WO2022051891A1

WO2022051891A1 - Glucose biosensor

Info

Publication number: WO2022051891A1
Application number: PCT/CN2020/113938
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 glucose biosensor in the technical field of medical diagnosis. The biosensor comprises a matrix (4) and an electrode (5) which is positioned on the matrix (4). The electrode (5) is coated with a glucose sensing membrane (7). The glucose sensing membrane (7) is formed by cross-linking of free carboxyl groups on the surface of and inside glucose dehydrogenase molecules and glucose dehydrogenase with amino groups coupled with a ruthenium or osmium complex having free carboxyl groups or amino groups. The glucose dehydrogenase is chemically modified to directly exchange electrons with the electrode (5). When the glucose sensing membrane (7) made using the same is applied to a glucose biosensor, the constraint of oxygen on glucose detection is fundamentally eliminated, the sensitivity, accuracy, reproducibility, consistency, specificity and interference resistance of dynamic glucose detection are enhanced, and the service life of an implanted continuous glucose monitoring system is extended.

Description

A glucose biosensor

technical field

The invention relates to the technical field of medical diagnosis, in particular to a glucose biosensor.

Background technique

With the development of the economy and the improvement of life, more and more people in the world have high blood sugar or diabetes. Diabetes has become a common disease in the world. According to the statistics of the International Diabetes Federation, there are more than 400 million diabetic patients in the world. , the number of diabetic patients in China exceeds 100 million. Diabetes is a chronic disease. Due to the decline of pancreatic function in diabetic patients, enough insulin cannot be produced or insulin can no longer be produced to effectively regulate blood sugar, resulting in excessive blood sugar in diabetic patients. Chronic high blood sugar can cause irreparable damage to human organs, causing complications such as skin ulcers, blindness, kidney failure and cardiovascular disease. Although the existing medical technology has not been able to cure diabetes, a large number of clinical data have proved that it is an effective treatment method to control the blood sugar of diabetic patients within the clinical normal range as much as possible. The goal is to maximize the use of drugs or dietary adjustments to control blood sugar within the normal range as much as possible, in order to reduce and delay the occurrence of diabetic complications. Therefore, daily self-monitoring of blood glucose becomes a part of life for people with diabetes.

The traditional self-monitoring of blood glucose is realized by finger stick blood collection. However, blood glucose monitoring by finger stick blood collection has great limitations except for the pain caused by acupuncture blood collection to diabetic patients. Blood sugar monitoring by acupuncture blood collection can only provide the blood sugar value at a certain time point in the day. In order to perform more reliable blood sugar monitoring, diabetic patients need to perform very frequent finger blood blood sugar testing every day, which gives them Work and life have brought great inconvenience. Therefore, the implantable continuous glucose monitoring system came into being, which has brought good news to thousands of diabetic patients. The implantable glucose continuous monitoring system can make the diabetic patients more convenient and effective to control blood sugar. It can continuously detect blood sugar in real time, and has gradually become a powerful tool for blood sugar control. Such as Dexcom's Dexcom G5 and G6 and Medtronic's Guardian and iPro2. They work by electrochemically detecting the hydrogen peroxide generated when the oxygen is reduced during the catalytic oxidation of glucose by glucose oxidase. Monitoring, they rely on oxygen in body fluids such as tissue fluid or blood, the natural mediator of glucose oxidase-catalyzed oxidation of glucose, to monitor glucose, and the oxygen content in body fluids (0.2-0.3 mmol/L) is much lower than Glucose (4-11 mmol/L), therefore, the oxygen content in body fluids becomes the main factor limiting the performance of such implantable continuous glucose monitoring systems, which are less efficient than other implantable continuous glucose monitoring systems. Sensitivity is generally low. Additionally, their glucose biosensors must be able to inhibit the passage of glucose to the greatest extent possible while allowing the passage of oxygen to the greatest extent possible on a highly biocompatible basis. It is well known that oxygen is hydrophobic compared to glucose, so their biocompatible membranes must also be highly hydrophobic. However, the requirement of high hydrophobicity has brought great challenges to the design of biocompatible membranes. Although after more than 20 years of research and exploration, its performance is still far from meeting the needs of continuous glucose monitoring. For example, Medtronic's Guardian and iPro2 also need to be calibrated twice a day, and their working life is only one week.

The artificial redox mediator technology developed at the end of the last century (Acc.Chem.Res.23(1990)128-134) has been widely used in biosensors, especially glucose biosensors, including various disposable blood glucose detection tests. Glucose biosensors for implantable continuous glucose monitoring systems, such as Abbott Diabetes Care's FreeStyleLibre. Since this type of glucose monitoring system performs direct electrochemical detection of glucose through artificial redox mediators, its sensitivity is significantly improved. On the other hand, 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. In order to eliminate the interference of oxygen, people have to eliminate the interference of oxygen to the maximum extent through various algorithms and the introduction of various biocompatible membranes that can effectively simulate oxygen.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a glucose biosensor, so that the biosensor does not require oxygen to participate in the reaction (relative to glucose oxidase) through glucose dehydrogenase, and naturally has the ability to avoid oxygen. It has better anti-interference ability to monitor the interference and restriction of glucose, and at the same time, it further modifies and cross-links glucose dehydrogenase, so that it has better electrochemical performance and can better respond to glucose concentration, Promote biosensors to improve the accuracy, reproducibility, stability and specificity of glucose detection, and extend the life of glucose sensors.

In order to achieve the above object, the present invention provides the following technical solutions:

A glucose biosensor, comprising a substrate and an electrode on the substrate; wherein, the working electrode is coated with a glucose sensing membrane; the glucose sensing membrane is prepared according to the following preparation method:

Glucose dehydrogenase is incubated in a buffer containing urea or guanidine hydrochloride, and the glucose dehydrogenase is fully expanded to expose free carboxyl and amino groups on the surface and inside of its molecule, and then add ruthenium or osmium with free carboxyl or amino groups The complex and the first cross-linking agent are reacted to obtain chemically modified glucose dehydrogenase, so that the glucose dehydrogenase can perform rapid and direct electron exchange with the electrode, and then further cross-link through the second cross-linking agent, An electrochemically active glucose sensing membrane was obtained.

In order to eliminate the restriction of oxygen on glucose detection, the present invention proposes an electrochemical activation technology of glucose dehydrogenase, so that glucose dehydrogenase can directly exchange electrons with electrodes. First, the glucose dehydrogenase is denatured in high concentration of urea-to open the glucose dehydrogenase, and then by chemically modifying the free carboxyl and amino groups on the surface and inside of the glucose dehydrogenase molecule-the electrochemical performance is excellent with free The ruthenium or osmium complexes of carboxyl or amino groups are covalently bonded to glucose dehydrogenase, and an electron channel is established from the inside to the outside, and the catalytic active center of glucose dehydrogenase can directly conduct very fast electrons with the electrode. exchange.

To further enhance the faster electron exchange between the glucose dehydrogenase and the electrode, the denatured glucose dehydrogenase can be modified again with ruthenium or osmium complexes with free carboxyl or amino groups before cross-linking. The specific method is to incubate the first chemically modified glucose dehydrogenase in a buffer containing urea or guanidine hydrochloride, and fully expand the glucose dehydrogenase to expose the free carboxyl and amino groups on its molecular surface and inside, and then Adding a ruthenium or osmium complex with a free amino group or a carboxyl group and a first cross-linking agent to react to obtain a second chemically modified glucose dehydrogenase, which can be compared with the first chemically modified glucose dehydrogenase. The electrode performs faster electron exchange; if the first chemical modification uses a ruthenium or osmium complex with a free amino group, the second modification uses a ruthenium or osmium complex with a free carboxyl group; if The first chemical modification uses a ruthenium or osmium complex with a free carboxyl group, and the second modification uses a ruthenium or osmium complex with a free amino group.

Preferably, the cross-linking is to cross-link the modified glucose dehydrogenase with a second cross-linking agent in a buffer solution.

Preferably, the first cross-linking agent can be selected from carbodiimide and N-hydroxysuccinimide, and the second cross-linking agent is a bifunctional chemical cross-linking agent such as glutaraldehyde, 1,4-butane Glycol diglycidyl ether, poly(dimethylsiloxane)-diglycidyl ether, tetraglycidyl-4,4-diaminodiphenylmethane, polyethylene glycol diglycidyl ether and 4-( 2,3-glycidoxy)-N,N-bis(2,3-glycidoxy)aniline and the like one or more.

Preferably, the concentration of the bifunctional cross-linking agent is 0.1-5%, more preferably 1%, and the cross-linking reaction time is 30-180 min, more preferably 60 min; in the cross-linking process, a buffer solution such as PBS can be used as a crosslinking reaction medium.

In a specific embodiment of the present invention, the cross-linking process of the glucose sensing membrane is to fully mix 2-20 mg/ml of modified glucose dehydrogenase in PBS buffer solution and 0.1-5% glutaraldehyde solution, After 30-180 minutes, a stable cross-linked glucose sensing membrane was obtained.

Preferably, in the first modification and the second modification, the concentration of the glucose dehydrogenase is 1-10 mg/mL, more preferably 5 mg/mL; the buffer is PBS buffer; the urea concentration is 1-6mol/L, more preferably 3mol/L; the incubation is at 4°C for 8-24h, more preferably at 4°C for 12h; the complex of ruthenium or osmium with free carboxyl or amino groups The concentration of the compound is 1-10mg/mL, more preferably 5mg/mL; the first cross-linking agent can be selected as carbodiimide and N-hydroxysuccinimide, and the concentrations of the two are 0.2-10mmol/L in turn and 0.1-1mmol/L, more preferably 1mmol/L and 0.5mmol/L; the reaction is 8-24h at 4°C, more preferably 12h at 4°C;

In addition, in the chemical modification process, it also includes using ultrafiltration bag dialysis to separate and purify the modified glucose dehydrogenase after the reaction; the ultrafiltration bag dialysis cut molecular weight is 1000-30000, more preferably 10000 molecular weight;

Experiments show that the glucose biosensor based on electrochemically activated glucose dehydrogenase not only maintains its catalytic oxidation performance for glucose, but also monitors glucose at a very low potential (20-200 mV). . Moreover, the direct electrochemistry of glucose dehydrogenase greatly simplifies the design and manufacture of glucose biosensors, while also significantly improving the sensitivity, accuracy, stability, specificity, and anti-interference ability of glucose biosensors. More importantly, the process of oxidizing glucose catalyzed by glucose dehydrogenase does not require the participation of oxygen, which fundamentally solves the problem of oxygen in the implantable continuous glucose monitoring system. On the other hand, it also greatly simplifies the design and fabrication of a permselective/biocompatible membrane for glucose biosensors. In addition to being highly biocompatible, the permselective membrane only needs to be able to effectively Regulating glucose suffices.

The present invention utilizes cyclic voltammetry to characterize the glucose sensing membrane containing the modified glucose dehydrogenase, and the results show that the ruthenium or osmium complexes with free carboxyl or amino groups with excellent electrochemical performance have been successfully bonded to glucose dehydrogenase. The cyclic voltammogram of the glucose sensing membrane clearly demonstrated a typical electrochemical catalytic process when 5 mmol/L glucose was added to the PBS buffer solution. In contrast, in the absence of the carbodiimide/N-hydroxysuccinimide chemical cross-linking agent, the glucose dehydrogenase treated as above did not have any electrochemical activity.

In order to further improve the linear response range and stability of the biosensing electrode of the present invention, the present invention also covers the glucose sensing membrane with a biocompatible membrane. In a specific embodiment of the present invention, the biocompatible film is a copolymer prepared by one or a combination of acrylate and acrylate derivatives. For example, hydroxyethyl methacrylate is used to polymerize polyhydroxyethyl methacrylate. Ethyl methacrylate, the specific method is: under anaerobic conditions, hydroxyethyl methacrylate, ethanol solution and Na ₂ S ₂ O ₈ react in a closed environment to generate polyhydroxyethyl methacrylate, and then add acetone to precipitate polymethyl methacrylate Hydroxyethyl acrylate is then repeatedly processed by means of ethanol dissolution and acetone precipitation, and finally the obtained polyhydroxyethyl methacrylate is dried and separated. Preferably, the biocompatible membrane is uniformly coated on the surface of the glucose sensing membrane by dipping and pulling. Specifically, the biocompatible membrane solution was immersed in an environment containing saturated alcohol vapor by immersion pulling method (decline speed: 100-5000 μm/s, optimal value: 2000, pulling speed 20-500 μm/s , optimal value: 200) uniformly coated on the glucose biosensors containing electrochemically activated glucose dehydrogenase, and then these glucose biosensors were dried to form films. After the solvent has completely evaporated, the surface of these glucose biosensors has been completely covered by a thin biocompatible film. In order to increase the thickness of the biocompatible film, the above process can be repeated many times, usually 2-8 times (optimal value: 4) can reach the required thickness. Since this biocompatible membrane is formed through multiple film-forming processes, its final glucose-regulating properties can be easily and effectively determined by the thickness of the membrane (number of dips and pulls) and the biocompatible membrane solution. The formula is optimized to achieve the desired effect.

Using a biosensor coated with a biocompatible membrane, the glucose monitoring range is 0-35 mmol/L, and after a week of continuous testing, its current signal has less than 2% attenuation, and oxygen does not interfere with glucose detection. At the same time, the anti-interference ability of acetaminophen is also improved. It can be seen that the biosensor of the present invention can bring extremely high stability and anti-interference ability, which fully meets the needs of an implantable glucose continuous monitoring system. In addition, the attached biocompatible membrane can not only regulate the rate of glucose in the blood entering the sensor, avoid entering too fast and shorten the service life of the biosensor, and play the role of osmotic regulation, and at the same time, it can achieve a high degree of biocompatibility.

Preferably, the electrode is coated with a glucose sensing film by a drop coating method or a dip-pulling method and is coated on the electrode. In the current glucose biosensors, the layout of each electrode (including the conductive layer) and the substrate is described in detail, such as Abbott, DEXCOM's CGM sensor, various commercially available products will set up different types of electrodes according to needs, which is in the It is within the capabilities of those skilled in the art. The biosensor of the present invention is provided with a working electrode, a reference electrode, a counter electrode and a conductive layer on the substrate; preferably, the working electrode is a carbon working electrode, the counter electrode is a carbon counter electrode, and the reference electrode It is a silver/silver chloride reference electrode, the conductive layer is a carbon conductive layer, and the matrix is a polyethylene terephthalate matrix.

In the present invention, the working electrode of the glucose biosensor is coated with a glucose sensing membrane, which is located on the same electrode layer as the counter electrode, and is located on both sides of the base layer with the conductive layer, and the reference electrode layer is located on the first side of the conductive layer. Then the whole is wrapped by a biocompatible film. The schematic diagram of the structure is shown in Figure 1.

It can be seen from the above technical solutions that the present invention chemically modifies glucose dehydrogenase so that it can directly exchange electrons with electrodes, and the glucose sensing membrane prepared based on it is used in glucose biosensors, which can fundamentally eliminate the The restriction of oxygen on glucose detection improves the sensitivity, accuracy, reproducibility, stability, specificity and anti-interference ability of glucose dynamic detection, and prolongs the service life of the implantable continuous glucose monitoring system, while greatly reducing glucose Cost of biosensors.

Description of drawings

Figure 1 shows a schematic structural diagram of the glucose biosensor of the present invention;

Figure 2 shows the electrochemical characterization of the glucose sensing membrane containing modified glucose dehydrogenase; among them, a is the cyclic voltammogram without adding glucose, and b is the cyclic voltammogram after adding 5 mmol/L glucose ;

Figure 3 shows the glucose concentration-current curve of the glucose biosensor; wherein, a is the glucose concentration-current curve of the glucose biosensor not covered with poly(hydroxyethyl methacrylate), and b is the glucose concentration-current curve of the glucose biosensor covered with poly(hydroxyethyl methacrylate) Glucose concentration-current curves of ester-based glucose biosensors;

Figure 4 shows the stability test results of the glucose biosensor; wherein, a is a glucose biosensor covered with a polyhydroxyethyl methacrylate film, and b is a glucose biosensor without a polyhydroxyethyl methacrylate film;

FIG. 5 shows the detection result of the anti-interference performance of the glucose biosensor of the present invention.

detailed description

The embodiment of the present invention discloses a glucose biosensor, 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 apparent to those skilled in the art are deemed to be included in the present invention. The glucose biosensor of the present invention has been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the glucose biosensor described herein without departing from the content, spirit and scope of the present invention. and application of the technology of the present invention.

The biocompatible film of the present invention adopts polyhydroxyethyl methacrylate, which can be prepared with reference to existing methods or purchased directly. The present invention also provides a method for preparing polyhydroxyethyl methacrylate. Hydroxyethyl acrylate, anhydrous ethanol, and water react with Na ₂ S ₂ O ₈ in an oxygen-free state to generate poly(hydroxyethyl methacrylate); One or more purifications are carried out, and finally it is precipitated with acetone and dried to obtain polyhydroxyethyl methacrylate.

When using poly(hydroxyethyl methacrylate) to coat the biosensor, the alcohol solution of poly(hydroxyethyl methacrylate) is uniformly coated on the glucose containing the chemically modified glucose dehydrogenase of the present invention by dipping and pulling method. The biosensor is then dried to form a film. After the solvent has completely evaporated, the surface of these glucose biosensors has been completely covered by a thin biocompatible film (see Figure 1).

In order to increase the thickness of the biocompatible film, the above process can be repeated many times, usually 2-8 times to achieve the required thickness. Since this biocompatible membrane is formed through multiple film-forming processes, its final glucose-regulating properties can be easily and effectively determined by the thickness of the membrane (number of dips and pulls) and the biocompatible membrane solution. The formulation is optimized to achieve the desired desired effect.

The following further describes a glucose biosensor provided by the present invention.

Example 1: Chemical modification of glucose dehydrogenase

Incubate 1-10 mg/mL glucose dehydrogenase (optimal value: 5) in PBS buffer solution containing 1-6 mol/L urea (optimal value: 3) at 4 degrees for 8-24 hours (optimal value: 3). value: 12), the glucose dehydrogenase was fully developed. Then 1-10 mg/ml (optimal value: 5) of ruthenium or osmium complexes with free amino groups are thoroughly mixed with 1-10 mg/ml glucose dehydrogenase (optimal value: 5), Then add 0.2-10 mmol/L carbodiimide (optimal value: 1) and 0.1-1 mmol/L N-hydroxysuccinimide (optimal value: 0.5) in sequence, after thorough mixing, React at 4 degrees for 8-24 hours (optimal value: 12). Then, the modified glucose dehydrogenase was separated and purified by ultrafiltration bag dialysis (molecular weight cut: 1000-30000, optimal value: 10000). The purified glucose dehydrogenase can directly exchange electrons with the electrode effectively;

To further improve the faster electron exchange between the glucose dehydrogenase and the electrode, we can add the purified glucose dehydrogenase to the PBS buffer solution containing 1-6 mol/L urea (optimal value: 3) again Incubate at 4 degrees for 8-24 hours (optimal value: 12), and once again fully develop the glucose dehydrogenase. Then, 1-10 mg/ml (optimum value: 5) of ruthenium or osmium complexes with free carboxyl groups and 0.2-10 mmol/l carbodiimide (optimal value: 5) were added to the solution sequentially. 1) and 0.1-1 mmol/L of N-hydroxysuccinimide (optimal value: 0.5), and after thorough mixing, react again at 4 degrees for 8-24 hours (optimal value: 12). After the reaction, the modified glucose dehydrogenase was separated and purified by ultrafiltration bag dialysis (molecular weight: 1000-30000, optimal value: 10000)).

Example 2: Preparation of glucose sensing membrane

1. Preparation

Mix 2-20 mg/ml (optimal value: 5) modified glucose dehydrogenase in PBS buffer with 0.1-5% (optimal value: 1) glutaraldehyde solution for 30-180 minutes After (optimal value: 60), the chemically cross-linked glucose dehydrogenase was coated on the electrode surface by drop coating method or dip-pull method to form a glucose sensing film.

2. Characterization of electrochemical activity

The glucose sensing membrane containing modified glucose dehydrogenase was characterized by cyclic voltammetry, and the results are shown in Figure 2. Curve a in Figure 2 clearly shows that after the above treatments, redox small molecules with superior electrochemical performance have been successfully bonded to glucose dehydrogenase. The cyclic voltammogram of the glucose-sensing membrane clearly demonstrated a typical electrochemical catalytic process when 5 mmol/L glucose was added to the PBS buffer solution (Fig. 2, curve b). In contrast, in the absence of the carbodiimide/N-hydroxysuccinimide coupling agent in Example 1, the above-treated glucose dehydrogenase did not have any electrochemical activity.

Example 3: Preparation of Biocompatible Membranes and Glucose Biosensors

1. Preparation of Biocompatible Films

(1) Synthesis of polyhydroxyethyl methacrylate: 5-500 ml of hydroxyethyl methacrylate (best value: 100) + 10-1000 ml of absolute ethanol (best value: 300) and 1- 100 ml (best value: 30) of water, deoxygenated with argon for 10-60 minutes (best value: 30). Then add 10-1000 mg (best value: 200) of Na ₂ S ₂ O ₈ , place in a closed container, and react at 50-75°C (best value: 70) for 10-40 hours (best value: 24 ). Then add 500-5000 ml of acetone (optimal value: 2500) to precipitate the poly(hydroxyethyl methacrylate) and separate by centrifugation. Add ethanol to dissolve, then add 500-5000 ml (optimal value: 2500) acetone to precipitate, and centrifuge. This was repeated several times, and finally the precipitate was vacuum dried at 60-120°C (optimal value: 100) for at least 8-24 hours (optimal value: 12).

(2) Coating of poly(hydroxyethyl methacrylate): Put the biocompatible film solution (viscosity at 25°C: 50-1000mPa.S) (best value: 500) in a 100,000-class clean room and contain saturated In the environment of alcohol vapor, the alcohol solution of 50-300 mg/ml (optimum value: 200) of poly(hydroxyethyl methacrylate) was immersed in the pulling method (fall speed: 100-5000 μm/sec, optimum value) : 2000; pulling speed 20-500 μm/s, optimal value: 200) uniformly coated on the glucose biosensors containing electrochemically activated glucose dehydrogenase, and then these glucose biosensors were placed in a strictly controlled In the environment (temperature: 22-30° C., optimum value: 25; relative humidity 30-60%, optimum value: 45; 30-120 minutes, optimum value: 60) to dry and form a film. After the solvent has completely evaporated, the surface of these glucose biosensors has been completely covered by a thin biocompatible film. In order to increase the thickness of the biocompatible film, the above process can be repeated many times, usually 2-8 times (optimal value: 4) can reach the required thickness. Since this biocompatible membrane is formed through multiple film-forming processes, its final glucose-regulating properties can be easily and effectively determined by the thickness of the membrane (number of dips and pulls) and the biocompatible membrane solution. The formulation is optimized to achieve the desired desired effect.

2. Preparation of glucose biosensors

The carbon counter electrode is coated with the glucose sensing film of the present invention, the silver/silver chloride reference electrode and the carbon conductive layer, and then prepared according to the layout shown in Figure 1, and then polyhydroxyethyl methacrylate is coated on the the entire outer surface.

3. Electrochemical characterization of glucose biosensors

Electrochemical characterization of glucose biosensors (control sensors provided with uncoated polyhydroxyethyl methacrylate) using cyclic voltammetry (change in glucose concentration: 0-5 mmol/L), results are shown in Figure 3; Figure 3 It was shown that the glucose biosensor without the poly(hydroxyethyl methacrylate) coating had a linear response in the range of 0-3 mmol/L, and its stability was also not ideal (curve a). Curve b is the result of the glucose biosensor covered with poly(hydroxyethyl methacrylate), compared with curve a, its response time to glucose is extended from 30 seconds to 4 minutes, but its current signal is greatly affected by this biocompatible film. well regulated, and the stability of the glucose biosensor was significantly improved. At the same time, the monitorable range of glucose was greatly extended from 0-3 mmol/L to 0-35 mmol/L.

In addition, the glucose biosensors covered with the poly(hydroxyethyl methacrylate) film and the glucose biosensor not covered with the poly(hydroxyethyl methacrylate) film were tested by cyclic voltammetry in PBS buffer solution containing 5 mmol/L glucose. stability, the results are shown in Figure 4.

Figure 4 shows that, after one week of continuous testing, the current signal of the glucose biosensor covered with poly(hydroxyethyl methacrylate) film showed less than 2% attenuation (Fig. 4, curve a), compared to that without The current signal of the glucose biosensor of the hydroxyethyl methacrylate thin film decayed by more than 60% during continuous testing over one week (Fig. 4, curve b).

4. Test of anti-interference ability

The existing glucose biosensors are all prepared on the basis of glucose oxidase. When detecting glucose, oxygen becomes a major factor restricting the performance of the implanted glucose continuous monitoring system. However, the glucose dehydrogenase in the glucose biosensor of the present invention does not require the participation of oxygen when oxidizing glucose. This is tested by cyclic voltammetry to see if this is the desired effect.

The current of the glucose biosensor covered with a biocompatible membrane did not have any decay when oxygen was bubbled through a PBS buffer solution containing 10 mmol/L glucose and when the oxygen in the solution was completely removed by argon. . In addition, since the detection of glucose was performed at a very low potential (20-200 mV), the anti-interference ability of acetaminophen was very significantly improved (Fig. 5).

The above is only for understanding the method of the present invention and its core idea. It should be pointed out that for those skilled in the art, the present invention can be improved and modified without departing from the principle of the present invention. Improvements and modifications also fall within the protection scope of the present invention.

Claims

A glucose biosensor is characterized in that it comprises a substrate and an electrode located on the substrate; the electrode is coated with a glucose sensing membrane; the glucose sensing membrane is prepared according to the following preparation method:

Glucose dehydrogenase is incubated in a buffer containing urea or guanidine hydrochloride, and the glucose dehydrogenase is fully expanded to expose free carboxyl and amino groups on the surface and inside of its molecule, and then add ruthenium or osmium with free carboxyl or amino groups The complex and the first cross-linking agent are reacted to obtain chemically modified glucose dehydrogenase, so that the glucose dehydrogenase can perform rapid and direct electron exchange with the electrode, and then further cross-link through the second cross-linking agent, An electrochemically active glucose sensing membrane was obtained.
The biosensor according to claim 1, further comprising modifying the obtained chemically modified glucose dehydrogenase again before cross-linking by the second cross-linking agent:

The first chemically modified glucose dehydrogenase was incubated in a buffer containing urea or guanidine hydrochloride, and the glucose dehydrogenase was fully expanded to expose the free carboxyl and amino groups on its molecular surface and interior, and then added with free carboxyl and amino groups. The ruthenium or osmium complex of amino or carboxyl group and the first cross-linking agent are reacted to obtain the second chemically modified glucose dehydrogenase, which can interact with the electrode more quickly than the first chemically modified glucose dehydrogenase electronic exchange;
The biosensor according to claim 1 or 2, further comprising separating and purifying the modified glucose dehydrogenase using ultrafiltration bag dialysis after reacting with the first cross-linking agent.
The biosensor according to claim 1 or 2, wherein the first cross-linking agent is carbodiimide and N-hydroxysuccinimide.
The biosensor according to claim 1, wherein the second cross-linking agent is a bifunctional chemical cross-linking agent.
The biosensor according to claim 5, wherein the bifunctional chemical cross-linking agent is selected from the group consisting of glutaraldehyde, 1,4-butanediol diglycidyl ether, poly(dimethylsiloxane)-diglycidyl Glycidyl ether, tetraglycidyl-4,4-diaminodiphenylmethane, polyethylene glycol diglycidyl ether and 4-(2,3-glycidoxy)-N,N-bis(2 , one or more of 3-epoxypropyl)aniline.
The biosensor according to any one of claims 1-6, further comprising a biocompatible membrane covering the surface of the glucose sensing membrane.
The biosensor according to claim 7, wherein the biocompatible film is a copolymer prepared by one or a combination of acrylate and acrylate derivatives.
The biosensor according to claim 8, wherein the biocompatible film is poly(hydroxyethyl methacrylate).
According to the biosensor of claim 9, the biocompatible film is prepared by the following method:

Under anaerobic conditions, hydroxyethyl methacrylate, ethanol solution and Na 2 S 2 O 8 react in a closed environment to generate poly(hydroxyethyl methacrylate), and then add acetone to precipitate poly(hydroxyethyl methacrylate), and then use The method of ethanol dissolution and acetone precipitation is repeated for many times, and finally the obtained polyhydroxyethyl methacrylate is dried and separated.