WO2023024495A1 - Biosensor and preparation method therefor - Google Patents

Abstract

The present invention provides a biosensor and a preparation method therefor. The biosensor comprises: an electrode structure, which comprises 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 causes part of an area of the first conductive layer to be exposed to form a working electrode, the second conductive layer is formed on the first insulating layer, the third conductive layer is formed on part of an area of the second conductive layer, and the second insulating layer is at least formed on the second conductive layer and causes part of an area of the second conductive layer to be exposed to form a counter electrode and causes at least part of an area of the third conductive layer to be exposed to form a reference electrode; and a reaction membrane layer, which is formed on the working electrode and used for electrochemical reaction with a target object. The biosensor is simple and logical in structure, and facilitates batch production.

Description

A kind of biosensor and preparation method thereof technical field

The invention relates to the technical field of medical devices, in particular to a biosensor and a preparation method thereof.

Background technique

The definition of a biosensor is "a device that uses immobilized biomolecular binding transducers to detect environmental chemicals inside or outside the organism or interact specifically with them to generate a response." According to the differences of living substances used in the sensors, biosensors can be divided into tissue sensors, cell sensors, enzyme sensors, etc. , insulin sensors, etc., and biosensors can also be divided into immune sensors, drug sensors, etc. according to their uses. So far, the most commercially successful biosensor is the glucose sensor, which is used to monitor glucose in body fluids such as human blood, interstitial fluid, and sweat. Due to the huge population of diabetic patients worldwide, glucose sensors have a huge market share.

Glucose monitoring requires the use of a dedicated glucose detector, the core component of which is the glucose sensor. Glucose sensors can be divided into in vitro sensors, fully implanted sensors and subcutaneously implanted sensors. Among them, the in vitro sensor monitors the blood glucose data of a single point through fingertip blood collection. The fully implanted sensor has biocompatibility problems, and the surgical implantation and removal are more complicated. The subcutaneous implanted sensor is quickly implanted in a minimally invasive way. In or out, also capable of continuous glucose monitoring. However, the subcutaneous implantable glucose sensor in the prior art has problems such as complex preparation process, high cost, short life, poor anti-interference ability, and sensitivity attenuation.

Contents of the invention

The object of the present invention is to provide a biosensor and a preparation method thereof, the biosensor has a simple structure and is convenient to prepare.

To achieve the above object, the invention provides a biosensor, comprising:

The electrode structure includes 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 An 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 A layer is formed on a partial area of the second conductive layer; the second insulating layer is formed at least on the second conductive layer, and a partial area of the second conductive layer is exposed to form a counter electrode, and the At least a portion of the third conductive layer is exposed to form a reference electrode; and,

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

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

Optionally, the metal complexes include transition metal complexes.

Optionally, the biological reaction enzyme includes any one of glucose oxidase, lactate oxidase, L-glutamic acid oxidase or xanthine oxidase.

Optionally, the reaction reagent also includes a stabilizer, based on the total amount of the metal complex, biological reaction enzyme, polypeptide macromolecule, stabilizer and first crosslinking agent as 100%, the mass of the metal complex The percentage 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%, and the mass percentage of the first crosslinking agent is 0.1% ~10%, and the balance is the biological reaction enzyme.

Optionally, the stabilizer includes a polymer prepolymerization solution.

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

Optionally, the anti-interference film layer includes at least one of naphthol, cellulose acetate, polylysine, polyvinylpyridine and its modified copolymer, and polyurethane.

Optionally, the functional film layer further includes an adjustment film layer, the adjustment film layer 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.

Optionally, the regulating film layer includes a hydrophilic polymer, a hydrophobic polymer and a second crosslinking agent.

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 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.

Optionally, in the first regulating film layer, the weight ratio of the hydrophilic polymer to the hydrophobic polymer is 1:9 to 1:1.1, and in the second regulating film layer, The weight ratio of the hydrophilic polymer to the hydrophobic polymer is 9:1˜1:1.

Optionally, the hydrophilic polymer includes polyethylene glycol, polyhydroxyethyl methacrylate, polyacrylic acid, polypropylene alcohol, chitosan, hydrophilic cellulose, and hydrophilic modified silane condensation polymers , Hydrophilic modified polyurethane, polyDMAEMA, polyNIPAM, polymethacrylamide, polydopamine, alginic acid, hyaluronic acid, sodium polystyrene sulfonate, polyethylene glycol modified vinylpyridine, polysulfonate At least one of acid-modified vinylpyridine and polycarboxy-modified 4-vinylpyrrolidone; the hydrophobic polymer includes polystyrene, polymethyl methacrylate, polyvinylpyridine, polyvinylpyrrolidone , polysilanes, polyurethanes, and polycarbonates; and/or,

The molecular weight distribution of the hydrophilic polymer and the hydrophobic polymer is 10000Da-1000000Da.

Optionally, the biosensor includes an implant part, the implant part is used to implant a target object, and the working electrode, the counter electrode, the reference electrode, and the reaction film layer are all located on On the implanted part; the biosensor also includes a biocompatible layer, the biocompatible layer is located at the implanted part, and the biocompatible layer is used to cover the functional film layer together the surface of the implant.

In order to achieve the above object, the present invention also provides a method for preparing a biosensor, which is used to prepare the biosensor as described in any one of the preceding items, and the preparation method includes the following steps:

providing said 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 on at least the second conductive layer;

The reaction film layer is formed on the working electrode.

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

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

Optionally, the biosensor includes an implant, and the working electrode, the counter electrode, the reference electrode, and the reaction film layer are located on the implant, and the preparation method further includes : the biosensor includes an implant, the working electrode, the counter electrode, the reference electrode, and the reaction film layer are located on the implant, and the preparation method further includes: A biocompatible layer is formed on the implanted part, and the biocompatible layer is used to cover the surface of the implanted part together with the functional film layer.

Compared with the prior art, the biosensor of the present invention and its preparation method have the following advantages:

The aforementioned biosensor comprises a substrate, an electrode structure and a reaction membrane layer; the electrode structure comprises a first conductive layer, a first insulating layer, a second conductive layer, a third conductive layer and a second insulating layer, and the first conductive layer 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, and the second conductive layer is formed on the a first insulating layer, the third conductive layer is formed on a partial area of the second conductive layer, the second insulating layer is formed at least on the second conductive layer, and makes the second conductive layer A part of the area is exposed to form a counter electrode, and at least a part 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 electrochemically react with the target, The target object is, for example, any one of glucose, lactic acid, xanthine, and L-glutamic acid in body fluids. The biosensor can be sequentially coated with coatings and then cured to shape. The preparation method is simple and low in cost.

Further, the reaction membrane layer includes metal complexes, bioreaction enzymes, polypeptide macromolecules, and a first crosslinking agent, wherein groups such as amino groups, carboxyl groups, and hydroxyl groups on the polypeptide macromolecules can be combined with the metal complexes and the first cross-linking agent. The bioreaction enzyme reaction forms a covalent bond, and the core-shell structure is formed by the action of the stabilizer, which can improve the stability and lifespan of the biosensor.

Still further, the biosensor also includes a functional film layer, the functional film layer may include an anti-interference film layer and an adjustment film layer, and the anti-interference film layer is used to prevent interfering substances from passing through the functional film layer, reducing the impact on The detection interference of the target object, the adjustment film layer is used to adjust the pass rate of the target object on the functional film layer, and then adjust the amount of the target object entering the reaction film layer, and improve the biosensor sensitivity.

Description of drawings

The accompanying drawings are used to better understand the present invention, and do not constitute improper limitations to the present invention. in:

Fig. 1 is a schematic diagram of the overall structure of a biosensor provided by the present invention according to an embodiment;

Fig. 2 is a cross-sectional view of a biosensor provided by the present invention according to an embodiment;

Fig. 3 is a schematic diagram of the core-shell structure in the reaction membrane layer of the biosensor according to an embodiment of the present invention;

Fig. 4 is a flow chart of the preparation of the biosensor provided by the present invention according to an embodiment;

Fig. 5 is a flow chart of the preparation of the electrode structure of the biosensor according to an embodiment of the present invention;

Figure 6a to Figure 6f show a schematic diagram of the preparation process of the electrode structure of the biosensor;

Fig. 7 is a graph of response current curves of the biosensor provided by an embodiment of the present invention to different concentrations of glucose;

Fig. 8 is a graph showing the linear relationship between the response current and the glucose concentration of the biosensor provided by an embodiment of the present invention.

[the reference signs are explained as follows]:

1100-electrode structure, 1101-implantation part, 1110-base, 1111-bonding 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 terminal, 1141b-fourth terminal, 1142-fifth part, 1142a-fifth terminal, 1142b-sixth terminal, 1143-sixth part, 1150-third conductive layer, 1160-second insulation layer, 1001-working electrode, 1002-counter electrode, 1003-reference electrode;

1200-reaction film layer;

1300-functional film layer, 1310-anti-interference film layer, 1320-regulating film layer, 1321-first regulating film layer, 1322-second regulating film layer;

1400 - Biocompatible layer.

Detailed ways

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the diagrams provided in this embodiment are only schematically illustrating the basic idea of the present invention, and only the components related to the present invention are shown in the diagrams rather than the number, shape and shape of the components in actual implementation. Dimensional drawing, the type, quantity and proportion of each component can be changed arbitrarily during actual implementation, and the component layout type may also be more complicated.

In addition, each embodiment of the content described below has one or more technical features respectively, but this does not mean that the inventor must implement all the technical features in any embodiment at the same time, or can only implement different embodiments separately. Some or all of the technical features. In other words, on the premise that the implementation is possible, those skilled in the art can selectively implement some or all of the technical features in any embodiment according to the disclosure of the present invention and depending on design specifications or implementation requirements, or Selectively implement a combination of some or all of the technical features in multiple embodiments, thereby increasing the flexibility of the implementation of the present invention.

As used in this specification, the singular forms "a", "an" and "the" include plural objects, and the plural form "a plurality" includes two or more objects, unless the content clearly states otherwise. As used in this specification, the term "or" is generally used in the sense including "and/or", unless the content clearly indicates otherwise, and the terms "install", "connect" and "connect" should be To understand it 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 intermediary, and it can be the internal communication of two elements or the interaction relationship between two elements. Those of ordinary skill in the art can understand the specific meanings of the above terms in the present invention according to specific situations.

In order to make the purpose, advantages and features of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings. It should be noted that all the drawings are in a very simplified form and use imprecise scales, and are only used to facilitate and clearly assist the purpose of illustrating the embodiments of the present invention. The same or similar reference numerals in the drawings represent the same or similar components.

FIG. 1 shows a schematic diagram of the overall structure of a biosensor provided by an embodiment of the present invention, and FIG. 2 is a cross-sectional view of the biosensor.

Please refer to FIG. 1 and FIG. 2 , the biosensor includes an electrode structure 1100 and a reaction membrane layer 1200 . Wherein, 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 exposes a part of the first conductive layer 1120 to form the 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 partial area of the second conductive layer. The second insulating layer 1160 is formed at least on the second conductive layer 1140, and exposes a part of the second conductive layer 1140 to form the counter electrode 1002, and at least a part of the third conductive layer 1150 exposed to form a reference electrode 1003. The reaction film layer 1200 is formed on the working electrode 1001 and used for electrochemical reaction with the target. Here, the "bare" means not covered, that is, the area where the working electrode 1001 is formed on the first conductive layer 1120 is not covered by the first insulating layer 1130, and the second conductive layer The area where the counter electrode 1002 is formed on 1140 is not covered by the third conductive layer 1150 and the second insulating layer 1160, and the area where the reference electrode 1003 is formed on the third conductive layer 1150 is not covered by the third conductive layer 1150. Covered by the second insulating layer 1160 , in this embodiment, preferably, the third conductive layer 1150 is completely exposed to the second insulating layer 1160 and forms the reference electrode 1003 .

At least a part of the structure of the biosensor can be used to implant a target object, and the target object can be subcutaneous of a patient. The part of the biosensor used to implant subcutaneous of a patient is referred to as an implant part 1101 herein, specifically as For the part shown in FIG. 1 , those skilled in the art should understand that the implanted part 1101 is the end of the electrode region 1112 as shown in FIG. The end of the electrode region 1112 away from the junction region 1111 (as described later, see the part shown in FIG. 6a ), the implanted part 1101 includes the part where the electrode structure is arranged in this position region and the 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 implanted portion 1101 . In addition, the surface of the implanted part 1101 has the reaction film layer 1200, the counter electrode 1002, the reference electrode 1003, and the second electrode for separating the counter electrode 1002 and the reference electrode 1003. The second insulating layer 1160 and/or the first insulating layer 1130 include the base 1110 on other parts of the surface of the implanted portion 1101 . In this way, when the implant part 1101 is implanted under the patient's skin, the biosensor can monitor the concentration of the target substance in the 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 shaped by coating a paste containing conductive substances on corresponding positions and curing. Similarly, the reaction film layer 1200 can also be formed by coating the coating containing the reactive substance on 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 to avoid reducing the working area of the electrode due to deviations in subsequent processes, and each film layer can have a smaller thickness, thereby reducing the size of the biosensor This will reduce the foreign body sensation when the partial structure of the biosensor is 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.

Optionally, the substrate 1110 is made of a flexible material such as PET film, PI film, PE film or PP film, and its thickness is preferably 50um to 150um, so as to further reduce the foreign body sensation brought by the biosensor implanted subcutaneously. . Please refer back to FIG. 1 in conjunction with FIG. 6 a , the substrate 1110 may be in a “convex” structure, including a substantially rectangular bonding region 1111 and an elongated electrode region 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 second pin 1111b electrically connected to the reference electrode 1003. The connected third pin 1111c, the formation of each pin will be described in detail below. At least a part of the electrode area 1112 is located on the implanted part 1101 of the electrode structure 1100, that is, the working electrode 1001, the counter electrode 1002 and the reference electrode 1003 are arranged on the electrode area 1112 .

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

Optionally, as shown in FIG. 1 and FIG. 6b, the first conductive layer 1120 includes a first part 1121, a second part 1122 and a third part 1123 separated from each other, wherein the first part 1121 is separated from the electrode region The free end of 1112 (that is, the end of the electrode region 1112 away from the bonding region 1111) extends to the bonding region 1111, and has an opposite first end 1121a and a second end 1121b, and the first end 1121a is located at The working electrode 1001 is formed on the electrode region 1112 . The second end 1121b is located on the bonding area 1111 and is used to form the first pin 1111a. A region of the first portion 1121 between the first end 1121 a and the second end 1121 b constitutes a 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 setting the second part 1122 and the third part 1123 is to raise the lead area, so as to facilitate subsequent coating of the second conductive layer 1140 on the lead area.

Next, a first insulating layer 1130 is disposed on the first conductive layer 1120 , and the embodiment of the present invention has no special limitation on the specific material and forming method of the first insulating layer 1130 . As shown in FIG. 6c, the first insulating layer 1130 covers the area where the first conductive layer 1120 is not provided on the substrate 1110, and the area covering the first portion 1121 of the first conductive layer 1120 located on the The area between the first end 1121a and the second end 1121b (that is, the wire portion of the working electrode 1001 ).

Next, as shown in FIG. 6d, the second conductive layer 1140 is formed. The second conductive layer 1140 is coated on the first insulating layer 1130 and the first part 1121 of the first conductive layer 1120 in a predetermined shape by using conductive paste through screen printing or any other suitable process. On the two ends 1121b, the second portion 1122 and the third portion 1123 . The conductive paste includes carbon paste. The second conductive layer 1140 includes a fourth portion 1141 , a fifth portion 1142 and a sixth portion 1143 separated from each other. Wherein, both the fourth portion 1141 and the fifth portion 1142 extend from the electrode region 1112 to the bonding region 1111, and the fourth portion 1141 has a third end 1141a and a fourth end 1141b opposite to each other, The third end 1141 a is located on the electrode region 1112 , and the fourth end 1141 b covers the second portion 1122 of the first conductive layer 1120 . The fifth part 1142 has opposite fifth end 1142a and sixth end 1142b, the fifth end 1142a is located on the electrode region 1112, and the sixth end 1142b covers the first conductive layer 1120. on the third part 1123 described above. The sixth portion 1143 covers the second end 1121b of the first portion 1121 of the first conductive layer 1120 .

Those skilled in the art should understand that, forming the second conductive layer 1140 may be through the process of screen printing as described above, or the fourth part 1141 and the fifth part 1141 may be integrally printed or coated first. part 1142, and then remove part of the second conductive layer 1140 between the fourth part 1141 and the fifth part 1142 to complete the second conductive layer separated by the third end 1141a and the fifth end 1142a Part 1140 is used as two wires respectively connected to the fourth end 1141b and the sixth end 1142b.

Next, as shown in FIG. 6e, the third conductive layer 1150 is formed on the fifth end 1142a of the fifth portion 1142 of the second conductive layer 1140 as the reference electrode 1003, also That is, the third conductive layer 1150 is connected to the fifth portion 1142 of the second conductive layer 1140 . Not only that, 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 screen printing process of conductive paste. The conductive paste includes silver silver chloride.

Next, as shown in Figures 6e and 6f, the second insulating layer 1160 is formed, and a part of the second conductive layer 1140 is covered by the second insulating layer 1160, wherein the fourth part 1141 is located in the The region 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 the arrangement of the second insulating layer 1160 in FIG. The area of the fifth portion 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). layer 1160 coverage). Wherein, the exposed area of the fourth part 1141 located on the electrode region 1112 forms the counter electrode 1002 (so that the counter electrode 1002 is isolated from the reference electrode 1003), the fourth part The fourth end 1141b of the first conductive layer 1141 and the second portion 1122 of the first conductive layer 1120 jointly form the second pin 1111b, and the sixth end 1142b of the fifth portion 1142 forms the second pin 1111b together with the first The third portion 1123 of a conductive layer 1120 jointly forms the third pin 1111c, and the sixth portion 1143 and the second end 1121b of the first portion 1121 of the first conductive layer 1120 jointly form the first pin 1111a. That is to say, each pin has a two-layer structure, and the advantage of this arrangement is that the printing effect of the second conductive layer 1140 is better, and each film layer of the biosensor is more uniform.

Since the third conductive layer 1150 is formed on the fifth end 1142a of the fifth portion 1142 of the second conductive layer 1140, the fifth end 1142a of the fifth portion 1142, and the fifth end 1142a of the fifth portion 1142, and the first The part between the fifth end 1142a and the sixth end 1142b is also used as the wire of the reference electrode 1003, that is, the wire of the reference electrode 1003 is located on the same plane as the counter electrode 1002 and formed at the same time, so Doing is beneficial to maintain the consistency of the reference electrode 1003, and can simplify the production process and reduce the cost. Those skilled in the art can also understand that when the implant part 1101 is implanted under the patient's skin, since a fixed potential difference is formed between the reference electrode 1003 and the interstitial fluid, when the biosensor passes through the first lead 1111a, the second pin 1111b and the third pin 1111c are connected to an external electroanalyzer, a voltage loop is formed between the working electrode 1001 and the reference electrode 1003, and the working electrode 1001 and A current loop is formed between the counter electrodes 1002 . When the operator sets the potential difference between the working electrode 1001 and the reference electrode 1003 through the electroanalyzer, for example, to 0V, the potential difference between the working electrode 1001 and the interstitial fluid can be determined, which has Help to improve the detection accuracy of the biosensor.

Please refer back to FIG. 2 , the reaction film layer 1200 can be formed by coating the outer surface of the working electrode 1001 with a reaction reagent and curing it. The reaction reagent is coated on the working electrode 1001 by dispensing or inkjet process, which is beneficial to ensure the accuracy of the dosage of the reaction reagent and improve the consistency of mass production of the biosensor.

The reaction reagents include biological reaction enzymes, metal complexes, polypeptide macromolecules, stabilizers and first cross-linking agents. Among them, amino groups, carboxyl groups, hydroxyl groups and other groups on the polypeptide macromolecules can react with metal complexes and biological oxidases to form covalent bonds, and undergo free radical polymerization under the action of the first cross-linking agent to form The shown core-shell structure improves the stability of the reaction membrane layer 1200 and prolongs the life of the biosensor. Active groups capable of reacting with the target exist on the core-shell structure. The stabilizer can stabilize the performance of the reaction reagent.

The specific type of the biological reaction enzyme is determined according to the target. For example, when the target substance is glucose, the bioreaction enzyme is glucose oxidase. When the target object is lactic acid, the biological reaction enzyme is lactate oxidase. When the target object is L-glutamic acid, the biological reaction enzyme is L-glutamic acid oxidase. When the target object is xanthine, the biological reaction enzyme is xanthine oxidase.

The metal complex may 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. Ligands can be high-molecular ligands or small-molecule ligands. Optional polymer ligands include but not limited to polymethacrylates, polyacrylamides, polyvinylpyrrolidones and other long-chain branched structures; optional small molecule ligands include but not limited to nitrogen-containing heterocyclic small Molecules such as pyridine, imidazole, etc. Appropriate ligands can reduce the oxidation-reduction potential of the metal, thereby reducing the operating voltage for the biological reaction enzyme to recognize and oxidize the target, and improve the reaction sensitivity of the biosensor. Not only that, a suitable ligand can also reduce the interference of some electroactive substances on the response current of the target under high working voltage, and improve the detection accuracy of the biosensor.

The polypeptide macromolecules include but are not limited to at least one of bovine serum albumin, human serum albumin, and citrulline.

The first crosslinking agent can be a multifunctional small molecule compound, including but not limited to a small molecule compound including a diisocyanate group and a polyisocyanate group, or at least one of a small molecule compound including a diepoxide group and a polyepoxy group. kind.

The stabilizer is a polymer prepolymer solution, including but not limited to polyacrylamide prepolymer solution, polyacrylate prepolymer solution, polyhydroxyethyl methacrylate prepolymer solution, poly NIPAM prepolymer solution, polyDMAEMA prepolymer solution at least one of polysolutions.

Optionally, in the reaction reagent, the total amount of the metal complex, the biological reaction enzyme, the stabilizer, the polypeptide macromolecule, and the first cross-linking agent is 100%. Standard, 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%, and the first The mass fraction of the cross-linking agent is 0.1%-10%, and the balance is the biological reaction enzyme.

Further, please refer back to FIG. 2 , the biosensor also includes a functional film layer 1300, the functional film layer 1300 means a film layer that promotes the electrochemical reaction of the reaction film layer 1200, and the functional film layer 1300 covers at least the reaction film layer 1200 , the counter electrode 1002 and the reference electrode 1003 for further improving 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 1112 .

Optionally, the functional film layer 1300 may include an anti-interference film layer 1310, and the anti-interference film layer 1310 is used to prevent interfering substances from passing through the functional film layer 1300, so as to reduce the impact of the interfering substances on the working electrode 1001. Produce adverse effects and reduce the monitoring accuracy of biosensors. The interfering substances mentioned here include at least one of acetaminophen, vitamin C, ascorbic acid and the like that may exist in body fluids. The anti-interference film layer 1310 covers at least 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 any other suitable method and Curing and forming. It can be understood that the anti-interference film layer 1310 can also cover the counter electrode 1002 and the reference electrode 1003. At this time, the anti-interference coating is also coated on the outer surface of the counter electrode 1002 and the reference electrode 1003. The outer surface of the electrode 1003. The composition of the anti-interference film layer 1310 includes at least one of naphthol, cellulose acetate, polylysine, polyvinylpyridine and its modified copolymer, and polyurethane, and the molecular weight distribution is 10000Da-1000000Da.

Further, the functional film layer 1300 also includes an adjustment film layer 1320 for regulating the passing rate of the target 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 completely covers the anti-interference film layer 1310 .

The adjustment film layer 1320 mainly includes a hydrophilic polymer, a hydrophobic polymer and a second cross-linking agent, and the hydrophilic polymer and the hydrophobic polymer are under the action of the second cross-linking agent A cross-linking reaction occurs to form a three-dimensional network structure. When the regulating film layer 1320 is in contact with body fluid, the regulating film layer 1320 swells to form a hydrogel, 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 After 1310, it contacts with the reaction film layer 1200 and an electrochemical reaction occurs. By adjusting the ratio of the hydrophilic polymer to the hydrophobic polymer, the density of the pores in the three-dimensional network structure and the size of the pores can be adjusted, thereby realizing the passage of the target object rate regulation.

Further, the adjustment film layer 1320 includes a first adjustment film layer 1321 and a second adjustment film layer 1322, the first adjustment film layer 1321 is coated and cured on the anti-interference film layer 1310 by a first adjustment paint , the second adjusting film layer 1322 is coated and cured on the first adjusting film layer 1321 by a second adjusting paint. The content of the hydrophobic polymer in the first regulating film layer 1321 is greater than the content of the hydrophobic polymer in the second regulating film layer 1322 . Specifically, in the first adjusting 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 adjusting layer, the The weight ratio of the hydrophilic polymer to the hydrophobic polymer is 9:1˜1:1. In this way, the first regulating film layer 1321 can have a certain mechanical strength, and it can react to the reaction film layer 1200, the counter electrode 1002, the reference electrode 1003 and the anti-interference film layer 1310 in body fluid. play a protective role. The hydrophilic performance of the second regulating membrane layer 1322 is better, which improves the biocompatibility of the biosensor.

In this embodiment, the hydrophilic polymer includes but not limited to polyethylene glycol, polyhydroxyethyl methacrylate, polyacrylic acid, polypropylene alcohol, chitosan, hydrophilic cellulose, hydrophilic modified Silane polycondensate, hydrophilic modified polyurethane, polyDMAEMA, polyNIPAM, polymethacrylamide, polydopamine, alginic acid, hyaluronic acid, sodium polystyrene sulfonate, polyethylene glycol modified vinyl At least one of pyridine, polysulfonic acid-modified vinylpyridine, and polycarboxy-modified 4-vinylpyrrolidone. The hydrophobic polymer includes but not limited to at least one of polystyrene, polymethylmethacrylate, polyvinylpyridine, polyvinylpyrrolidone, polysilanes, polyurethane, and polycarbonate. Preferably, the molecular weight distributions of the hydrophilic polymer and the hydrophobic polymer are both 10000Da-1000000Da.

Further preferably, please refer to FIG. 2 , in order to improve the biocompatibility of the biosensor described in the present application when it is applied to the human body, the biosensor further includes a biocompatible layer 1400, which is used for Coat the surface of the implant part 1101 together with the functional film layer 1300 .

Further, the embodiment of the present invention also provides a preparation method of the aforementioned biosensor, as shown in Fig. 4, Fig. 5 and Fig. 6a to Fig. 6f, the preparation method includes the following steps:

Step S100: preparing the electrode structure 1100.

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

Wherein, the step S100 includes:

Step S110: providing the substrate 1110;

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

Step S130 : forming the first insulating layer 1130 on the first conductive layer 1120 , and exposing a part of the first conductive layer 1120 to serve as the working electrode 1001 .

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

Step S150 : forming the third conductive layer 1150 on a partial area of the second conductive layer 1140 , and the third conductive layer serves as the reference electrode 1003 .

Step S160: forming the second insulating layer on the second conductive layer 1140, and exposing a part of the second conductive layer 1140 as the counter electrode 1002, and the counter electrode 1002 is connected with the reference electrode 1002. The specific electrodes 1003 are isolated from each other.

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 .

Further, 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 .

The step S300 specifically includes:

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

Step S320: coating the first adjusting paint on the anti-interference film layer 1310, and forming the first adjusting film layer 1321 after curing.

Step S320: coating a second adjusting paint on the first adjusting film layer 1321, and forming the second adjusting film layer 1322 after curing.

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 for The functional film layer 1300 covers the surface of the implant part 1101 together. The biocompatible layer 1400 can be coated with any biocompatible paint and cured.

Next, the biosensor and its effect will be introduced in conjunction with a specific embodiment.

In this embodiment, the conductive paste used to form the first conductive layer includes carbon paste. And forming the first conductive layer, the second conductive layer and the third conductive layer by screen printing process.

The reaction reagent is drop-coated on the working electrode through a dispensing process. In this embodiment, the biological reaction enzyme is glucose oxidase. The metal complex is an osmium metal complex, specifically a graft polymerized complex, and the main chain of the polymer is an acrylate copolymer, and the ligand is biimidazole. The polypeptide macromolecule is human serum albumin (HSA). The stabilizer is polyacrylamide prepolymerization solution. The first cross-linking agent is bis-shrunk ethylene glycol ester with a molecular weight of 1000 Da. Based on the total amount of solute as 100%, the mass percentage of glucose oxidase is 37.5%, the mass percentage of osmium metal complex is 45%, the mass percentage of human serum protein is 5%, the mass percentage of polyacrylamide prepolymerization solution The percentage is 10%, and the mass percentage of bis-shrunk ethylene glycol ester is 2.5%. And in the reaction reagent, the total solid content is 10%.

After the reaction reagent is cured to form the reaction film layer 1200 , an anti-interference coating is coated on the reaction film layer 1200 , the counter electrode 1002 , and the reference electrode 1003 by dip coating. The anti-jamming paint includes nafine, and the mass fraction of nafine in the anti-jamming paint is 5%.

After the anti-jamming paint is cured to form the anti-jamming film layer, a first adjusting paint is coated on the anti-jamming film layer by dip coating. Wherein, the hydrophobic polymer includes polystyrene and polyvinylpyridine, the hydrophilic polymer includes polycarboxy-modified vinylpyridine, and the second crosslinking agent is bis-shrunk ethylene glycol ester. In the first adjustment paint, the total mass fraction of the hydrophobic polymer and the hydrophilic polymer is 20%, and in percentage by weight, the hydrophobic polymer and the hydrophilic polymer The ratio of polystyrene to polyvinylpyridine is 1:1. The molecular weight of the bis-shrunk ethylene glycol ester is 500, and the mass fraction is 2%.

After the first adjustment paint is dried to form the first adjustment layer, a second adjustment paint is coated on the first adjustment layer by means of dip coating. Wherein, the hydrophobic polymer of the second adjustment coating includes polysiloxane, the hydrophilic polymer includes polyethylene glycol modified vinylpyridine and polysulfonic acid modified vinylpyridine, and the second The crosslinking agent is glycidyl ester. In the second adjustment paint, 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 The weight ratio of polyethylene glycol-modified vinylpyridine to polysulfonic acid-modified vinylpyridine is 1:2. The mass fraction of glycidyl ester is 1%.

The biosensor provided in this embodiment is used for performance testing. The test steps are as follows:

The biosensor was submerged in standard PBS buffer solution and soaked for 30 minutes. Those skilled in the art know how to prepare standard PBS buffer solution. The remaining measurements on the biosensor were then performed at 0V. Wait for 10 minutes to allow the biosensor to reach a constant background, 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, 30mM , to measure the linearity of the biosensor response. The solution was equilibrated for 5 minutes after each addition of glucose, and the solution should be continuously stirred during the measurement to make the concentration of the measurement solution uniform. The test results are shown in Figure 7 and Figure 8.

Figure 7 shows the current curve of the biosensor's response to glucose when the glucose of different concentrations is continuously added to the test solution under the 0V potential, and the interval between two adjacent samples is 10 seconds, where the abscissa is the number of sampling points . Figure 8 shows the linear relationship between the response current of the biosensor and the glucose concentration. It can be seen from Figure 7 that the oxidation peak current increases stepwise with the addition of glucose, and it can be found from the figure that the biosensor prepared by using the scheme of the present application can change accordingly at the next sampling time when the glucose solution is added, indicating that the biosensor The response time is within 10 seconds. Compared with some existing biosensors, it shortens the feedback time of sensing environmental solutions. Specifically, after being applied to the human body, it can also reflect changes in the user's biological value faster, with high sensitivity. . This also shows that the regulating membrane layer of the present application has a good ability to regulate the diffusion of glucose, and the reaction membrane layer has a good catalytic oxidation performance on glucose, and can quickly establish a redox balance. As can be seen from Figure 8, within the test concentration range, the response current of the biosensor has a linear relationship with the concentration of glucose, and its linear equation is I(nA)=0.4929C+0.0146, where C represents the concentration of glucose, and the linearity The goodness of fit of the equation was 0.9894.

Although the present invention is disclosed above, it is not limited thereto. Those skilled in the art can 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 equivalent technologies, the present invention also intends to include these modifications and variations.

Claims (18)

1. A biosensor, characterized in that, comprising:

   The electrode structure includes 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 An 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 A layer is formed on a partial area of the second conductive layer; the second insulating layer is formed at least on the second conductive layer, and a partial area of the second conductive layer is exposed to form a counter electrode, and the At least a portion of the third conductive layer is exposed to form a reference electrode; and,

   The reaction film layer is formed on the working electrode and is used for electrochemical reaction with the target.
2. The biosensor according to claim 1, wherein the reaction film layer is formed by coating the working electrode with a reaction reagent and curing; the reaction reagent includes a metal complex, a bioreaction enzyme, a polypeptide macromolecule and the first crosslinking agent.
3. The biosensor according to claim 2, wherein the metal complex comprises a transition metal complex.
4. The biosensor according to claim 2, wherein the bioreaction enzyme comprises any one of glucose oxidase, lactate oxidase, L-glutamic acid oxidase or xanthine oxidase.
5. The biosensor according to claim 2, wherein the reaction reagent also includes a stabilizer, and the total amount of the metal complex, bioreaction enzyme, polypeptide macromolecule, stabilizer and first cross-linking agent is 100%. As a benchmark, 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%, and the first The mass percentage of a cross-linking agent is 0.1%-10%, and the balance is the biological reaction enzyme.
6. The biosensor according to claim 5, wherein the stabilizer comprises a polymer pre-polymerization solution.
7. The biosensor according to claim 1, characterized in that, the biosensor also includes a functional film layer; the functional film layer includes an anti-interference film layer, and the anti-interference film layer is at least arranged on the reaction film layer, The counter electrode and the reference electrode are used to prevent interfering substances from passing through the functional film layer.
8. The biosensor according to claim 7, wherein the anti-interference film layer comprises at least one of naphthol, cellulose acetate, polylysine, polyvinylpyridine and its modified copolymer, and polyurethane .
9. The biosensor according to claim 7, wherein the functional film layer also includes an adjustment film layer, the adjustment film layer is arranged on the anti-interference film layer, and is used to regulate the target object in the The pass rate on the functional film layer.
10. The biosensor according to claim 9, wherein the regulating membrane layer comprises a hydrophilic polymer, a hydrophobic polymer and a second cross-linking agent.
11. The biosensor according to claim 10, wherein the regulating film layer comprises a first regulating film layer and a second regulating film layer, the first regulating film layer is formed on the anti-interference film layer, the The second regulating film layer is formed on the first regulating film layer; the hydrophobic polymer content of the first regulating film layer is greater than the hydrophobic polymer content of the second regulating film layer.
12. The biosensor according to claim 11, characterized in that, in the first regulating film layer, the weight ratio of the hydrophilic polymer to the hydrophobic polymer is 1:9 to 1:1.1, In the second regulating film layer, the weight ratio of the hydrophilic polymer to the hydrophobic polymer is 9:1˜1:1.
13. The biosensor according to any one of claims 10-12, wherein the hydrophilic polymer comprises polyethylene glycol, polyhydroxyethyl methacrylate, polyacrylic acid, polypropylene alcohol, chitosan Sugar, hydrophilic cellulose, hydrophilic modified silane polycondensate, hydrophilic modified polyurethane, polyDMAEMA, polyNIPAM, polymethacrylamide, polydopamine, alginic acid, hyaluronic acid, polystyrene At least one of sodium sulfonate, polyethylene glycol-modified vinylpyridine, polysulfonic acid-modified vinylpyridine, and polycarboxy-modified 4-vinylpyrrolidone; the hydrophobic polymer includes polystyrene, At least one of polymethylmethacrylate, polyvinylpyridine, polyvinylpyrrolidone, polysilanes, polyurethane, and polycarbonate; and/or,

    The molecular weight distribution of the hydrophilic polymer and the hydrophobic polymer is 10000Da-1000000Da.
14. The biosensor according to claim 7, wherein the biosensor comprises an implant part for implanting a target object, the working electrode, the counter electrode, the reference Both electrodes and the reaction film layer are located on the implanted part; the biosensor further includes a biocompatible layer, and the biocompatible layer is used to cover the implanted part together with the functional film layer surface of the part.
15. A preparation method of a biosensor, for preparing the biosensor according to any one of claims 1-14, characterized in that the preparation method comprises the following steps:

    providing said 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 on at least the second conductive layer;

    The reaction film layer is formed on the working electrode.
16. The preparation method of the biosensor according to claim 15, characterized in that, the reaction film layer is formed on the working electrode by dispensing or inkjet process.
17. The method for preparing a biosensor according to claim 15, wherein a functional film layer is formed at least on the reaction film layer, the counter electrode and the reference electrode.
18. The method for preparing a biosensor according to claim 17, wherein the biosensor comprises an implant, and the working electrode, the counter electrode, the reference electrode, and the reaction film layer are all located at On the implanted part, the preparation method further includes: forming a biocompatible layer on the implanted part, and the biocompatible layer is used to cover the implanted part together with the functional film layer s surface.

Images