1130 is formed on the first conductive layer 1120, and a portion of the first conductive layer 1120 is exposed to form a working electrode 1001. The second conductive layer 1140 is formed on the first insulating layer 1130, and the third conductive layer 1150 is formed on a portion of the second conductive layer. The second insulating layer 1160 is at least formed on the second conductive layer 1140, and a portion of the second conductive layer 1140 is exposed to form a counter electrode 1002, and at least a portion of the third conductive layer 1150 is exposed to form a reference electrode 1003. The reaction film layer 1200 is formed on the working electrode 1001 and is used to react electrochemically with the target. Here, the meaning of "exposed" refers to being uncovered, that is, the area on the first conductive layer 1120 where the working electrode 1001 is formed is not covered by the first insulating layer 1130, the area on the second conductive layer 1140 where the counter electrode 1002 is formed is not covered by the third conductive layer 1150 and the second insulating layer 1160, and the area on the third conductive layer 1150 where the reference electrode 1003 is formed is not covered by the second insulating layer 1160. In this embodiment, it is preferred that the third conductive layer 1150 is completely exposed to the second insulating layer 1160 and forms the reference electrode 1003.

[0057] At least a portion of the structure of the biosensor can be implanted into a target object, which can be a subcutaneous portion of a patient. In this document, the portion of the biosensor that is implanted into the subcutaneous portion of a patient is referred to as an implantation portion 1101. Figure 1 The implant portion 1101 is as shown in FIG. Figure 6a When the electrode area 1112 is in the shape of a strip, the implanted portion 1101 is the electrode area 1112 away from the bonding area 1111 (as described later, see Figure 6aThe implant part 1101 is an end portion on one side of the implant part 1101 (shown in the figure), and the implant part 1101 includes a portion where the electrode structure is arranged in this position area and a reaction film layer 1200. The working electrode 1001, the counter electrode 1002, the reference electrode 1003, and the reaction film layer 1200 are all located on the implant part 1101. In addition, the surface of the implant part 1101 has the reaction film layer 1200, the counter electrode 1002, the reference electrode 1003, and the second insulating layer 1160 and / or the first insulating layer 1130 for separating the counter electrode 1002 and the reference electrode 1003, and the surface of other parts of the implant part 1101 includes a substrate 1110. In this way, when the implant part 1101 is implanted subcutaneously in a patient, the biosensor can monitor the concentration of a target in a body fluid, such as glucose, lactose, L-glutamic acid or xanthine. The first conductive layer 1120, the second conductive layer 1140 and the third conductive layer 1150 are all thin layer structures, which can be formed by applying a slurry containing a conductive substance to the corresponding position and curing it. Similarly, the reaction film layer 1200 can also be formed by applying a coating containing a reactive substance to the outer surface of the working electrode 1001 and curing it. In this way, the preparation process of the biosensor is simple, and the size of each electrode can be guaranteed, avoiding the reduction of the working area of ​​the electrode due to deviations in subsequent processes, and each film layer can have a smaller thickness, thereby reducing the volume of the biosensor, which will reduce the foreign body sensation of some structures of the biosensor when implanted under the patient's skin. Moreover, the arrangement of the electrode structure also improves the performance structure and performance consistency of the biosensor when the biosensor is mass-produced.

[0058] Optionally, the substrate 1110 is made of a flexible material such as a PET film, a PI film, a PE film or a PP film, and its thickness is preferably 50 um to 150 um, so as to further reduce the foreign body sensation caused by the biosensor when implanted subcutaneously. Figure 1 Combined with Figure 6a , the substrate 1110 may be a "convex" structure, including a roughly rectangular bonding area 1111 and an elongated electrode area 1112. The bonding area 1111 is provided with a first pin 1111a electrically connected to the working electrode 1001, a second pin 1111b electrically connected to the counter electrode 1002, and a third pin 1111c electrically connected to the reference electrode 1003, and the formation of each pin will be described in detail below. At least a portion of the electrode area 1112 is located on the implantation portion 1101 of the electrode structure 1100, that is, the working electrode 1001, the counter electrode 1002 and the reference electrode 1003 are provided on the electrode area 1112.

[0059] The first conductive layer 1120 is formed by coating the conductive paste on the substrate 1110 in a predetermined shape by any suitable process such as screen printing, inkjet printing, laser etching, etc. and curing. The conductive paste includes at least one of carbon paste, gold paste and platinum black (i.e., very fine platinum powder). For the working electrode 1001 including carbon paste, its cost is relatively low.

[0060] Optionally, combined Figure 1 He Ru Figure 6b As shown, the first conductive layer 1120 includes a first portion 1121, a second portion 1122 and a third portion 1123 separated from each other, wherein the first portion 1121 extends from the free end of the electrode region 1112 (i.e., the end of the electrode region 1112 away from the bonding region 1111) to the bonding region 1111, and has a first end 1121a and a second end 1121b opposite to each other, the first end 1121a is located on the electrode region 1112, and forms the working electrode 1001. The second end 1121b is located on the bonding region 1111, and is used to form the first pin 1111a. The area of ​​the first portion 1121 located between the first end 1121a and the second end 1121b constitutes the wire of the working electrode 1001. The second portion 1122 is used to form the second pin 1111b. The third portion 1123 is used to form the third pin 1111c. The purpose of providing the second portion 1122 and the third portion 1123 is to raise the pin area to facilitate the subsequent coating of the second conductive layer 1140 on the pin area.

[0061] Next, a first insulating layer 1130 is disposed on the first conductive layer 1120. The embodiment of the present invention has no particular restrictions on the specific material and molding method of the first insulating layer 1130. Figure 6c As shown, the first insulating layer 1130 covers the area on the substrate 1110 where the first conductive layer 1120 is not set, and covers the area of ​​the first part 1121 of the first conductive layer 1120 located between the first end 1121a and the second end 1121b (i.e., the wire part of the working electrode 1001).

[0062] Then, if Figure 6dAs shown, the second conductive layer 1140 is formed. The second conductive layer 1140 is coated on the first insulating layer 1130, the second end 1121b of the first part 1121 of the first conductive layer 1120, the second part 1122 and the third part 1123 by screen printing or any other suitable process according to a predetermined shape using conductive paste. The conductive paste includes carbon paste. The second conductive layer 1140 includes a fourth part 1141, a fifth part 1142 and a sixth part 1143 separated from each other. The fourth part 1141 and the fifth part 1142 both extend from the electrode area 1112 to the bonding area 1111, and the fourth part 1141 has a third end 1141a and a fourth end 1141b opposite to each other, the third end 1141a is located on the electrode area 1112, and the fourth end 1141b covers the second part 1122 of the first conductive layer 1120. The fifth portion 1142 has a fifth end 1142a and a sixth end 1142b opposite to each other, wherein the fifth end 1142a is located on the electrode region 1112, and the sixth end 1142b covers the third portion 1123 of the first conductive layer 1120. The sixth portion 1143 covers the second end 1121a of the first portion 1121 of the first conductive layer 1120.

[0063] Those skilled in the art should understand that the formation of the second conductive layer 1140 may be through a screen printing process as described above, or may be through first printing or coating the fourth part 1141 and the fifth part 1142 as a whole, and then removing part of the second conductive layer 1140 between the fourth part 1141 and the fifth part 1142, so as to complete the third end 1141a and the fifth end 1142a with the separated second conductive layer 1140 parts as two wires to connect the fourth end 1141b and the sixth end 1142b respectively.

[0064] Then, if Figure 6e As shown, the third conductive layer 1150 is formed on the fifth end 1142a of the fifth portion 1142 of the second conductive layer 1140 to serve as the reference electrode 1003, that is, the third conductive layer 1150 is connected to the fourth portion 1142 of the second conductive layer 1140. In addition, the third conductive layer 1150 is also separated from the fourth portion 1141 of the second conductive layer 1140. The third conductive layer 1150 is formed by a conductive paste through a screen printing process. The conductive paste includes silver-silver chloride.

[0065] Then, if Figure 6e and 6fAs shown, the second insulating layer 1160 is formed, and part of the second conductive layer 1140 is covered by the second insulating layer 1160, wherein the area of ​​the fourth portion 1141 located on the electrode region 1112 and close to the third conductive layer 1150 (including the third end 1141a not connected to the third conductive layer 1150, and the fifth end 1142a connected to the third conductive layer 1150), the fourth end 1141b of the fourth portion 1141, the sixth end 1142b of the fifth portion 1142, and the sixth portion 1143 are not covered by the second insulating layer 1160. In addition, it can be understood that, as Figure 6f The arrangement of the second insulating layer 1160 is for the convenience of quickly printing the second insulating layer 1160. Of course, when technicians in this field use other processes to arrange the second insulating layer 1160, the area of ​​the fifth part 1142 close to the third conductive layer 1150 can also be covered by the second insulating layer 1160 (that is, the fifth end 1142a connected to the third conductive layer 1150 can also be covered by the second insulating layer 1160). The fourth part 1141 is located in the exposed area on the electrode area 1112 to form the counter electrode 1002 (so that the counter electrode 1002 and the reference electrode 1003 are isolated from each other), the fourth end 1141b of the fourth part 1141 and the second part 1122 of the first conductive layer 1120 together form the second pin 1111b, the sixth end 1142b of the fifth part 1142 and the third part 1123 of the first conductive layer 1120 together form the third pin 1111c, and the sixth part 1143 and the second end 1121b of the first part 1121 of the first conductive layer 1120 together form the first pin 1111a. That is, each pin has a two-layer structure, and the advantage of such a setting is that the printing effect of the second conductive layer 1140 is better, and each film layer of the biosensor is more uniform.

[0066] Since the third conductive layer 1150 is formed on the fifth end 1142a of the fourth part 1142 of the second conductive layer 1140, the fifth end 1142a of the fourth part 1142 and the portion between the fifth end 1142a and the sixth end 1142b are also used as the conductive wire of the reference electrode 1003, that is, the conductive wire of the reference electrode 1003 and the counter electrode 1002 are located in the same plane and are formed at the same time, which is beneficial to maintaining the consistency of the reference electrode 1003, and can simplify the production process and reduce costs. Those skilled in the art may also understand that when the implantation portion 1101 is implanted subcutaneously in a patient, a fixed potential difference is formed between the reference electrode 1003 and the tissue fluid, so when the biosensor is connected to an external electrical analyzer through the first pin 1111a, the second pin 1111b, and the third pin 1111c, a voltage loop is formed between the working electrode 1001 and the reference electrode 1003, and a current loop is formed between the working electrode 1001 and the counter electrode 1002. When the operator sets the potential difference between the working electrode 1001 and the reference electrode 1003 through the electrical analyzer, for example, to 0V, the potential difference between the working electrode 1001 and the tissue fluid can be determined, which helps to improve the detection accuracy of the biosensor.

[0067] Please refer back to Figure 2 The reaction film layer 1200 can be formed by coating the reaction reagent on the outer surface of the working electrode 1001 and curing. The reaction reagent is coated on the working electrode 1001 by dispensing or inkjet process, which is conducive to ensuring the accuracy of the amount of the reaction reagent and improving the consistency of the mass production of the biosensor.

[0068] The reaction reagents include bioreaction enzymes, metal complexes, polypeptide macromolecules, stabilizers and first crosslinking agents. Among them, amino, carboxyl, hydroxyl and other groups on the polypeptide macromolecules can react with the metal complexes and biooxidases to form covalent bonds, and free radical polymerization occurs under the action of the first crosslinking agent to form Figure 3 The core-shell structure shown improves the stability of the reaction membrane layer 1200 and prolongs the life of the biosensor. The core-shell structure has active groups that can react with the target. The stabilizer can stabilize the performance of the reaction reagent.

[0069] The specific type of the bioreaction enzyme is determined according to the target. For example, when the target is glucose, the bioreaction enzyme is glucose oxidase. When the target is lactic acid, the bioreaction enzyme is lactate oxidase. When the target is L-glutamic acid, the bioreaction enzyme is L-glutamate oxidase. When the target is xanthine, the bioreaction enzyme is xanthine oxidase.

[0070] The metal complex can be a transition metal complex, including but not limited to at least one of an osmium (OS) complex, a rhodium (Rh) complex, and a cobalt (Co) complex. The ligand can be a polymer ligand or a small molecule ligand. Optional polymer ligands include but are not limited to long branched chain structures such as polymethacrylates, polyacrylamides, and polyvinyl pyrrolidones, and optional small molecule ligands include but are not limited to nitrogen-containing heterocyclic small molecules such as pyridine, imidazole, etc. Suitable ligands can reduce the redox potential of the metal, thereby reducing the operating voltage of the bioreaction enzyme to recognize and oxidize the target, and improving the reaction sensitivity of the biosensor. Not only that, suitable ligands can also reduce the interference of electroactive substances on the response current of the target under some high operating voltages, thereby improving the detection accuracy of the biosensor.

[0071] The polypeptide macromolecule includes but is not limited to at least one of bovine serum albumin, human serum albumin and citrulline.

[0072] The first cross-linking agent may be a multifunctional small molecule compound, including but not limited to a small molecule compound including a diisocyanate group, a polyisocyanate group, or at least one of a small molecule compound including a diepoxy group and a polyepoxy group.

[0073] The stabilizer is a polymer prepolymer solution, including but not limited to at least one of polyacrylamide prepolymer solution, polyacrylate prepolymer solution, polyhydroxyethyl methacrylate prepolymer solution, polyNIPAM prepolymer solution, and polyDMAEMA prepolymer solution.

[0074] Optionally, in the reaction reagent, based on the total amount of the metal complex, the bioreaction enzyme, the stabilizer, the polypeptide macromolecule, and the first cross-linking agent as 100%, the mass fraction of the metal complex is 5% to 50%, the mass fraction of the stabilizer is 1% to 20%, the mass fraction of the polypeptide macromolecule is 1% to 20%, the mass fraction of the first cross-linking agent is 0.1% to 10%, and the remainder is the bioreaction enzyme.

[0075] Further, please refer back to Figure 2 The biosensor further comprises a functional film layer 1300, which is a film layer that promotes the electrochemical reaction of the reaction film layer 1200. The functional film layer 1300 at least covers the reaction film layer 1200, the counter electrode 1002 and the reference electrode 1003, and is used to further improve the reaction sensitivity of the biosensor. Optionally, the functional film layer 1300 also covers the exposed area of ​​the fifth portion 1142 of the second conductive layer 1140 in the electrode region 1111.

[0076] Optionally, the functional film layer 1300 may include an anti-interference film layer 1310, which is used to prevent interfering substances from passing through the functional film layer 1300, so as to reduce the adverse effects of the interfering substances on the working electrode 1001 and reduce the monitoring accuracy of the biosensor. The interfering substances described herein include at least one of acetaminophen, vitamin C, ascorbic acid, etc. that may be present in body fluids. The anti-interference film layer 1310 at least covers the working electrode 1001, and the anti-interference film layer 1310 is coated on the outer surface of the reaction film layer 1200 by dipping, spraying or other any suitable methods by anti-interference coating and cured. It is understandable that the anti-interference film layer 1310 can also cover the counter electrode 1002 and the reference electrode 1003, and at this time, the anti-interference coating is also coated on the outer surface of the counter electrode 1002 and the outer surface of the reference electrode 1003. The anti-interference film layer 1310 includes at least one of naphthol, cellulose acetate, polylysine, polyvinyl pyridine and its modified copolymer, and polyurethane, and has a molecular weight distribution of 10000Da to 1000000Da.

[0077] Further, the functional film layer 1300 further includes an adjustment film layer 1320 for adjusting the passing rate of the target object on the functional film layer 1300. The adjustment film layer 1320 covers the reaction film layer 1200, the counter electrode 1002 and the reference electrode 1003. It can be understood that when the interference film layer 1310 covers the counter electrode 1002 and the reference electrode 1003, the adjustment film layer 1320 is completely covered on the anti-interference film layer 1310.

[0078] The adjustment membrane layer 1320 mainly includes a hydrophilic polymer, a hydrophobic polymer and a second cross-linking agent, and the hydrophilic polymer and the hydrophobic polymer undergo a cross-linking reaction under the action of the second cross-linking agent to form a three-dimensional network structure. When the adjustment membrane layer 1320 contacts the body fluid, the adjustment membrane layer 1320 swells to form a hydrosol gel, and the target in the body fluid passes through the pores of the three-dimensional network structure, and further passes through the anti-interference layer 1310 and contacts with the reaction membrane layer 1200 and undergoes an electrochemical reaction. By adjusting the ratio of the hydrophilic polymer and the hydrophobic polymer, the density of the pores in the three-dimensional network structure and the size of the pores can be adjusted, thereby achieving the regulation of the pass rate of the target.

[0079] Further, the adjustment film layer 1320 includes a first adjustment film layer 1321 and a second adjustment film layer 1322, wherein the first adjustment film layer 1321 is coated with a first adjustment coating and cured on the anti-interference film layer 1310, and the second adjustment film layer 1322 is coated with a second adjustment coating and cured on the first adjustment film layer 1321. The content of the hydrophobic polymer in the first adjustment film layer 1321 is greater than the content of the hydrophobic polymer in the second adjustment film layer 1322. Specifically, in the first adjustment film layer 1321, the weight ratio of the hydrophilic polymer to the hydrophobic polymer is 1:9 to 1:1.1, and in the second adjustment layer, the weight ratio of the hydrophilic polymer to the hydrophobic polymer is 9:1 to 1:1. In this way, the first adjustment film layer 1321 can have a certain mechanical strength, and play a protective role in the body fluid for the reaction film layer 1200, the counter electrode 1002, the reference electrode 1003 and the anti-interference film layer 1310. The second adjustment film layer 1322 has better hydrophilicity, which improves the biocompatibility of the biosensor.

[0080] In the present embodiment, the hydrophilic polymer includes but is not limited to polyethylene glycol, polyhydroxyethyl methacrylate, polyacrylic acid, polypropylene alcohol, chitosan, hydrophilic cellulose, hydrophilic modified silane condensation polymer, hydrophilic modified polyurethane, poly-DMAEMA, poly-NIPAM, polymethacrylamide, polydopamine, alginic acid, hyaluronic acid, sodium polystyrene sulfonate, polyethylene glycol-modified vinyl pyridine, polysulfonic acid-modified vinyl pyridine, and at least one of polycarboxyl-modified 4-vinyl pyrrolidone. The hydrophobic polymer includes but is not limited to at least one of polystyrene, polymethyl methacrylate, polyvinyl pyridine, polyvinyl pyrrolidone, polysilanes, polyurethane, and polycarbonate. Preferably, the molecular weight distribution of the hydrophilic polymer and the hydrophobic polymer is 10000Da to 1000000Da.

[0081] For further preference, please refer to Figure 2 In order to improve the biocompatibility of the biosensor described in the present application when implanted in a human body, the biosensor further includes a biocompatible layer 1400 , and the biocompatible layer 1400 is used to cover the surface of the implant part 1101 together with the functional film layer 1300 .

[0082] Furthermore, the present invention also provides a method for preparing the aforementioned biosensor, such as Figure 4 , Figure 5 and Figures 6a to 6f As shown, the preparation method comprises the following steps:

[0083] Step S100: preparing the electrode structure 1100 .

[0084] Step S200 : forming a reaction film layer 1200 on the working electrode 1001 .

[0085] Wherein, the step S100 includes:

[0086] Step S110: providing the substrate 1110;

[0087] Step S120 : forming the first conductive layer 1120 on the substrate 1110 .

[0088] Step S130 : forming the first insulating layer 1130 on the first conductive layer 1120 , and leaving a portion of the first conductive layer 1120 exposed to serve as the working electrode 1001 .

[0089] Step S140 : forming the second conductive layer 1140 on the first insulating layer 1130 .

[0090] Step S150 : forming the third conductive layer 1150 on a partial area of ​​the second conductive layer 1140 , wherein the third conductive layer serves as the reference electrode 1003 .

[0091] Step S160 : forming the second insulating layer on the second conductive layer 1140 , and leaving a portion of the second conductive layer 1140 exposed to serve as the counter electrode 1002 , and the counter electrode 1002 and the reference electrode 1003 are isolated from each other.

[0092] The step S200 specifically includes: coating the reaction reagent on the working electrode 1001 by dispensing or inkjet process, and curing to form the reaction film layer 1200 .

[0093] Furthermore, the preparation method further includes step S300 : forming a functional film layer 1300 at least on the reaction film layer 1200 , the counter electrode 1002 and the reference electrode 1003 .

[0094] The step S300 specifically includes:

[0095] Step S310 : coating anti-interference coating at least on the reaction film layer 1200 , the counter electrode 1002 , and the reference electrode 1003 , and curing to form the anti-interference film layer 1310 .

[0096] Step S320: coating a first adjustment coating on the anti-interference film layer 1310 , and forming the first adjustment film layer 1321 after curing.

[0097] Step S320: coating the second adjustment coating on the first adjustment film layer 1321 , and forming the second adjustment film layer 1322 after curing.

[0098] In addition, when the biosensor further includes a biocompatible layer 1400, the preparation method further includes step S400: forming a biocompatible layer on the implant part 1101, that is, the biocompatible layer 1200 is used to cover the surface of the implant part 1101 together with the functional film layer 1300. The biocompatible layer 1400 can be obtained by coating and curing with any biocompatible coating.

[0099] Next, the biosensor and its effects are introduced in conjunction with a specific embodiment.

[0100] In this embodiment, the conductive paste used to form the first conductive layer includes carbon paste, and the first conductive layer, the second conductive layer and the third conductive layer are formed by a screen printing process.

[0101] The reaction reagent is drop-coated on the working electrode by a dispensing process. In this embodiment, the bioreaction enzyme is glucose oxidase. The metal complex is an osmium metal complex, specifically a grafted polymer complex, and the polymer main chain is an acrylic copolymer, and the ligand is bisimidazole. The polypeptide macromolecule is human serum albumin (HAS). The stabilizer is a polyacrylamide prepolymer solution. The first cross-linking agent is diethylene glycol ester, and its molecular weight is 1000Da. Taking the total amount of solute as 100% as a benchmark, the mass percentage of glucose oxidase is 37.5%, the mass percentage of osmium metal complex is 45%, the mass percentage of human serum albumin is 5%, the mass percentage of polyacrylamide prepolymer solution is 10%, and the mass percentage of diethylene glycol ester is 2.5%. And in the reaction reagent, the total solid content is 10%.

[0102] After the reaction reagent is cured to form the reaction film layer 1200, an anti-interference coating is applied on the reaction film layer 1200, the counter electrode 1002, and the reference electrode 1003 by dip coating. The anti-interference coating includes naphthalene, and the mass fraction of naphthalene in the anti-interference coating is 5%.

[0103] After the anti-interference coating is cured to form the anti-interference film layer, a first adjustment coating is applied on the anti-interference film layer by dip coating. Wherein, the hydrophobic polymer includes polystyrene and polyvinyl pyridine, the hydrophilic polymer includes polycarboxyl-modified vinyl pyridine, and the second cross-linking agent is diethylene glycol ester. In the first adjustment coating, the total mass fraction of the hydrophobic polymer and the hydrophilic polymer is 20%, and the ratio of the hydrophobic polymer to the hydrophilic polymer is 3:1 by weight percentage, and the ratio of polystyrene to polyvinyl pyridine is 1:1. The molecular weight of diethylene glycol ester is 500, and the mass fraction is 2%.

[0104] After the first adjustment coating is dried to form the first adjustment layer, a second adjustment coating is applied on the first adjustment layer by dipping. The hydrophobic polymer of the second adjustment coating includes polysiloxane, the hydrophilic polymer includes polyethylene glycol-modified vinyl pyridine and polysulfonic acid-modified vinyl pyridine, and the second cross-linking agent is glycidyl ester. In the second adjustment coating, the total mass fraction of the hydrophobic polymer and the hydrophilic polymer is 20%, and the weight ratio of the hydrophobic polymer to the hydrophilic polymer is 1:3, and the weight ratio of polyethylene glycol-modified vinyl pyridine to polysulfonic acid-modified vinyl pyridine is 1:2. The mass fraction of glycidyl ester is 1%.

[0105] The biosensor provided in this embodiment was used to perform a performance test. The test steps are as follows:

[0106] Immerse the biosensor in a standard PBS buffer solution and soak for 30 minutes. Those skilled in the art know how to prepare a standard PBS buffer solution. Then perform the remaining measurements on the biosensor at 0V. Wait for 10 minutes to allow the biosensor to reach a constant background, and then add 5mM of glucose to the tested solution every 5 minutes, so that the glucose content in the tested solution is 0mM, 5mM, 10mM, 15mM, 20mM, 25mM, and 30mM, respectively, and measure the linearity of the biosensor reaction. After each addition of glucose, allow the solution to equilibrate for 5 minutes, and the solution should be stirred continuously during the measurement process to make the concentration of the measured solution uniform. The test results are as follows: Figure 7 and Figure 8 shown.

[0107] Figure 7 The graph shows the current curve of the biosensor responding to glucose when different concentrations of glucose are continuously added to the test solution at a potential of 0 V, with the interval between two adjacent samplings being 10 seconds, wherein the abscissa represents the number of sampling points. Figure 8 The linear relationship between the response current of the biosensor and the glucose concentration is shown. Figure 7 It can be seen that the oxidation peak current increases in a step-like manner with the addition of glucose. It can be found from the figure that the biosensor prepared using the scheme of the present application will change accordingly at the next sampling moment when the glucose solution is added, indicating that the response time of this biosensor is within 10 seconds. Compared with some existing biosensors, the feedback time of the sensing environmental solution is shortened. Specifically, after being applied to the human body, it can also reflect the changes in the user's biological values ​​more quickly and has high sensitivity. This also indirectly shows that the regulating membrane layer of the present application has good regulating diffusion ability for glucose, and the reaction membrane layer has good catalytic oxidation performance for glucose, and can quickly establish redox balance. Figure 8It can be seen that within the test concentration range, the response current of the biosensor is linearly related to the concentration of glucose, and its linear equation is I(nA)=0.4929C+0.0146, where C represents the concentration of glucose, and the fitting degree of the linear equation is 0.9894.

[0108] Although the present invention is disclosed as above, it is not limited thereto. Those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A biosensor, It is characterized in that include: An electrode structure, comprising a substrate, a first conductive layer, a first insulating layer, a second conductive layer, a third conductive layer, and a second insulating layer; wherein the first conductive layer is formed on the substrate; the first insulating layer is formed on the first conductive layer, and a portion of the first conductive layer is exposed to form a working electrode; the second conductive layer is formed on the first insulating layer; the third conductive layer is formed on a portion of the second conductive layer; the second insulating layer is at least formed on the second conductive layer, and a portion of the second conductive layer is exposed to form a counter electrode, and at least a portion of the third conductive layer is exposed to form a reference electrode; and, The reaction film layer is formed by coating a reaction reagent on the working electrode and curing it, and is used to undergo an electrochemical reaction with a target; the reaction reagent includes a metal complex, a bioreaction enzyme, a polypeptide macromolecule, a stabilizer, and a first cross-linking agent; wherein the stabilizer is a polymer prepolymer solution, and the first cross-linking agent includes a small molecule compound with a diisocyanate group or a polyisocyanate group, or is at least one of a small molecule compound with a diepoxy group and a polyepoxy group.

2. The biosensor according to claim 1, It is characterized in that The metal complex includes a transition metal complex.

3. The biosensor according to claim 1, It is characterized in that The bioreaction enzyme includes any one of glucose oxidase, lactate oxidase, L-glutamate oxidase or xanthine oxidase.

4. The biosensor according to claim 1, It is characterized in that Taking the total amount of the metal complex, the bioreaction enzyme, the polypeptide macromolecule, the stabilizer and the first cross-linking agent as 100%, the mass percentage of the metal complex is 5% to 50%, the mass percentage of the stabilizer is 1% to 20%, the mass percentage of the polypeptide macromolecule is 1% to 20%, the mass percentage of the first cross-linking agent is 0.1% to 10%, and the remainder is the bioreaction enzyme.

5. The biosensor according to claim 1, It is characterized in that The biosensor also includes a functional membrane layer; the functional membrane layer includes an anti-interference membrane layer, the anti-interference membrane layer is at least arranged on the reaction membrane layer, the counter electrode and the reference electrode, and is used to prevent interfering substances from passing through the functional membrane layer.

6. The biosensor according to claim 5, It is characterized in that The anti-interference film layer comprises at least one of naphthol, cellulose acetate, polylysine, polyvinyl pyridine and its modified copolymer, and polyurethane.

7. The biosensor according to claim 5, It is characterized in that The functional film layer further comprises a regulating film layer, which is arranged on the anti-interference film layer and is used for regulating the passing rate of the target object on the functional film layer.

8. The biosensor according to claim 7, It is characterized in that The adjustment membrane layer includes a hydrophilic polymer, a hydrophobic polymer and a second cross-linking agent.

9. The biosensor according to claim 8, It is characterized in that The adjustment film layer includes a first adjustment film layer and a second adjustment film layer, the first adjustment film layer is formed on the anti-interference film layer, and the second adjustment film layer is formed on the first adjustment film layer; the content of the hydrophobic polymer in the first adjustment film layer is greater than the content of the hydrophobic polymer in the second adjustment film layer.

10. The biosensor according to claim 9, It is characterized in that In the first adjustment film layer, the weight ratio of the hydrophilic polymer to the hydrophobic polymer is 1:9 to 1:1.1, and in the second adjustment film layer, the weight ratio of the hydrophilic polymer to the hydrophobic polymer is 9:1 to 1:

1.

11. The biosensor according to any one of claims 8 to 10, It is characterized in that The hydrophilic polymer includes at least one of polyethylene glycol, polyhydroxyethyl methacrylate, polyacrylic acid, polypropylene alcohol, chitosan, hydrophilic cellulose, hydrophilic modified silane condensation polymer, hydrophilic modified polyurethane, polyDMAEMA, polyNIPAM, polymethacrylamide, polydopamine, alginic acid, hyaluronic acid, sodium polystyrene sulfonate, polyethylene glycol-modified vinyl pyridine, polysulfonic acid-modified vinyl pyridine, and polycarboxyl-modified 4-vinyl pyrrolidone; the hydrophobic polymer includes at least one of polystyrene, polymethyl methacrylate, polyvinyl pyridine, polyvinyl pyrrolidone, polysilanes, polyurethane, and polycarbonate; and / or, The molecular weight distribution of the hydrophilic polymer and the hydrophobic polymer is 10000Da to 1000000Da.

12. The biosensor according to claim 5, It is characterized in that The biosensor includes an implantation part, which is used to be implanted into a target object, and the working electrode, the counter electrode, the reference electrode, and the reaction film layer are all located on the implantation part; the biosensor also includes a biocompatible layer, and the biocompatible layer is used to cover the surface of the implantation part together with the functional film layer.

13. A method for preparing a biosensor, for preparing the biosensor according to any one of claims 1 to 12, It is characterized in that The preparation method comprises the following steps: providing the substrate; forming the first conductive layer on the substrate; forming the first insulating layer on the first conductive layer; forming the second conductive layer on the first insulating layer; forming the third conductive layer on the second conductive layer; forming the second insulating layer at least on the second conductive layer; The reaction film layer is formed on the working electrode.

14. The method for preparing the biosensor according to claim 13, It is characterized in that The reaction film layer is formed on the working electrode by dispensing or inkjet process.

15. The method for preparing the biosensor according to claim 13, It is characterized in that A functional film layer is formed at least on the reaction film layer, the counter electrode and the reference electrode.

16. The method for preparing the biosensor according to claim 15, It is characterized in that The biosensor includes an implant part, the working electrode, the counter electrode, the reference electrode, and the reaction film layer are all located on the implant part, and the preparation method also includes: forming a biocompatible layer on the implant part, and the biocompatible layer is used to cover the surface of the implant part together with the functional film layer.

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