WO2022051888A1

WO2022051888A1 - Film-forming composition for biosensor, and preparation method therefor

Info

Publication number: WO2022051888A1
Application number: PCT/CN2020/113934
Authority: WO
Inventors: 高志强
Original assignee: 三诺生物传感股份有限公司
Priority date: 2020-09-08
Filing date: 2020-09-08
Publication date: 2022-03-17

Abstract

A film-forming composition for a biosensor, and a preparation method therefor, which belong to the field of electrochemistry. A glucose biosensor developed on the basis of third-generation biosensing technology can effectively and accurately perform real-time in-vivo monitoring on glucose; moreover, due to the presence of a 4-vinylpyridine-acetylcholine copolymer thin film, the monitoring range of glucose is significantly extended, and the stability of the sensor is greatly improved. Preliminary in-vivo experiments indicate that the third-generation glucose biosensor has excellent biocompatibility and an ultra-long service life, and is, so far, a glucose biosensor which has the longest service life and can be used for an implantable continuous glucose monitoring system.

Description

Film-forming composition of biosensor and preparation method thereof

technical field

The invention relates to the field of electrochemistry, in particular to a film-forming composition of a biosensor and a preparation method thereof.

Background technique

Since Clark and Lyon successfully developed the first biosensor in 1962, after more than 50 years of development, biosensors have been widely used in environmental detection, food industry, clinical medicine and other fields. For example, various glucose sensors developed based on biosensing technology have benefited millions of diabetic patients. Among them, the implantable continuous glucose monitoring system, which has developed rapidly in recent years, is favored by more and more diabetic patients, especially type I diabetic patients, due to its convenient use and real-time monitoring. As the core component of the implantable continuous glucose monitoring system, the performance of the glucose biosensor directly determines the performance and service life of the implantable continuous glucose monitoring system. The glucose biosensors used in the existing implantable continuous glucose monitoring systems are developed based on the first and second generation biosensing technologies. The first generation of biosensing technology is to monitor glucose indirectly by electrochemically detecting the hydrogen peroxide generated or the oxygen consumed during glucose oxidation. For example, Medtronic's Guardian and iPro2 and Dexcom's Dexcom G5 and G6 are developed based on the first-generation biosensing technology. They detect the hydrogen peroxide generated during the catalytic oxidation of glucose by electrochemical methods. Glucose is monitored. Since the electrochemical detection of hydrogen peroxide requires very strict electrodes, only a few materials such as platinum and platinum alloys can be used for the fabrication of such glucose biosensors, which greatly increases the number of implantable continuous glucose monitoring systems. the cost of the sensor. In addition, the electrochemical detection of hydrogen peroxide requires a high detection potential, which greatly reduces the anti-interference ability of the implantable continuous glucose monitoring system, especially the anti-interference ability of commonly used antipyretics such as acetaminophen.

The second-generation biosensing technology realizes direct electrochemical detection of glucose by introducing redox mediators in glucose biosensors. Different from ordinary protein molecules, the molecular weight of glucose oxidase is very large (160KDa), its molecular structure, especially the three-dimensional structure of the catalytic active center is very complex, and it is located inside the glucose oxidase and is deeply penetrated by various peptide chains. wrapped. Therefore, glucose oxidase cannot directly exchange electrons with electrodes. Heller et al. (Acc. Chem. Res. 23 (1990) 128-134) found that the introduction of redox species—redox mediators (small redox molecules such as ferricyanide or redox macromolecules) in glucose biosensors, glucose Oxidase can exchange electrons with electrodes through these mediators. The second-generation biosensing technology developed based on this principle has been widely used in biosensors, especially glucose biosensors, including various disposable blood glucose detection test strips and implantable continuous glucose monitoring systems, such as Abbott Diabetes Care FreeStyle Libre. Through molecular design and optimization of redox mediators, glucose detection can be achieved at lower potentials, thus greatly improving the anti-interference ability of implantable continuous glucose monitoring systems, especially for commonly used antipyretics such as acetyl Anti-interference ability of aminophenols. Since this type of glucose monitoring system performs direct electrochemical detection of glucose through redox mediators, its sensitivity is also significantly improved. However, because the redox mediator is a small molecule or a polymer material, it is difficult to accurately control the preparation, and there is also the possibility of the redox mediator leaking out of the implantable glucose biosensor, which is very difficult for the implantable continuous glucose biosensor. The performance of monitoring systems introduces considerable uncertainty.

When the glucose biosensing membrane was tested repeatedly by cyclic voltammetry, because the binding between this layer of glucose biosensing membrane and the substrate electrode was only by physical adsorption, and there was no strong binding mechanism, some biosensing membranes were It inevitably falls off the electrode, resulting in a significant attenuation of its catalytic oxidation current for glucose. To be applied to an implantable continuous glucose monitoring system, its stability needs to be greatly enhanced.

On the other hand, similar to the second-generation biosensing technology, oxygen, as the natural mediator of glucose oxidase catalyzed oxidation of glucose, inevitably participates in the catalytic oxidation of glucose and becomes an important interference factor in glucose monitoring. Although the catalytic oxidation efficiency of glucose by direct electrochemistry is much higher than that of glucose oxidase through its natural mediator oxygen, to fundamentally eliminate the interference of oxygen, it is necessary to coat the glucose biosensor with a layer capable of Selectively permeable membranes that effectively eliminate oxygen interference. In addition, due to the high sensitivity of direct electrochemistry for glucose detection, this permselective membrane must also be able to efficiently regulate glucose. That is, this permselective membrane must be dual-functional—one that can greatly improve the longevity of the glucose biosensor while efficiently regulating both oxygen and glucose. There is a chemical cross-linking reaction in the formulation of the biocompatible membrane of the existing implantable continuous glucose monitoring system, which greatly shortens the service life of the biocompatible membrane solution and virtually increases the implantation. cost of production of a continuous glucose monitoring system. More seriously, with the increase of use time, more and more chemical cross-linking reactions occur, and the viscosity of the biocompatible film solution also increases, which seriously affects the consistency of the product.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a biosensor film-forming composition and a preparation method thereof. By adjusting the composition of the permselective membrane and the ratio between each component, such as the type and ratio of hydrophobic and hydrophilic components in the polymer molecule, the hydrophobic polymer and hydrophilic polymer are in the biocompatible membrane solution. The ratio of oxygen and glucose can be controlled at the same time. After detailed research and experiments, it is found that the above purpose can be achieved by covering a layer of 4-vinylpyridine-acetylcholine copolymer and film on the electrochemically activated glucose oxidase biosensing membrane.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides a biosensor film-forming composition, which is characterized by comprising: a hydrophilic polymer, a hydrophobic polymer, an initiator and a solvent;

the hydrophilic polymer includes acetylcholine;

The hydrophobic polymer includes one or a combination of two or more of 4-vinylpyridine, styrene, acrylamide and derivatives thereof, acrylates and derivatives thereof;

The initiator comprises one or more compositions of sodium persulfate, azobisisobutyronitrile or dibenzoyl peroxide;

The solvent includes a combination of two or more of absolute ethanol, water, and acetone.

In some specific embodiments of the present invention, in g/mg/mL/mL, the ratio of the hydrophilic polymer, the initiator, the hydrophobic polymer and the solvent is (2-10): (20-300): (5-100): (1031-10530).

In some specific embodiments of the present invention, in the solvent, the volume ratio of ethanol, water and acetone is (30-500):(1-30):(1000-10000).

In some specific embodiments of the present invention, the film-forming composition includes the following components:

In some specific embodiments of the present invention, the hydrophilic polymer further includes polyethylene oxide, copolymers containing polyethylene oxide, polypropylene oxide, copolymers containing polypropylene oxide, polyethylene oxide One or more combinations of pyrrolidone and polyvinyl alcohol; or

The hydrophobic polymer also includes one or more combinations of styrene and vinylpyridine copolymer, styrene and vinylpyrrole copolymer, and styrene and acrylamide copolymer.

In some specific embodiments of the present invention, the solvent further includes one or a combination of two or more of methanol, propanol, and isopropanol.

In some embodiments of the present invention, the film-forming composition further comprises one or a mixture of polyvinyl alcohol or Nafion.

In some specific embodiments of the present invention, the concentration of the polyvinyl alcohol is 10-100 mg/mL; the concentration of Nafion is 5% (v/v); the volume ratio of polyvinyl alcohol to Nafion in the mixture is 1 :1.

On the basis of the above research, the present invention also provides a method for preparing the film-forming composition. The hydrophilic polymer, the hydrophobic polymer, anhydrous ethanol and water are mixed, and oxygen is removed by argon; Mixing with the initiator, closed reaction; acetone precipitation, centrifugation, collecting precipitation, dissolving in absolute ethanol, acetone precipitation, centrifuging, collecting precipitation, and vacuum drying.

In some specific embodiments of the present invention, the mass volume of the hydrophilic polymer, the initiator, the hydrophobic polymer, absolute ethanol, water and acetone in g/mg/mL/mL/mL The ratio is (2-10):(20-300):(5-100):(30-500):(1-30):(1000-10000).

In some specific embodiments of the present invention, the argon deoxygenation time is 20-60 min; the temperature of the airtight reaction is 50-75°C, and the time is 12-24 h; the vacuum drying temperature is 60- 120℃, the degree of vacuum is -1.0Bar.

In some specific embodiments of the present invention, the specific steps are as follows: take 2-10 g of acetylcholine (MPC), 5-100 mL of 4-vinylpyridine, 20-300 mL of absolute ethanol and 1-30 mL of water, deoxygenate with argon 20 to 60 minutes. Then add 20-300 mg of Na ₂ S ₂ O ₈ , place in a closed container, and react at 50-75° C. for 12-24 h. Then add 500-5000 mL of acetone to precipitate 4-vinylpyridine-acetylcholine copolymer, centrifuge; collect the precipitate, add 10-200 mL of ethanol to dissolve, then add 500-5000 mL of acetone to precipitate, centrifuge, collect the precipitate and vacuum dry at 60-120 °C for at least 12 hours .

The present invention also provides the application of the film-forming composition or the film-forming composition prepared by the preparation method in the preparation of thin films, biosensors and/or biomonitoring systems.

Based on the above research, the present invention also provides a biosensor coated with the film-forming composition or the film-forming composition prepared by the preparation method.

The present invention also provides the preparation method of the biosensor. The ethanol solution of the film-forming composition of 100-300 mg/mL is uniformly coated on the biosensor film by dipping and pulling method, and dried at room temperature. , repeated 3 to 6 times to form a biocompatible film and prepare a biosensor.

In some specific embodiments of the present invention, after repeating 3 to 6 times, it also includes adding 10-100 mg/mL polyvinyl alcohol and/or 5% Nafion (the volume ratio of polyvinyl alcohol to Nafion in the mixture). 1:1), uniformly coated on the biocompatible film by dip-pull method. Among them, the Chinese name of Nafion is perfluorosulfonic acid type polymer solution, and 1/1 volume refers to the volume ratio of polyvinyl alcohol and 4-vinylpyridine-acetylcholine copolymer.

Based on the above, the present invention also provides a biomonitoring system, including the biosensor or the biosensor prepared by the preparation method.

The glucose biosensor developed by the present invention based on the third-generation biosensing technology can very effectively and accurately perform real-time in vivo monitoring of glucose. At the same time, the existence of the 4-vinylpyridine-acetylcholine copolymer film also significantly expands glucose The monitorable range is greatly improved, and the stability of the sensor is greatly improved. Preliminary in vivo experiments show that our third-generation glucose biosensor has excellent biocompatibility and ultra-long working life, and is the longest working life glucose biosensor so far that can be used in implantable continuous glucose monitoring systems.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required in the description of the embodiments or the prior art.

Figure 1 shows the schematic diagram of the third-generation glucose biosensor; 1- biocompatible glue; 2- silver/silver chloride reference electrode; 3- carbon conductive layer; 4- polyethylene terephthalate substrate; 5 - Carbon working electrode; 6- Carbon counter electrode; 7- Glucose sensing membrane;

Figure 2 shows the cyclic voltammogram of the glucose biosensor covered with 4-vinylpyridine-acetylcholine copolymer film (a) in PBS buffer solution and (b) after adding 20 mmol/L glucose;

Figure 3 shows the glucose concentration-current curve of the glucose biosensor covered with 4-vinylpyridine-acetylcholine copolymer film, detection potential: 0.1 V (silver/silver chloride reference electrode);

Figure 4 shows (a) a glucose biosensor covered with a 4-vinylpyridine-acetylcholine copolymer film and (b) a glucose biosensor not covered with a 4-vinylpyridine-acetylcholine copolymer film in the presence of 10 mmol/L glucose. Stability in PBS buffer solution; detection potential: 0.1 V (silver/silver chloride reference electrode);

Figure 5 shows the experimental results of two implantable continuous glucose monitoring systems containing the electrochemically activated glucose oxidase glucose biosensor implanted in the upper arm of the same person.

detailed description

The invention discloses a film-forming composition of a biosensor and a preparation method thereof, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

The film-forming composition of the biosensor provided by the present invention and the raw materials and reagents used in the preparation method thereof can be purchased from the market.

Below in conjunction with embodiment, the present invention is further elaborated:

Example 1 Synthesis of 4-vinylpyridine-acetylcholine copolymer

2 g of acetylcholine (MPC) + 5 ml of 4-vinylpyridine + 300 ml of absolute ethanol and 10 ml of water, deoxygenated with argon for 40 minutes. Then, 20 mg of Na ₂ S ₂ O ₈ was added, and the mixture was placed in a closed vessel and reacted at 60° C. for 18 hours. The 4-vinylpyridine-acetylcholine copolymer was then precipitated by adding 500 ml of acetone and centrifuged. Add 100 ml of ethanol to dissolve, and then add 5000 ml of acetone to precipitate and centrifuge. This was repeated several times and finally the precipitate was vacuum dried at 90°C for at least 12 hours. The 4-vinylpyridine in the 4-vinylpyridine-acetylcholine copolymer may also be substituted by styrene, acrylamide and its derivatives, acrylates and its derivatives, and the like.

Example 2 Synthesis of 4-vinylpyridine-acetylcholine copolymer

10 g of acetylcholine (MPC) + 100 ml of 4-vinylpyridine + 150 ml of absolute ethanol and 1 ml of water, deoxygenated with argon for 60 minutes. Then, 150 mg of Na ₂ S ₂ O ₈ was added, placed in a closed vessel, and reacted at 75° C. for 12 hours. The 4-vinylpyridine-acetylcholine copolymer was then precipitated by adding 2500 ml of acetone and centrifuged. Add 200 ml of ethanol to dissolve, and then add 500 ml of acetone to precipitate and centrifuge. This was repeated several times and finally the precipitate was vacuum dried at 120°C for at least 12 hours. The 4-vinylpyridine in the 4-vinylpyridine-acetylcholine copolymer may also be substituted by styrene, acrylamide and its derivatives, acrylates and its derivatives, and the like.

Example 3 Synthesis of 4-vinylpyridine-acetylcholine copolymer

5 g of acetylcholine (MPC) + 50 ml of 4-vinylpyridine + 20 ml of absolute ethanol and 15 ml of water, deoxygenated with argon for 20 minutes. Then, 300 mg of Na ₂ S ₂ O ₈ was added, and the mixture was placed in a closed vessel and reacted at 50° C. for 12 hours. Then 5000 ml of acetone was added to precipitate the 4-vinylpyridine-acetylcholine copolymer and centrifuged. Add 10 ml of ethanol to dissolve, and then add 2500 ml of acetone to precipitate and centrifuge. This was repeated several times, and finally the precipitate was vacuum dried at 60°C for at least 12 hours. The 4-vinylpyridine in the 4-vinylpyridine-acetylcholine copolymer may also be substituted by styrene, acrylamide and its derivatives, acrylates and its derivatives, and the like.

Example 4 Coating of 4-vinylpyridine-acetylcholine copolymer film

The 100 mg/ml ethanol solution of 4-vinylpyridine-acetylcholine copolymer (prepared in Example 1) was evenly coated on the biosensing membrane by dipping and pulling method, and then dried at room temperature to form a membrane, repeated 3 to 6 times, the glucose biosensor was obtained (Figure 4).

Example 5 Coating of 4-vinylpyridine-acetylcholine copolymer film

The ethanol solution of 300 mg/ml 4-vinylpyridine-acetylcholine copolymer (prepared in Example 2) was evenly coated on the biosensing membrane by dipping and pulling method, and then dried at room temperature to form a membrane. 3 to 6 times to get the glucose biosensor.

Example 6 Coating of 4-vinylpyridine-acetylcholine copolymer film

The 200 mg/ml ethanol solution of 4-vinylpyridine-acetylcholine copolymer (prepared in Example 3) was evenly coated on the biosensing membrane by dipping and pulling method, and then dried at room temperature to form a membrane, repeated 3 to 6 times to get the glucose biosensor.

Effect example 1

The product prepared in Example 4 is shown in Figure 2. Although the glucose biosensor is completely wrapped by the 4-vinylpyridine-acetylcholine copolymer film, its catalytic oxidation performance of glucose by direct electrochemistry is not greatly affected. Voltammetry test showed that the biosensing membrane still showed good electrochemical performance in PBS buffer solution (pH 7.4) (Fig. 2, curve a), when 20 mmol/L glucose was added to this buffer solution , the cyclic voltammogram of this biosensing membrane clearly demonstrates a typical electrochemical catalytic process (Fig. 2, curve b).

Effect example 2

When the surface of the glucose biosensor prepared in Example 4 was covered with a 4-vinylpyridine-acetylcholine copolymer film, compared with the glucose biosensor without any membrane, the monitorable range of glucose was from 8 to 10 mmol/L. It has been successfully expanded to 30-40 mmol/L, which fully meets the glucose monitoring needs of diabetics. Its response time to glucose is 2-3 minutes. While broadening the monitorable range of glucose, its current signal was also well regulated by this biocompatible membrane (Figure 3). Since the detection of glucose was performed at very low potentials (50-150 mV), the anti-interference ability of acetaminophen was very significantly improved (Figure 3).

Effect example 3

The stability of the glucose biosensor prepared in Example 4 was also significantly improved. For example, after a 20-day continuous test experiment, the current signal decreased by less than 5% (Fig. 4, curve a), compared to the current signal of the glucose biosensor without the 4-vinylpyridine-acetylcholine copolymer film Its current decayed by more than 90% in 20 days of continuous testing (Fig. 4, curve b).

Example 7

Surface coating: The 4-vinylpyridine-acetylcholine copolymer film of the glucose biosensor prepared in Example 4 was uniformly coated on the 4-vinylpyridine-acetylcholine copolymer film containing 10 mg/ml of polyvinyl alcohol by dipping and pulling method to completely cover it. It was then dried to form a film at room temperature. Polyvinyl alcohol may also be substituted with polyethylene oxide, polypropylene oxide, polyvinylpyrrolidone, and the like. In addition, hydrophilic polymers such as polyethylene oxide, copolymers containing polyethylene oxide, polypropylene oxide, and polypropylene oxide can also be directly added to the 4-vinylpyridine-acetylcholine copolymer solution. Copolymer, polyvinylpyrrolidone, polyvinyl alcohol, etc. In addition to adjusting the content of 4-vinylpyridine in the 4-vinylpyridine-acetylcholine copolymer, hydrophobic polymers such as 4-vinylpyridine, styrene and Vinylpyridine copolymers, styrene and vinylpyrrole copolymers, styrene and acrylamide copolymers, etc., in order to obtain the desired hydrophilic or hydrophobic properties of the outer membrane. The purpose of adding hydrophilic polymer and hydrophobic polymer in the above operation is to adjust the content of 4-vinylpyridine, and adjust the content until the current is sufficiently small and the oxygen interference is sufficiently small. All of the above biocompatible membrane formulations are based on synthesized and purified polymers, as long as they are dissolved in a suitable solvent such as methanol, ethanol, propanol, isopropanol, etc., so the prepared solution Can be used indefinitely.

Example 8

Surface coating: the 4-vinylpyridine-acetylcholine copolymer film of the glucose biosensor prepared in Example 5 was uniformly coated on the 4-vinylpyridine-acetylcholine copolymer film of the glucose biosensor prepared in its completely covered. It was then dried to form a film at room temperature. Polyvinyl alcohol may also be substituted with polyethylene oxide, polypropylene oxide, polyvinylpyrrolidone, and the like. In addition, hydrophilic polymers such as polyethylene oxide, copolymers containing polyethylene oxide, polypropylene oxide, and polypropylene oxide can also be directly added to the 4-vinylpyridine-acetylcholine copolymer solution. Copolymer, polyvinylpyrrolidone, polyvinyl alcohol, etc. In addition to adjusting the content of 4-vinylpyridine in the 4-vinylpyridine-acetylcholine copolymer, hydrophobic polymers such as 4-vinylpyridine, styrene and Vinylpyridine copolymers, styrene and vinylpyrrole copolymers, styrene and acrylamide copolymers, etc., in order to obtain the desired hydrophilic or hydrophobic properties of the outer membrane. The purpose of adding hydrophilic polymer and hydrophobic polymer in the above operation is to adjust the content of 4-vinylpyridine, and adjust the content until the current is sufficiently small and the oxygen interference is sufficiently small. All of the above biocompatible membrane formulations are based on synthesized and purified polymers, as long as they are dissolved in a suitable solvent such as methanol, ethanol, propanol, isopropanol, etc., so the prepared solution Can be used indefinitely.

Example 9

Surface coating: The solution of Nafion containing 50 mg/ml polyvinyl alcohol and 5% perfluorosulfonic acid type polymer (the volume ratio of polyvinyl alcohol and Nafion is 1:1) was evenly coated on the surface by dipping and pulling method. On the 4-vinylpyridine-acetylcholine copolymer film of the glucose biosensor prepared in Example 6, it was completely covered. It was then dried to form a film at room temperature. Polyvinyl alcohol may also be substituted with polyethylene oxide, polypropylene oxide, polyvinylpyrrolidone, and the like. In addition, hydrophilic polymers such as polyethylene oxide, copolymers containing polyethylene oxide, polypropylene oxide, and polypropylene oxide can also be directly added to the 4-vinylpyridine-acetylcholine copolymer solution. Copolymer, polyvinylpyrrolidone, polyvinyl alcohol, etc. In addition to adjusting the content of 4-vinylpyridine in the 4-vinylpyridine-acetylcholine copolymer, hydrophobic polymers such as 4-vinylpyridine, styrene and Vinylpyridine copolymers, styrene and vinylpyrrole copolymers, styrene and acrylamide copolymers, etc., in order to obtain the desired hydrophilic or hydrophobic properties of the outer membrane. The purpose of adding hydrophilic polymer and hydrophobic polymer in the above operation is to adjust the content of 4-vinylpyridine, and adjust the content until the current is sufficiently small and the oxygen interference is sufficiently small. All of the above biocompatible membrane formulations are based on synthesized and purified polymers, as long as they are dissolved in a suitable solvent such as methanol, ethanol, propanol, isopropanol, etc., so the prepared solution Can be used indefinitely.

Effect example 4

The glucose biosensor (made in Example 7) containing electrochemically activated glucose oxidase was applied to an implantable continuous glucose monitoring system. Preliminary test results show that its working curve has a good linearity between 1.0 and 30 mmol/L, and it is the continuous glucose monitoring system with the widest linear range at present. Stability was also significantly improved, with no significant change in sensitivity over 20 consecutive days of human trials (Figure 5).

The film-forming composition of the biosensor provided by the present invention and the preparation method thereof are described in detail above. The principles and implementations of the present invention are described herein by using specific examples, and the descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

A film-forming composition for a biosensor, comprising: a hydrophilic polymer, a hydrophobic polymer, an initiator and a solvent;

the hydrophilic polymer includes acetylcholine;

The hydrophobic polymer includes one or a combination of two or more of 4-vinylpyridine, styrene, acrylamide and derivatives thereof, acrylates and derivatives thereof;

The initiator comprises one or more compositions of sodium persulfate, azobisisobutyronitrile or dibenzoyl peroxide;

The solvent includes a combination of two or more of absolute ethanol, water, and acetone.
The film-forming composition of claim 1, wherein, in g/mg/mL/mL, the ratio of the hydrophilic polymer, the initiator, the hydrophobic polymer to the solvent is (2 to 10): (20 to 300): (5 to 100): (1031 to 10530).
The film-forming composition according to claim 1 or 2, wherein in the solvent, the volume ratio of ethanol, water and acetone is (30-500):(1-30):(1000-10000).
The film-forming composition according to any one of claims 1 to 3, characterized in that it comprises the following components:
The film-forming composition according to any one of claims 1 to 4, wherein the hydrophilic polymer further comprises polyethylene oxide, a copolymer containing polyethylene oxide, polypropylene oxide, A composition containing one or more of polypropylene oxide copolymer, polyvinylpyrrolidone and polyvinyl alcohol; or

The hydrophobic polymer also includes one or more combinations of styrene and vinylpyridine copolymer, styrene and vinylpyrrole copolymer, and styrene and acrylamide copolymer.
The film-forming composition according to any one of claims 1 to 5, wherein the solvent further comprises one or more compositions of methanol, propanol, and isopropanol.
The film-forming composition of any one of claims 1 to 6, further comprising one or a mixture of polyvinyl alcohol or Nafion.
The film-forming composition according to claim 7, wherein the concentration of the polyvinyl alcohol is 10-100 mg/mL; the concentration of Nafion is 5% (v/v); The volume ratio of Nafion is 1:1.
The method for preparing a film-forming composition according to any one of claims 1 to 8, wherein the hydrophilic polymer, the hydrophobic polymer, absolute ethanol and water are mixed, and oxygen is removed by argon; It is then mixed with the initiator to react in a closed manner; precipitated with acetone, centrifuged, collected the precipitate, dissolved in anhydrous ethanol, then precipitated with acetone, centrifuged, collected the precipitate, and dried in vacuum.
The preparation method according to claim 9, wherein, in g/mg/mL/mL/mL, the hydrophilic polymer, the initiator, the hydrophobic polymer, absolute ethanol, water and The mass-volume ratio of acetone is (2-10):(20-300):(5-100):(30-500):(1-30):(1000-10000).
The preparation method according to claim 9 or 10, characterized in that, the time for deoxygenation of the argon gas is 20-60min; the temperature of the closed reaction is 50-75°C, and the time is 12-24h; the vacuum The drying temperature is 60-120°C, and the vacuum degree is -1.0Bar.
The preparation method according to any one of claims 9 to 11, characterized in that: taking 2-10 g of acetylcholine (MPC), 5-100 mL of 4-vinylpyridine, 20-300 mL of absolute ethanol and 1 ~30mL of water, deoxygenated by argon for 20~60min; then add 20~300mg of Na 2 S 2 O 8 , place in a closed container, react at 50~75°C for 12~24h; then add 500~5000mL of acetone to precipitate 4 -Vinylpyridine-acetylcholine copolymer, centrifuge; collect the precipitate, add 10-200 mL of ethanol to dissolve, then add 500-5000 mL of acetone to precipitate, centrifuge, collect the precipitate and vacuum dry at 60-120°C for at least 12 hours.
The film-forming composition according to any one of claims 1 to 8 or the film-forming composition prepared by the preparation method according to any one of claims 9 to 12 is used in the preparation of thin films, biosensors and/or biomonitoring systems applications in .
A biosensor, characterized in that it is coated with the film-forming composition according to any one of claims 1 to 8 or the film-forming composition prepared by the preparation method according to any one of claims 9 to 12.
The method for preparing a biosensor according to claim 14, wherein 100-300 mg/mL of the ethanol solution of the film-forming composition is uniformly coated on the biosensor film by a dip-pulling method, Drying at room temperature was repeated 3 to 6 times to form a biocompatible film to prepare a biosensor.
The preparation method according to claim 15, characterized in that, after repeating 3 to 6 times, it further comprises adding 10-100 mg/mL polyvinyl alcohol and/or 5% Nafion, the volume ratio of polyvinyl alcohol and Nafion 1:1; uniformly coated on the biocompatible film by dip-pull method.
The biological monitoring system is characterized by comprising the biosensor as claimed in claim 14 or the biosensor prepared by the preparation method as claimed in claim 15 or 16 .