Polymers, preparation methods, electrodes and components for biosensors and sensing membranes

Abstract

The present invention provides a biosensor and a polymer for a sensing membrane, a preparation method, an electrode, and an assembly. The polymer includes a matrix and a metal complex unit grafted onto the matrix. The matrix is ​​obtained by polymerizing at least a first monomer and a second monomer. The first monomer conforms to the general formula I: #imgabs0#R1 includes a functional group, and the functional group has anti-interference, low-temperature sensitivity, or biocompatibility. The second monomer conforms to the general formula II: #imgabs1#R2 is hydrogen or methyl, and R3 is hydrogen, alkyl, halogen atom, nitro, cyano, aminoacyl, aminosulfonyl, C2-C 10 Alkyl, C2-C 10 Hydroxyalkyl, C2-C 10 Ester alkyl, C2-C 10 haloalkyl, C2-C 10 Alkoxy, C3-C 10 Olefin groups, C6-C 12 Aryl, C5-C 12 Heteroaryl, C6-C 12 Aryloxy, C3-C 12 Cycloalkyl, C3-C 12 Heterocycloalkyl, C2-C 10 The polymer can be applied to a biosensor and endow the biosensor with specific properties.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and in particular to a polymer, a preparation method, an electrode and an assembly for a biosensor and a sensing membrane. Background Art

[0002] Biosensors have developed rapidly in recent years. Biosensors incorporate bioactive materials such as enzymes, microorganisms, plant and animal tissues, organelles, antigens, and antibodies. Their principle of operation is that when a target substance enters the sensor, it undergoes molecular recognition, triggering a biological reaction and generating corresponding information. This information is then converted by a transducer (chemical or physical) into acoustic, optical, or electrical signals, which are then output to determine the concentration of the substance being measured.

[0003] Biosensors, due to their strong specificity, high sensitivity, and rapid response, are playing an increasingly important role in the medical field, providing a rapid and convenient new method for basic medical research and clinical diagnosis. As devices that respond characteristically to biochemical substances, biosensors possess receptors or sensing membranes similar to human taste buds or olfactory cells to capture target substances. When the sensing membrane comes into contact with the analyte, certain functional substances or biochemically active substances on the membrane interact with it, enabling the biosensor to achieve selective recognition.

[0004] To date, the most commercially successful biosensor is the glucose sensor, which is used to measure glucose concentrations in body fluids such as blood, interstitial fluid, and sweat. Due to the large global diabetic population, glucose sensors have a significant market share. However, existing glucose sensors suffer from a series of issues, including limited functionality, complex preparation, high cost, and poor sensitivity. Summary of the Invention

[0005] The purpose of the present invention is to provide a polymer, preparation method, electrode and assembly for a biosensor and a sensing membrane, aiming to improve at least some of the problems in the functional selection, sensitivity, preparation complexity and cost of the active ingredients of the sensing membrane of the biosensor.

[0006] To achieve the above objectives, the present invention provides a polymer for a biosensor sensing membrane, the polymer comprising a substrate and a metal complex unit grafted onto the substrate; the metal complex unit comprises an osmium complex unit and / or a ruthenium complex unit; the substrate is obtained by free radical polymerization of monomers, the monomers comprising a first monomer and a second monomer, and the first monomer conforms to the following general formula I:

[0007]

[0008] In Formula I, R1 includes a functional group, and the functional group has anti-interference, low-temperature sensitivity, or biocompatibility;

[0009] The second monomer complies with the following general formula II:

[0010]

[0011] In formula II, R2 is a hydrogen atom or a methyl group, and R3 is a hydrogen atom, an alkyl group, a halogen atom, a nitro group, a cyano group, an aminoacyl group, an aminosulfonyl group, a C2-C 10 Alkyl, C2-C 10 Hydroxyalkyl, C2-C 10 Ester alkyl, C2-C 10 Halogenated alkyl, C2-C 10 Alkoxy, C3-C 10 Alkenyl, C6-C 12 Aryl, C5-C 12 Heteroaryl, C6-C 12 Aryloxy, C3-C 12 Cycloalkyl, C3-C 12 Heterocycloalkyl, C2-C 10 Any of the alkyl groups.

[0012] Optionally, the monomer further comprises a third monomer, and the third monomer conforms to the following general formula III:

[0013]

[0014] In formula III, R4 is any one of a hydrogen atom, a methyl group, a halogen atom, a nitro group, an aminoacyl group, an aminosulfonyl group, an amino group, a C2-C10 alkyl group, a C2-C10 hydroxyalkyl group, a C2-C10 esteralkyl group, a C2-C10 haloalkyl group, a C2-C10 alkoxy group, a C6-C12 aryl group, a C5-C12 heteroaryl group, a C6-C12 aryloxy group, a 3-C12 cycloalkyl group, and a C3-C12 heterocycloalkyl group.

[0015] Optionally, when the functional group has anti-interference property, the functional group includes a sulfonate anion or a carboxylate anion.

[0016] Optionally, when the functional group is low-temperature sensitive, the functional group includes an amide group.

[0017] Optionally, when the functional group is biocompatible, the functional group includes a siloxy group or a polyethylene glycol group.

[0018] Optionally, the ligand of the metal complex unit includes a nitrogen-containing heterocycle, and the nitrogen-containing heterocycle is derived from any one of imidazole, biimidazole, pyridine or bipyridine.

[0019] Furthermore, the present invention also provides an electrode comprising a conductive substrate and a sensing membrane disposed on the surface of the conductive substrate, wherein the sensing membrane comprises a bioreaction enzyme and the polymer for the biosensor sensing membrane as described above.

[0020] Optionally, the bioreaction enzyme includes any one of glucose oxidase, lactate oxidase, L-glutamate oxidase or xanthine oxidase.

[0021] Optionally, the sensing film further comprises a cross-linking agent, and the cross-linking agent is polyethylene glycol diglycidyl ether, which is obtained from its ethanol solution.

[0022] Optionally, the step of providing a sensing film on the surface of the conductive substrate includes:

[0023] preparing a reaction reagent, wherein the reaction reagent at least includes the bioreaction enzyme, the polymer for the biosensor sensing membrane and the cross-linking agent; and

[0024] The reaction reagent is coated on the surface of the conductive substrate, and the reaction reagent is solidified.

[0025] Furthermore, the present invention also provides an electrode assembly, comprising a working electrode, a counter electrode and a reference electrode, wherein the working electrode is an electrode as described in any one of the items.

[0026] Furthermore, the present invention also provides a biosensor, comprising an electrode and a control component as described in any of the preceding items, wherein the control component is connected to the electrode, the control component is used to supply power to the electrode so that the electrode captures the target substance and undergoes an electrochemical reaction, and the control component is also used to receive the electrical signal generated by the electrode when the electrochemical reaction occurs.

[0027] Compared with the prior art, the biosensor and the polymer used for the sensing membrane, the preparation method, the electrode and the assembly of the present invention have the following advantages:

[0028] The aforementioned polymer for biosensor sensing membranes comprises a matrix and a metal complex unit; the metal complex unit comprises an osmium complex unit and / or a ruthenium complex unit; the matrix is ​​obtained by free radical polymerization of monomers, the monomers comprising a first monomer and a second monomer, the first monomer being an olefin compound and comprising a functional group having anti-interference properties, low-temperature sensitivity, or biocompatibility, and the second monomer being an acrylic compound containing a carbon-carbon double bond or an acrylic ester compound containing a carbon-carbon double bond. The osmium complex unit and the ruthenium complex unit can be used as redox mediators, allowing the polymer to be disposed on the surface of a conductive matrix to undergo an electrochemical reaction. When used in conjunction with a bioreaction enzyme in a biosensor, the polymer can rapidly sense the concentration of a target substance, such as glucose, with high sensitivity. Furthermore, a wide variety of first and second monomers can be used to polymerize the matrix, allowing the matrix structure to be rationally designed according to actual needs. By selecting suitable first and second monomers for polymerization, the matrix can meet the desired performance, thereby obtaining a polymer for biosensor sensing membranes having the desired performance. Moreover, the free radical polymerization between the first monomer and the second monomer is relatively easy to achieve, and a matrix with a relatively high molecular weight can be obtained through a one-step polymerization. The reaction is simple, the parameters are easy to control, and the yield is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings are used to better understand the present invention and are not intended to constitute an improper limitation of the present invention.

[0030] Figure 1 1 is a graph showing the response current of the first biosensor provided by the first embodiment of the present invention and a comparison sensor to glucose in the presence of interfering substances.

[0031] Figure 2 1 is a graph showing the response current of the second biosensor provided by the second embodiment of the present invention and a comparison sensor to glucose at different temperatures.

[0032] Figure 3 1 is a graph showing the response current of the third biosensor provided by the third embodiment of the present invention and a comparison sensor pair to glucose in simulated tissue fluid. DETAILED DESCRIPTION

[0033] The following describes the embodiments of the present invention through specific specific examples. 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 this embodiment only illustrate the basic concept of the present invention in a schematic manner, and the drawings only show components related to the present invention rather than being drawn according to the number, shape and size of components during actual implementation. During actual implementation, the type, quantity and proportion of each component can be changed at will, and the component layout type may also be more complicated.

[0034] In addition, each embodiment described below has one or more technical features, but this does not mean that users of the present invention must implement all technical features in any embodiment at the same time, or that they can only implement some or all technical features in different embodiments separately. In other words, under the premise that implementation is possible, those skilled in the art can, based on the disclosure of the present invention and depending on design specifications or implementation requirements, selectively implement some or all technical features in any embodiment, or selectively implement a combination of some or all technical features in multiple embodiments, thereby increasing the flexibility of the implementation of the present invention.

[0035] An embodiment of the present invention provides a polymer for a biosensor sensing membrane, comprising a substrate and a metal complex unit grafted onto the substrate complex unit. The metal complex unit comprises an osmium complex unit and / or a ruthenium complex unit. The substrate is obtained by free radical polymerization of monomers. The monomers comprise a first monomer and a second monomer, wherein the first monomer conforms to the following general formula I:

[0036]

[0037] In Formula I, R1 includes a functional group, and the functional group has anti-interference, low-temperature sensitivity, or biocompatibility. The second monomer conforms to Formula II:

[0038]

[0039] In formula II, R2 is a hydrogen atom or a methyl group, and R3 is a hydrogen atom, an alkyl group, a halogen atom, a nitro group, a cyano group, an aminoacyl group, an aminosulfonyl group, a C2-C 10 Alkyl, C2-C 10 Hydroxyalkyl, C2-C 10 Ester alkyl, C2-C 10 Halogenated alkyl, C2-C 10 Alkoxy, C3-C 10 Alkenyl, C6-C12 Aryl, C5-C 12 Heteroaryl, C6-C 12 Aryloxy, C3-C 12 Cycloalkyl, C3-C 12 Heterocycloalkyl, C2-C 10 Any of the alkyl groups.

[0040] The polymer of the biosensor sensing membrane can be applied to the sensing membrane of the working electrode of the biosensor, wherein the osmium complex unit or the ruthenium complex unit can participate in the electrochemical reaction as a redox mediator and can generate a response current signal to the target substance, so that the biosensor has good sensitivity.

[0041] The monomer of the matrix provided in the embodiment of the present invention has a carbon-carbon double bond (C=C). When the monomer is polymerized, it mainly polymerizes between the carbon-carbon double bonds to form a longer carbon-containing main chain. At the same time, the functional groups included in R1 form side chains and / or side groups of the matrix, so that the functional groups are retained and the matrix has corresponding specific functions.

[0042] Compared with the existing glucose sensor, the active ingredient in the sensing membrane has a series of problems such as single function, complex preparation, high cost, and poor sensitivity. For example, the active ingredient in a commonly used sensing membrane includes a pyridine ring in a matrix polymer and a metal complex, and the metal complex is connected to the nitrogen on the pyridine ring. The active ingredient is prepared by a multi-step reaction, specifically first polymerizing a small molecule pyridine compound to obtain a macromolecular polymer including a pyridine ring, and then grafting is performed on the macromolecular polymer to introduce a side chain with a reactive group, and then reacting with the metal complex through the reactive group. The structure and performance of this polymer skeleton are relatively simple and cannot meet the performance requirements of hydrophilicity, hydrophobicity, or other aspects. Moreover, the multi-step reaction causes the entire preparation process to be complicated, and the difficulty of parameter control in the preparation process is further increased. Furthermore, conventional polymers used in biosensor sensing membranes are typically obtained by grafting nitrogen-containing heterocyclic polymers (e.g., polyvinylpyridines and polythiophenes) onto metal complexes. This can be expensive, reaching as much as 100 yuan / g. However, the monomers selected in the present invention are inexpensive, effectively reducing the cost of the matrix polymer and the biosensor. Typically, the polymers prepared in accordance with the present invention can cost less than 10 yuan / g. This significantly reduces the production cost of the polymers used in biosensor sensing membranes and the biosensor itself.

[0043] However, the various types of first and second monomers employed in the embodiments of the present invention, along with the presence of functional groups on the first monomers, make the structure of the matrix unnecessary. When preparing the polymer, the matrix structure can be rationally designed based on the desired performance and suitable first and second monomers can be selected for polymerization. Furthermore, the first and second monomers can readily undergo a one-step polymerization reaction initiated by a free radical initiator to yield a matrix with a relatively high molecular weight. This results in a simple reaction process, high yield, and easily controlled reaction parameters.

[0044] In an embodiment of the present invention, when the functional group has anti-interference properties, the functional group includes a sulfonate anion or a carboxylate anion. In other words, when the functional group has anti-interference properties and the functional group includes a sulfonate anion, the first monomer is an organic sulfonate; when the functional group has anti-interference properties and includes a carboxylate anion, the first monomer is a carboxylate; when R1 includes both a sulfonate anion and a carboxylate anion, the first monomer is both an organic sulfonate and a carboxylate. When the functional group has low-temperature sensitivity, the functional group includes an amide group. When the functional group has biocompatibility, the functional group includes a siloxy group or a polyethylene glycol group.

[0045] It should be noted that when free polymerization is performed to obtain the matrix, at least one first monomer may be used for polymerization to obtain a matrix having at least one specific function. For example, when the first monomer is one and has only one functional group, for example, when R1 includes a sulfonate anion, the matrix obtained by polymerization has anti-interference properties. When the first monomer is one and includes two or more functional groups with the same properties but different structures, for example, when R1 of the first monomer includes both a sulfonate anion and a carboxylate anion, the matrix obtained by polymerization has anti-interference properties. When the first monomer is two and the functional groups of the two first monomers have the same function but different structures, for example, when one of the first monomers has a sulfonate anion and the other has a carboxylate anion, the matrix obtained by polymerization has anti-interference properties. When the first monomer is two and the functional groups of the two first monomers have different functions, for example, when one of the first monomers has a sulfonate anion and the other has an amide group, the matrix obtained by polymerization has both anti-interference properties and low-temperature sensitivity. When the first monomer is one type and has two functional groups with different functions, for example, the functional groups of the first monomer include both a sulfonyl anion and a silicon-oxygen bond, the matrix obtained by polymerization has anti-interference properties and good biocompatibility.

[0046] Furthermore, the monomer may further include a third monomer, and the third monomer conforms to the following general formula III:

[0047]

[0048] In formula III, R4 is a hydrogen atom, a methyl group, a halogen atom, a nitro group, an aminoacyl group, an aminosulfonyl group, an amino group, a C2-C 10 Alkyl, C2-C 10 Hydroxyalkyl, C2-C 10 Ester alkyl, C2-C 10 Halogenated alkyl, C2-C 10 Alkoxy, C6-C 12 Aryl, C5-C 12 Heteroaryl, C6-C 12 Aryloxy, C3-C 12 Cycloalkyl, C3-C 12 Any of the heterocycloalkyl groups.

[0049] It should be noted that the present invention does not impose any restrictions on the feed ratio of the monomers involved in the reaction and the reaction conditions. Instead, they are rationally designed and selected based on the desired properties of the substrate. Specifically, the types of monomers involved in the reaction and the feed ratio are selected based on the properties of the substrate, and the reaction conditions are determined based on the types of monomers involved in the reaction. Furthermore, the present invention does not impose any restrictions on the molecular weight of the substrate.

[0050] In addition, the metal complex unit preferably includes a nitrogen-containing heterocycle, and a primary amino group is attached to one of the nitrogen atoms of the nitrogen-containing heterocycle. The metal complex unit reacts with a group on the substrate via the primary amino group, so that the metal complex unit is grafted onto the substrate to form a polymer for the biosensor sensing membrane. The groups on the substrate that react with the primary amino group include, but are not limited to, epoxy groups and carboxyl groups. Nitrogen-containing heterocycles have a large number of coordination sites and are easy to form metal ligands to prepare redox mediators. When the polymer is used in a biosensor, the detection sensitivity of the biosensor can be further improved. Optional nitrogen-containing heterocycles are derived from imidazole, biimidazole, pyridine, bipyridine, etc.

[0051] The preparation method of the polymer for the biosensor sensing membrane comprises the following steps: firstly, subjecting the monomer to free radical polymerization to obtain the matrix; then, subjecting the primary amino group of the metal complex unit to a grafting reaction with the matrix to graft the metal complex unit onto the matrix.

[0052] The monomers can be polymerized conventionally to obtain the matrix. Free radical initiators used in the polymerization reaction include, but are not limited to, azo initiators such as azobisisobutylnitrile, peroxide initiators such as benzoyl peroxide, and persulfate initiators. The reaction can be carried out in an oxygen-free environment at 40°C to 100°C. Furthermore, the monomers can preferably be polymerized by atom transfer radical polymerization (ATRP) to obtain the matrix. The polymerization reaction is carried out in the presence of a haloalkyl initiator, a catalyst, and a ligand at an oxygen-free environment of 40°C to 100°C. The catalyst is, for example, a copper salt, and the ligand is, for example, bipyridine or pentamethyldiethylenetriamine. Atom transfer radical polymerization makes it easier to control the molecular weight of the matrix. In other words, using atom transfer radical polymerization can reduce the dispersion coefficient of the matrix, resulting in a more uniform molecular weight distribution. Regardless of the polymerization reaction used, the polymerization time is determined by the actual reaction temperature and the specific monomer type. Generally, the higher the reaction temperature, the shorter the reaction time.

[0053] Furthermore, an embodiment of the present invention also provides an electrode, the electrode comprising a conductive substrate and a sensing film coated on the conductive substrate, the sensing film comprising the aforementioned polymer and a bioreaction enzyme. The type of the bioreaction enzyme is determined according to the target substance. For example, when the target substance is glucose, the bioreaction enzyme is glucose oxidase (GOx). When the target substance is lactic acid, the bioreaction enzyme is lactate oxidase. When the target substance is L-glutamic acid, the bioreaction enzyme is L-glutamate oxidase. When the target substance is xanthine, the bioreaction enzyme is xanthine oxidase.

[0054] Preferably, the sensing film further comprises a cross-linking agent, and the cross-linking agent is polyethylene glycol diglycidyl ether, which is obtained from an ethanol solution thereof.

[0055] Thus, the step of setting a sensing film on the surface of the conductive substrate includes: preparing a reaction reagent, the reaction reagent at least including the bioreaction enzyme, the polymer for the biosensor sensing film and the cross-linking agent; and coating the reaction reagent on the surface of the conductive substrate and solidifying the reaction reagent.

[0056] Furthermore, an embodiment of the present invention also provides an electrode assembly, comprising a working electrode, a counter electrode and a reference electrode, wherein the working electrode is the aforementioned electrode.

[0057] Furthermore, an embodiment of the present invention also provides a biosensor, which includes the aforementioned electrode and a control component, wherein the control component is connected to the electrode, and the control component is used to supply power to the electrode so that the electrode captures the target substance and undergoes an electrochemical reaction, and the control component is also used to receive the electrical signal generated by the electrode when the electrochemical reaction occurs.

[0058] In order to make the objects, advantages and features of the present invention more clear, the present invention is further described in detail below with reference to comparative examples and specific embodiments.

[0059] First, a comparative example is provided. Specifically, a sensor is prepared using existing technology. For ease of distinction, the sensor provided in the comparative example is referred to herein as a comparative sensor, and the polymer used is referred to as a comparative polymer. The comparative polymer includes poly(4-vinylpyridine) and an osmium complex grafted onto poly(4-vinylpyridine). The structural formula of the poly(4-vinylpyridine) used in this comparative example is: It is a commercially available product manufactured by Sigma-Aldrich (Shanghai) Trading Co., Ltd., with a CAS number of 25232-41-1. The osmium complex is grafted onto poly(4-vinylpyridine) using conventional methods.

[0060] The preparation method of the comparison sensor is as follows:

[0061] First, prepare the reaction reagents, which include the contrast polymer, glucose oxidase, polypeptide macromolecules and cross-linking agents. The polymer used for the biosensor sensing membrane is taken from its ethanol / water solution, and the concentration of the polymer is 20 mg / ml. The glucose oxidase is taken from its PBS solution, and the concentration of the glucose oxidase is 60 mg / ml. The polypeptide macromolecule is citrulline, which is taken from its PBS solution at a concentration of 60 mg / ml. The cross-linking agent is polyethylene glycol diglycidyl ether, which is taken from its ethanol solution at a concentration of 80 mg / ml. Based on the total volume of the reaction reagents, the polymer solution accounts for 10%, the glucose oxidase solution accounts for 10%, the polypeptide macromolecule solution accounts for 5%, the cross-linking agent solution accounts for 1%, and the balance is PBS buffer.

[0062] Then, 1 μl of the reaction reagent was drop-coated on a standard working electrode and dried at 37°C for 24 hours to form a sensing film, thereby obtaining a working electrode coated with a sensing film. This working electrode was then applied to a comparative sensor in a conventional manner.

[0063] Next, examples are provided.

[0064] In the first embodiment of the present invention, the substrate is first prepared.

[0065] The matrix in this embodiment is obtained by free radical polymerization of three monomers, wherein the three monomers are sodium styrene sulfonate (ie, the first monomer), glycidyl methacrylate (the second monomer), and butyl acrylate (the second monomer).

[0066] Sodium styrene sulfonate, glycidyl methacrylate, and butyl acrylate were added to a reactor in a molar ratio of 8:1:1. A mixture of isopropyl alcohol and deionized water in a volume ratio of 10:7 was used as a solvent. Azobisisobutylnitrile (AIBN) was used as an initiator. Under the protection of an inert gas (e.g., nitrogen), the reaction of the following formula (1) was carried out at 70° C. for 24 hours to obtain the matrix. The matrix is ​​white crystals that are soluble in organic solvents such as isopropyl alcohol. Formula (1) is:

[0067]

[0068] As can be seen from formula (1), the carbon-carbon double bonds of the three monomers add to each other to form the main chain of the matrix, while the other parts of the three monomers form three side chains respectively. The benzene ring and sodium sulfonate of sodium styrene sulfonate, the ester group and epoxy group of glycidyl methacrylate, and the ester group of butyl acrylate are all retained. In formula (1), a, b, and c are all integers greater than 1.

[0069] During actual polymerization, the feed ratio of the three monomers can be adjusted as needed to adjust the relevant properties of the matrix. Specifically, increasing the amount of sodium styrene sulfonate can adjust the anti-interference properties of the matrix, adjusting the amount of glycidyl methacrylate can adjust the amount of subsequently grafted metal complex units, and adjusting the amount of butyl acrylate can change the molecular weight of the matrix.

[0070] Next, the polymer for the biosensor sensing membrane is prepared.

[0071] The substrate and the osmium-biimidazole complex with an amino group (i.e., the metal complex unit) are reacted as shown in formula (2) at 50° C. for 24 hours, so that the amino group reacts with the epoxy group on the substrate to form a -CH(OH)-CH2-NH- covalent bond (as shown in the following formula (3), wherein some groups are not shown), to obtain the polymer. Formulas (2) and (3) are respectively:

[0072]

[0073] In formula (2), a, b, c, and d are all integers greater than 1, and b is greater than or equal to d.

[0074] Next, the polymer for the biosensor sensing membrane is applied to a biosensor to obtain a first biosensor. The preparation method is as follows:

[0075] First, prepare the reaction reagents, which include the polymer for the biosensor sensing membrane, glucose oxidase, polypeptide macromolecules and cross-linking agents. The polymer for the biosensor sensing membrane is taken from its ethanol / water solution, and the concentration of the polymer is 20 mg / ml. The glucose oxidase is taken from its PBS solution, and the concentration of the glucose oxidase is 60 mg / ml. The polypeptide macromolecule is citrulline, which is taken from its PBS solution, and the concentration is 60 mg / ml. The cross-linking agent is polyethylene glycol diglycidyl ether, which is taken from its ethanol solution, and the concentration is 80 mg / ml. Based on the total volume of the reaction reagents, the polymer solution accounts for 10%, the glucose oxidase solution accounts for 10%, the polypeptide macromolecule solution accounts for 5%, the cross-linking agent solution accounts for 1%, and the balance is PBS buffer.

[0076] Then, 1 μl of the reaction reagent was drop-coated on the standard working electrode and dried at 37° C. for 24 h to form a sensing film, thereby obtaining a working electrode with a surface coated with a sensing film.

[0077] Then, the working electrode prepared in this example was applied to a biosensor to obtain a first biosensor. In the first biosensor, the working electrode, the counter electrode, and the reference electrode constitute an electrode assembly.

[0078] Finally, the response sensitivity of the first biosensor and the comparison sensor provided in this embodiment to the target substance in the presence of an interfering substance was tested. In this embodiment, the interfering substance was ascorbic acid and the target substance was glucose. The test process was as follows:

[0079] The first biosensor and the comparison sensor were immersed in a standard PBS buffer solution for 5 minutes, and at the same time, the standard PBS buffer solution was stirred. After the biosensor and the comparison sensor reached a constant background, a glucose solution was added to the PBS buffer solution to form a first test solution. In the first test solution, the glucose concentration was 11 mmol / L. After the current was balanced, a solution containing ascorbic acid was added to the first test solution to form a second test solution. In the second test solution, the concentration of ascorbic acid was 0.2 mmol / L. During this process, the current curves of the biosensor and the comparison sensor in response to glucose were measured, and the results are shown in Tables 1 and Figure 1 shown. Figure 1 Curve 1 in FIG. 1 is a current curve of the biosensor provided in this embodiment in response to glucose, and curve 2 is a current curve of the comparison sensor in response to glucose.

[0080] Table 1

[0081]

[0082] Through Table 1 and Figure 1 It can be seen that with the addition of ascorbic acid, the rates of change in the response currents to glucose of the first biosensor and the comparison sensor differ. Specifically, the rate of change in the response current of the first biosensor to glucose is significantly smaller than that of the comparison sensor. This confirms that the polymer used in the biosensor sensing membrane provided in the first embodiment has excellent anti-interference properties in practical use.

[0083] In the second embodiment of the present invention, the substrate is first prepared.

[0084] The matrix in this embodiment is obtained by free radical polymerization of four monomers, wherein the four monomers are vinyl pyrrolidone (third monomer), methacrylic acid (second monomer), vinyl acetate (second monomer) and dimethylacrylamide (first monomer).

[0085] Vinyl pyrrolidone, methacrylic acid, vinyl acetate, and dimethylacrylamide were added to a reactor in a molar ratio of 4:5:6:35. A mixture of methanol and deionized water in a volume ratio of 5:5 was used as a solvent. Azobisisobutylnitrile was used as an initiator. Under inert gas protection, the reaction of the following formula (4) was carried out at 60° C. for 24 hours to obtain the matrix. The matrix is ​​white crystals and is soluble in solvents such as water, methanol, and isopropanol. Formula (4) is:

[0086]

[0087] Wherein, a, b, c, and d are all integers greater than 1.

[0088] Next, the polymer for the biosensor sensing membrane is prepared.

[0089] The substrate and the amino-containing osmium-biimidazole complex react as shown in formula (5) at 50° C. for 24 hours to obtain the polymer used for the biosensor sensing membrane. Formula (5) is:

[0090]

[0091] Wherein, a, b, c, d, and e are all integers greater than 1, and b is greater than or equal to e.

[0092] Next, the polymer used for the biosensor sensing membrane is used to prepare a second biosensor. The preparation method is the same as that of the first embodiment and will not be repeated here.

[0093] Finally, the response sensitivity of the second biosensor provided in this embodiment and the comparison sensor to the target at different temperatures was tested. In this embodiment, the target was glucose.

[0094] The test method is as follows:

[0095] The second biosensor and the comparison sensor were immersed in a standard PBS buffer solution and balanced at 37°C for 5 minutes. At the same time, the PBS buffer solution was stirred. After the second biosensor and the comparison sensor reached a constant background, a glucose solution was added to the PBS buffer solution to form a third test solution. In the third test solution, the concentration of glucose was 11 mmol / L. After the response currents of the two sensors were balanced, the temperature of the third test solution was adjusted to 42°C. After the response currents of the two sensors were balanced again, the temperature of the third test solution was adjusted to 32°C. Then, after the response currents of the two sensors were balanced, the temperature of the test solution was adjusted to 37°C. The response current curves of the two sensors are shown in the figure. Figure 2 As shown, Figure 2 Curve 1 is the response current of the second sensor, and curve 2 is the response current curve of the comparison sensor. The response currents of the two sensors at various temperatures and the rates of change of the response currents within different temperature ranges are shown in Table 2.

[0096] Table 2

[0097]

[0098] Depend on Figure 2 As can be seen from Table 2, when the temperature of the third test liquid changes, the change rate of the response current of the second biosensor is smaller than that of the comparison sensor, that is, the second biosensor has better low-temperature sensitivity.

[0099] In the third embodiment of the present invention, the substrate is first prepared.

[0100] The matrix in this embodiment is obtained by free radical polymerization of two monomers, wherein the two monomers are methacrylic acid (second monomer) and PDMS methacrylic acid (first monomer).

[0101] Methacrylic acid and PDMS methacrylic acid were added to a reactor at a molar ratio of 9:1. A mixture of isopropyl alcohol and deionized water at a volume ratio of 6:4 was used as the solvent. Ammonium persulfate was used as the initiator. Under the protection of an inert gas (e.g., nitrogen), the reaction of the following formula (6) was carried out at 70°C for 24 hours to obtain the matrix. The matrix was white crystals that were soluble in solvents such as water, ethanol, and ether. Formula (6) is:

[0102]

[0103] Wherein, a and b are integers greater than 1.

[0104] Next, the polymer for the biosensor sensing membrane is prepared.

[0105] The substrate and the osmium-biimidazole complex with an amino group are reacted as shown in the following formula (7) at 50° C. for 24 hours, so that the amino group reacts with the carboxyl group on the substrate to obtain the polymer for the biosensor sensing membrane. Formula (7) is:

[0106]

[0107] In the formula, c is an integer greater than 1, and b is greater than or equal to c.

[0108] Next, a third biosensor is prepared using the polymer for the biosensor sensing membrane prepared in this embodiment. The preparation method is the same as that of the first embodiment and will not be repeated here.

[0109] Finally, the response sensitivity of the third biosensor and the comparison sensor to the target in the fourth test liquid is tested. In this embodiment, the target is glucose. The fourth test liquid includes multiple components, as shown in Table 3. The specific testing method is to control the temperature of the fourth test liquid to 37°C, immerse the third biosensor and the comparison sensor in the fourth test liquid respectively, measure the response current of the third biosensor and the comparison sensor to glucose for 14 consecutive days and calculate the coefficient of variation (CV) of the response current. The response current curves of the third biosensor and the comparison sensor to glucose are shown in Figure 3. Figure 3 As shown, Figure 3 Curve 1 is the response current curve of the third biosensor to glucose, and curve 2 is the response current curve of the comparison sensor to glucose. The coefficients of variation of the response currents of the third biosensor and the comparison sensor to glucose are shown in Table 4.

[0110] Table 3

[0111] Element concentration <![CDATA[NaH2PO4-2H20]]> 1.9mmol / L <![CDATA[Na2HPO4-12H20]]> 8.1mmol / L NaCl 138mmol / L KCL 2.7mmol / L EDTA 1mmol / L Bovine serum albumin 22 mg / mL glucose 11mmol / L

[0112] Table 4

[0113]

[0114] Depend on Figure 3 Table 4 shows that the response current of the third biosensor to glucose did not change much over 14 days, indicating good stability. This is because the polymer used in the biosensor membrane prepared in the third embodiment adsorbs less protein and has good biocompatibility.

[0115] It can be seen from the first to third embodiments above that when the polymer provided by the embodiment of the present invention is applied to a biosensor, the matrix can be obtained by selecting a first monomer having a functional group with different functions and a second monomer capable of reacting therewith to polymerize, thereby obtaining a polymer for a biosensor sensing membrane with a specific function. Compared with the prior art, the technical solution provided by the embodiment of the present invention maintains the detection sensitivity of the biosensor to the target while providing options for realizing multiple performances of the biosensor. In addition, the matrix provided by the embodiment of the present invention can be obtained by one-step polymerization of monomers, the synthesis process is simple, the reaction conditions are mild and controllable, and the yield is high. In addition, the matrix does not need to be additionally added with a cross-linking agent when grafted with the metal complex unit, further reducing the types of raw materials and reducing production costs.

[0116] While the present invention is disclosed above, it is not limited thereto. Those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, the present invention is intended to encompass such modifications and variations as long as they fall within the scope of the claims and their equivalents.

Claims

1. A polymer for a biosensor sensing membrane, characterized in that: The polymer includes a matrix and a metal complex unit grafted onto the matrix; the metal complex unit includes an osmium complex unit and / or a ruthenium complex unit; the matrix is ​​obtained by free radical polymerization of monomers, the monomers include a first monomer and a second monomer, and the first monomer conforms to the following general formula I: In Formula I, R1 includes a functional group, the functional group is biocompatible, and the functional group includes a silicon-oxygen bond or a polyethylene glycol group; The second monomer complies with the following general formula II: In formula II, R2 is a hydrogen atom or a methyl group, and R3 is a hydrogen atom or a C2-C 10 Alkyl; When the monomer is polymerized, the carbon-carbon double bonds on the monomer polymerize to form the carbon-containing main chain of the matrix, and the functional group included in R1 forms a branch chain and / or a side group of the matrix; The metal complex unit includes a nitrogen-containing heterocycle, and a primary amino group is connected to a nitrogen atom of the nitrogen-containing heterocycle; the metal complex unit is grafted onto the substrate through the reaction of the primary amino group with the epoxy group and / or carboxyl group on the substrate.

2. The polymer for biosensor sensing membrane according to claim 1, characterized in that The monomer further comprises a third monomer, and the third monomer conforms to the following general formula III: In formula III, R4 is a hydrogen atom, a methyl group, a halogen atom, a nitro group, an aminoacyl group, an aminosulfonyl group, an amino group, a C2-C 10 Alkyl, C2-C 10 Hydroxyalkyl, C2-C 10 Ester alkyl, C2-C 10 Halogenated alkyl, C2-C 10 Alkoxy, C6-C 12 Aryl, C5-C 12 Heteroaryl, C6-C 12 Aryloxy, C3-C 12 Cycloalkyl, C3-C 12 Any of the heterocycloalkyl groups.

3. The polymer for biosensor sensing membrane according to claim 1, characterized in that The nitrogen-containing heterocyclic ring of the metal complex unit is derived from any one of imidazole, biimidazole, pyridine or bipyridine.

4. An electrode, characterized in that The invention comprises a conductive substrate and a sensing membrane arranged on the surface of the conductive substrate, wherein the sensing membrane comprises a bioreaction enzyme and the polymer for the biosensor sensing membrane according to any one of claims 1 to 3.

5. The electrode according to claim 4, characterized in that The bioreaction enzyme includes any one of glucose oxidase, lactate oxidase, L-glutamate oxidase or xanthine oxidase.

6. The electrode according to claim 4, characterized in that The sensing film further comprises a cross-linking agent, which is polyethylene glycol diglycidyl ether and is taken from an ethanol solution thereof.

7. The electrode according to claim 6, characterized in that The step of providing a sensing film on the surface of the conductive substrate comprises: preparing a reaction reagent, wherein the reaction reagent at least includes the bioreaction enzyme, the polymer for the biosensor sensing membrane and the cross-linking agent; and The reaction reagent is coated on the surface of the conductive substrate, and the reaction reagent is solidified.

8. An electrode assembly, characterized in that: The method comprises a working electrode, a counter electrode and a reference electrode, wherein the working electrode is the electrode according to any one of claims 4 to 7.

9. A biosensor, characterized in that: The method comprises an electrode and a control component as described in any one of claims 4 to 7, wherein the control component is connected to the electrode, the control component is used to supply power to the electrode so that the electrode captures the target substance and generates an electrochemical reaction, and the control component is also used to receive the electrical signal generated by the electrode when the electrochemical reaction occurs.

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