CN113311054A

CN113311054A - Glucose biosensor

Info

Publication number: CN113311054A
Application number: CN202110475609.4A
Authority: CN
Inventors: 沈薇
Original assignee: Suzhou Zhongxing Medical Technology Co ltd
Current assignee: Suzhou Zhongxing Medical Technology Co ltd
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2021-08-27

Abstract

The invention discloses a glucose biosensor. The biosensor disclosed by the invention is characterized in that the surface of the biosensor film is covered with 1-10 layers of hydrophobic/hydrophilic films, each hydrophobic/hydrophilic film is a double-layer film consisting of a hydrophobic layer and a hydrophilic layer, and the hydrophobic layer and the hydrophilic layer have biocompatibility. According to the invention, 1-10 layers of hydrophobic/hydrophilic double-layer membranes with high biocompatibility are covered on the glucose biosensor developed based on the third-generation biosensing technology, so that oxygen and glucose can be effectively and accurately regulated and controlled simultaneously, meanwhile, the existence of the double-layer membranes also obviously expands the monitoring range of glucose and greatly improves the stability of the sensor.

Description

Glucose biosensor

Technical Field

The invention relates to the technical field of biological membranes, in particular to a glucose biosensor.

Background

An implanted human body continuous monitoring system, such as a dynamic blood glucose meter, brings good news to millions of diabetics. For diabetics, daily self-blood glucose monitoring is a part of their lives. However, the traditional finger blood and blood sugar test has great limitation, only blood sugar value at a certain time point in a day can be provided, and for reliable blood sugar monitoring, diabetics need to perform frequent finger blood and blood sugar tests every day, which brings great inconvenience to their work and life. On the other hand, the dynamic blood glucose meter can enable a diabetic patient to regulate and control blood glucose more conveniently and more effectively. The blood glucose detection instrument can continuously detect blood glucose in real time and gradually becomes a powerful tool for regulating and controlling blood glucose. As the main components of the biosensor of the dynamic blood glucose meter and the only interface which is directly contacted with a living body, the performance of the biocompatible membrane directly determines the biocompatibility, sensitivity, stability and anti-interference capability of the dynamic blood glucose meter and the working life of the dynamic blood glucose meter during living body monitoring. The existing dynamic blood glucose meters are developed based on the first or second generation biosensing technology. The working principle of continuous monitoring of glucose by using the first-generation biosensing technology is to indirectly monitor glucose by detecting hydrogen peroxide generated when oxygen is reduced in the catalytic oxidation process of glucose oxidase by an electrochemical method. Since the continuous glucose monitoring system developed based on the first generation biosensing technology relies on the natural mediator of oxygen-glucose oxidase in body fluid such as interstitial fluid or blood to catalyze and oxidize glucose, and the oxygen content (0.2-0.3mmol/L) in the body fluid is far lower than that (5-10mmol/L) of glucose, its biocompatible membrane must be capable of maximally allowing the passage of oxygen while effectively simulating the passage of glucose on the basis of high biocompatibility. It is well known that oxygen is hydrophobic compared to glucose, so its biocompatible membrane must also be highly hydrophobic. However, the requirement of being highly hydrophobic presents a significant challenge to the design of biocompatible membranes, since the main component of human interstitial fluid is water. Although they have been explored for more than 20 years, their performance is far from meeting the need for continuous glucose monitoring.

At the end of the last century, Heller et al discovered that by introducing a redox species, an artificial redox mediator (a redox small molecule such as ferricyanide, ferrocene and its derivatives or a redox polymer) into a biosensing membrane, glucose oxidase can exchange electrons with an electrode through the artificial mediator. Second generation biosensing technologies developed based on this principle are now widely used in biosensors, particularly glucose biosensors, including dynamic blood glucose meters. Because the second generation of biosensing technology realizes direct electrochemical detection of glucose by introducing an artificially synthesized redox mediator into the biosensor, the detection of glucose can be realized at a very low potential by molecular design and optimization of the redox mediator, thereby greatly improving the anti-interference capability of the dynamic blood glucose meter. Because the glucose monitoring system directly and electrochemically detects glucose through the artificial redox mediator, the sensitivity of the glucose monitoring system is also remarkably improved. On the other hand, although direct electrochemical detection of glucose is realized by introducing an artificially synthesized redox mediator, oxygen, which is a natural mediator for catalyzing and oxidizing glucose by glucose oxidase, inevitably participates in catalytic oxidation of glucose, and becomes an important interference factor for glucose monitoring. In order to further improve the performance of the dynamic blood glucose meter, various biocompatible membranes are introduced, so that the interference of oxygen is eliminated to the maximum extent, and the monitoring range of glucose is expanded. In view of the significant difference in hydrophilicity between glucose and oxygen, a high degree of hydrophilicity is an essential characteristic of such biocompatible membranes. Although they are very effective in eliminating oxygen interference, it is difficult to achieve effective and accurate simultaneous regulation of oxygen and glucose. To effectively regulate glucose, the thickness of the biocompatible membrane must be significantly increased. The excessively thick biocompatible film directly causes the response time of the dynamic blood glucose meter to glucose to be too long, so that a serious hysteresis phenomenon occurs, and the accuracy of the dynamic blood glucose meter is greatly reduced. In addition, the existing biocompatible membrane has a chemical crosslinking reaction in the formula, so that the service life of the biocompatible membrane solution is greatly shortened, and the production cost of the dynamic glucometer is invisibly increased. More seriously, as the using time is increased, the chemical crosslinking reaction is more and more, and the viscosity of the biocompatible film solution is more and more, thereby seriously influencing the consistency of the product.

The third generation biosensing technology is developed by direct electrochemistry using oxidoreductases. The biosensor technology can be used for manufacturing a high-performance glucose biosensor urgently needed by an implantable continuous glucose monitoring system, and can also be used for manufacturing other various biosensors containing oxidoreductase. Compared with the second generation of biosensing technology, the direct electrochemistry of the glucose oxidase greatly simplifies the design and manufacture of the glucose biosensor, and also obviously improves the sensitivity, accuracy, stability, specificity and anti-interference capability of the glucose biosensor. On the other hand, similar to the second generation biosensing technology, oxygen, which is a natural mediator for the catalytic oxidation of glucose by glucose oxidase, inevitably participates in the catalytic oxidation of glucose, and becomes an important interference factor for glucose monitoring. Although the efficiency of the catalytic oxidation of glucose by direct electrochemistry is much higher than the efficiency of the catalytic oxidation of glucose oxidase by its natural mediator oxygen, to eliminate the interference of oxygen fundamentally, a selective permeation membrane for eliminating oxygen must be coated on the glucose biosensor. In addition, this permselective membrane must also be able to effectively regulate glucose due to the high sensitivity of direct electrochemical detection of glucose. That is, this permselective membrane must be bifunctional: oxygen and glucose can be regulated simultaneously. Although the simultaneous regulation of oxygen and glucose can be achieved to some extent by adjusting the components of the permselective membrane and the ratios between the components, such as the types and ratios of hydrophobic and hydrophilic components, it is very difficult to achieve the simultaneous and accurate regulation of oxygen and glucose.

Disclosure of Invention

In order to solve the technical problems, the invention discovers through detailed research and experiments that the simultaneous regulation and control of oxygen and glucose can be effectively and accurately carried out by covering 1-10 layers of hydrophobic/hydrophilic double-layer membranes on a biosensing membrane which is developed based on a third-generation biosensing technology and contains glucose oxidase.

The first purpose of the invention is to provide a biosensor, wherein the surface of a biosensor membrane is covered with 1-10 layers of hydrophobic/hydrophilic membranes, each hydrophobic/hydrophilic membrane is a double-layer membrane consisting of a hydrophobic layer and a hydrophilic layer, and the hydrophobic layer and the hydrophilic layer have biocompatibility.

Further, the hydrophobic layer is composed of one or more of polyvinyl pyridine or its copolymer, polyvinyl imidazole or its copolymer, polyurethane or its copolymer, polyvinyl butyral or its copolymer, polyvinyl acetate or its copolymer.

Further, the hydrophilic layer is composed of one or more of polyacrylic acid or a copolymer thereof, polyethylene glycol or a copolymer thereof, and polyvinyl alcohol or a copolymer thereof.

Further, the biosensor is used for glucose detection.

Further, when the biosensor is used for detecting glucose, the biosensor comprises an electrode, a biosensor film containing oxidoreductase capable of exchanging electrons with the electrode, and 1-10 layers of hydrophobic/hydrophilic films covering the surface of the biosensor film.

Further, when the biosensor is used for detecting glucose, the biosensor film is prepared by crosslinking oxidoreductase through a crosslinking agent.

The second purpose of the invention is to provide a preparation method of the biosensor, which comprises the following steps:

s1, preparing a solution containing 10-500mg/mL of hydrophobic polymer material, uniformly coating the solution on the biosensor film, drying to form a film, and covering a layer of hydrophobic film on the surface of the biosensor film;

s2, preparing a solution containing 10-500mg/mL of hydrophobic high polymer material, uniformly coating the solution on the biosensor film covered with the hydrophobic film obtained in the step S1, drying to form a film, and covering the surface of the hydrophobic film with a hydrophilic film to obtain the biosensor with the surface covered with 1 layer of hydrophobic/hydrophilic film;

and S3, repeating the steps S1-S20-9 times to obtain the biosensor.

Furthermore, the coating mode is a dripping coating method, a spin coating method or a dip-draw method.

Further, in the step S1, the solvent of the solution containing 10-500mg/mL of the hydrophobic polymer material is ethanol or tetrahydrofuran; in step S2, the solvent of the solution containing 10-500mg/mL of hydrophobic polymer material is ethanol or water.

The third purpose of the invention is to provide the application of the biosensor in glucose detection.

Because the hydrophobic/hydrophilic double-layer membrane structure is formed by two membrane forming processes, the final hydrophobic/hydrophilic property and the regulation and control performance on oxygen and glucose can be conveniently and effectively regulated by respectively independently regulating the components and the thickness of the hydrophobic membrane and the hydrophilic membrane in the double-layer membrane, thereby achieving the expected effect. Specifically, the hydrophilic component allows glucose to pass through, the hydrophobic component limits oxygen to pass through, the hydrophobic component limits glucose to pass through, and the components and the thicknesses of the hydrophobic membrane and the hydrophilic membrane in the double-layer membrane are adjusted, so that the oxygen and the glucose are accurately regulated and controlled.

By the scheme, the invention at least has the following advantages:

according to the invention, 1-10 layers of hydrophobic/hydrophilic double-layer membranes with high biocompatibility are covered on the glucose biosensor developed based on the third-generation biosensing technology, so that oxygen and glucose can be effectively and accurately regulated and controlled simultaneously, meanwhile, the existence of the double-layer membranes also obviously expands the monitoring range of glucose and greatly improves the stability of the sensor.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

FIG. 1 is a schematic diagram of a dip-draw method for preparing a biocompatible membrane;

FIG. 2 is a cyclic voltammogram of a glucose biosensor (1) covered with a bilayer membrane (biocompatible membrane) in PBS buffer (pH 7.4) and (2) after addition of 10mmol/L glucose;

FIG. 3 is a graph showing the operating curves of (A) a glucose biosensor (1) without any double membrane and (2) covered with three double membranes. (B) Stability of glucose biosensors (1) without any double membrane and (2) covered with three layers of double membranes in PBS buffer solution (pH 7.4) containing 10mmol/L glucose;

FIG. 4 is a graph showing the effect of (A) air (oxygen) on the current signal in a PBS buffer solution (pH 7.4) containing 10mmol/L glucose for (1) a glucose biosensor without any double layer membrane and (2) a glucose biosensor covered with three layers of double layers.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1:

firstly, an ethanol solution containing 200mg/mL of polyvinyl pyridine hydrophobic polymer material with high biocompatibility is uniformly coated on a biosensing membrane by a dipping and pulling method, the biosensing membrane is completely covered, and then the sensor is dried in a strictly controlled environment to form a membrane. After the solvent is completely evaporated and the surface of the biosensing membrane is completely covered by a thin hydrophobic membrane, an ethanol solution containing 200mg/mL polyvinyl alcohol with high biocompatibility is uniformly coated on the hydrophobic membrane by a dip-coating method to completely cover the hydrophobic membrane, and then the sensor is dried again in a strictly controlled environment to form a membrane. After complete evaporation of the solvent, a first hydrophobic/hydrophilic bilayer membrane was formed (FIG. 1). Because the hydrophobic/hydrophilic double-layer membrane structure is formed by two membrane forming processes, the final hydrophobic/hydrophilic property and the regulation and control performance on oxygen and glucose can be conveniently and effectively regulated by respectively independently regulating the components and the thickness of the hydrophobic membrane and the hydrophilic membrane in the double-layer membrane, thereby achieving the expected effect.

As shown in FIG. 2, although the glucose biosensor is completely covered with a bilayer membrane, its catalytic oxidation performance for glucose by direct electrochemistry is not greatly affected. The cyclic voltammetry test shows that the biosensing membrane still has good electrochemical performance in PBS buffer solution (pH 7.4) (figure 2, curve 1), and the cyclic voltammogram of the biosensing membrane clearly shows a typical electrochemical catalysis process after 10mmol/L glucose is added into the buffer solution (figure 2, curve 2).

Example 2:

if the oxygen and the glucose are required to be more accurately regulated and controlled, the glucose biosensor with good accuracy, reproducibility and stability and stronger anti-interference capability is prepared, and multiple layers (2-10 layers) of double-layer films can be covered on a biosensor film. In this embodiment, after the surface of the implantable glucose biosensor is covered with three layers of polyvinyl pyridine/polyvinyl alcohol double-layer films, compared with the implantable glucose biosensor without any double-layer film, the monitoring range of glucose is successfully expanded from 10mmol/L to 40mmol/L, which completely meets the glucose monitoring requirement of the diabetic. Its response time to glucose is 2-3 minutes. While broadening the monitorable range of glucose, the current signal was well regulated by this layer of biocompatible membrane (fig. 3A). Meanwhile, the stability of the glucose biosensor is also remarkably improved. For example, the current signal decayed less than 1% over a 24 hour continuous test run (fig. 3B, curve 2). In contrast, the current signal of the glucose biosensor without any double membrane showed a significant decay of up to 15% in the continuous test over 24 hours (fig. 3B, curve 1).

In the case of a glucose biosensor developed based on the third-generation biosensing technology, when glucose is directly detected electrochemically, oxygen is a natural mediator for catalyzing and oxidizing glucose by glucose oxidase, so that oxygen in body fluid such as interstitial fluid or blood inevitably participates in the catalytic oxidation of glucose. If the glucose biosensor does not effectively regulate oxygen, the interference of oxygen will cause certain difficulties in accurate determination of glucose. As shown in FIG. 4, when air was introduced into the oxygen-free glucose solution, the current signal of the glucose biosensor without any double membrane was attenuated by 5-6%, and then returned to the original level when the oxygen in the solution was completely removed by the nitrogen (FIG. 4, Curve 1). In contrast, the glucose biosensor covered with a three-layer bilayer membrane showed only less than 1% attenuation when air was introduced (FIG. 4, curve 2).

In conclusion, 1-10 layers of hydrophobic/hydrophilic double-layer membranes with high biocompatibility are covered on a glucose biosensor developed based on a third-generation biosensing technology, so that oxygen and glucose can be effectively and accurately regulated and controlled simultaneously, meanwhile, the existence of the double-layer membranes also obviously expands the monitoring range of glucose and greatly improves the stability of the sensor.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims

1. The biosensor is characterized in that the surface of a biosensor film is covered with 1-10 layers of hydrophobic/hydrophilic films, each hydrophobic/hydrophilic film is a double-layer film consisting of a hydrophobic layer and a hydrophilic layer, and the hydrophobic layer and the hydrophilic layer are both biocompatible.

2. The biosensor of claim 1, wherein said hydrophobic layer is comprised of one or more of polyvinylpyridine or its copolymers, polyvinylimidazole or its copolymers, polyurethane or its copolymers, polyvinylbutyral or its copolymers, polyvinyl acetate or its copolymers.

3. The biosensor of claim 1, wherein the hydrophilic layer is composed of one or more of polyacrylic acid or its copolymer, polyethylene glycol or its copolymer, and polyvinyl alcohol or its copolymer.

4. The biosensor of claim 1, wherein the biosensor is used for glucose detection.

5. The biosensor of claim 4, wherein the biosensor comprises an electrode, a biosensor film comprising an oxidoreductase capable of exchanging electrons with the electrode, and 1-10 hydrophobic/hydrophilic layers covering the surface of the biosensor film, when the biosensor is used for detecting glucose.

6. The biosensor in accordance with claim 4, wherein the biosensor is used for glucose detection, and the biosensor membrane is prepared by crosslinking an oxidoreductase with a crosslinking agent.

7. A method for preparing the biosensor according to any one of claims 1 to 6, comprising the steps of:

and S3, repeating the steps S1-S20-9 times to obtain the biosensor.

8. The method according to claim 7, wherein the coating is performed by a dropping method, a spin coating method or a dip-draw method.

9. The method according to claim 7, wherein in the step S1, the solvent of the solution containing 10-500mg/mL of the hydrophobic polymer material is ethanol or tetrahydrofuran; in step S2, the solvent of the solution containing 10-500mg/mL of hydrophobic polymer material is ethanol or water.

10. Use of the biosensor according to any one of claims 1 to 6 for glucose detection.