Preparation method of biocompatible membrane for biosensor and glucose sensor

Abstract

The invention discloses a preparation method of a biocompatible membrane for a biosensor and a glucose sensor. The preparation method specifically comprises the following steps: sequentially carrying out main chain monomer polymerization reaction, side chain modification reaction, side chain coenzyme modification reaction and coating film formation to obtain the biocompatible film. The biocompatible membrane prepared by the preparation method can be applied to a biosensor, not only can realize accurate regulation and control of chemical substance permeation and expand the linear range of chemical substance detection by the biosensor, but also can provide cofactors for the biosensor, so that the stability and durability of the biosensor are improved.

Description

Technical Field

[0001] The invention relates to the technical field of biosensors, and in particular to a method for preparing a biocompatible membrane for a biosensor and a glucose sensor. Background Art

[0002] Electrochemical biosensors are analytical devices based on biomass and its derivative materials or biomimetic materials, and can combine multiple technologies such as electrochemistry, polymer chemistry, biochemistry and electronic circuits. They usually contain biological enzymes that can specifically identify target substances. They are used to quantitatively detect chemical substances in the human body, such as glucose and uric acid, by converting biochemical reaction signals into electrical signals for detection.

[0003] In the implantable continuous blood glucose detection sensor, the sensing layer of the working electrode directly contacts the chemical substance, and the enzyme on the electrode converts the chemical reaction signal into an electrical signal by reacting with the target glucose and exchanging electrons with the electrode. There is a certain linear relationship between this electrical signal and the concentration of the chemical substance, and then the concentration of the chemical substance can be determined according to the strength of the electrical signal. In the blood glucose reaction system of direct electron transfer of the enzyme electrode, the cofactor (coenzyme factor) contained in the sensing layer plays a key role, but the cofactor is an exogenous small molecule, which is lost during the multiple use of the sensor, resulting in insufficient amount of cofactor, affecting the accuracy of the detection; in addition, when the sensing layer of the working electrode directly contacts the normal blood glucose concentration of the human body, due to the high glucose concentration, the enzyme electrochemical kinetics is no longer limited by the glucose concentration, and the collected current value and the concentration of the chemical substance no longer have a corresponding relationship. Therefore, in the direct electron transfer blood glucose biosensor, how to stabilize the cofactor related to the biological enzyme and effectively limit the glucose concentration so as to be able to stably perform multiple long-term detections is a technical problem that needs to be solved by those skilled in the art. Summary of the invention

[0004] In view of the problem that cofactors are lost during multiple uses of existing biosensors, and that the sensor detection accuracy is affected when the concentration of chemical substances is too high, the present application proposes a method for preparing a biocompatible membrane for a biosensor and a glucose sensor to overcome the above problems.

[0005] In order to achieve the above purpose, this application adopts the following technical solutions:

[0006] On the one hand, the present application discloses a method for preparing a biocompatible membrane for a biosensor, the preparation method comprising the following steps:

[0007] Step 01, polymerizing the main chain monomer; mixing the main chain monomer and the initiator in a first solvent according to a certain ratio, reacting under a first reaction condition, and then separating and purifying the reaction product to obtain a first intermediate product;

[0008] Step 02, side chain modification: mixing the first intermediate product and the side chain modifier in a second solvent, reacting under second reaction conditions, and then separating and purifying the reaction product to obtain a second intermediate product;

[0009] Step 03, side chain coenzyme modification; mixing the second intermediate product and the side chain modified coenzyme in a third solvent, reacting under third reaction conditions, and then separating and purifying the reaction product to obtain a cofactor grafted polymer;

[0010] Step 04, coating to form a membrane; uniformly mixing the cofactor grafted polymer and the fourth solvent to form a membrane solution, and then using a coating process to coat the membrane solution to form the biocompatible membrane.

[0011] On the other hand, the present application discloses a glucose sensor, which includes an electrode, wherein the outer side of the sensing layer of the electrode is wrapped with a biocompatible film prepared by the above-mentioned method for preparing a biocompatible film for a biosensor.

[0012] In summary, the beneficial effects of this application are:

[0013] The biocompatible membrane prepared in the present application through main chain monomer polymerization reaction, side chain modification reaction, side chain coenzyme modification reaction and coating film formation can be applied to biosensors. It can not only realize the precise regulation of the penetration of chemical substances, expand the linear range of biosensors for chemical substance detection, and enable biosensors to accurately and sensitively detect the concentration of chemical substances, but also provide cofactors for biosensors, thereby increasing the stability and durability of biosensors, so that biosensors can perform multiple consecutive detections. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a diagram of the implementation steps of a method for preparing a biocompatible membrane for a biosensor in one embodiment of the present application;

[0015] Figure 2 A graph showing changes in relative current detected as a function of glucose concentration for a glucose sensor not covered with a biocompatible film and a glucose sensor covered with a biocompatible film;

[0016] Figure 3 A graph showing the change of current detected by the glucose sensor covered with a biocompatible film over time when detecting glucose concentration;

[0017] Figure 4 The figure is a graph showing the change of the current detected by the glucose sensor covered with a biocompatible membrane at different times as a function of the glucose concentration. DETAILED DESCRIPTION

[0018] In order to make the objectives, technical solutions and advantages of the present application clearer, the implementation methods of the present application will be further described in detail below with reference to the accompanying drawings.

[0019] In one embodiment of the present application, a method for preparing a biocompatible membrane for a biosensor is provided, such as Figure 1 As shown, the preparation method comprises the following steps:

[0020] Step 01, polymerizing the main chain monomer; mixing the main chain monomer and the initiator in a first solvent in a certain ratio, and reacting under the first reaction condition, and then separating and purifying the reaction product to obtain a first intermediate product; wherein, the reaction under the first reaction condition is specifically: reacting under an oxygen-free environment at 30°C to 80°C for 6h to 18h; the mass ratio of the main chain monomer to the initiator is 50 to 500:1. .

[0021] Step 02, side chain modification; mixing the first intermediate product and the side chain modifier in a second solvent, reacting under second reaction conditions, and then separating and purifying the reaction product to obtain a second intermediate product; wherein, reacting under the second reaction conditions is specifically: reacting in an anaerobic environment at 40 to 90°C for 12h to 24h; the mass ratio of the first intermediate product to the side chain modifier is 0.5 to 2:1.

[0022] Step 03, side chain coenzyme modification; mixing the second intermediate product and the side chain modified coenzyme in a third solvent, reacting under third reaction conditions, and then separating and purifying the reaction product to obtain a cofactor grafted polymer; wherein, reacting under the third reaction conditions is specifically: reacting in an anaerobic environment at 30 to 80°C for 6h to 18h; the mass ratio of the second intermediate product and the side chain modified coenzyme is 2 to 10:1.

[0023] Step 04, coating to form a membrane: uniformly mix the cofactor grafted polymer and the fourth solvent to form a membrane solution, and then use a coating process to coat the membrane solution to form a biocompatible membrane. The biocompatible membrane needs to be dried or left to dry after formation.

[0024] It should be noted that since the coating and film-forming process is independent of the synthesis process of the cofactor grafted polymer, the cofactor grafted polymer is separated and preserved by purification of the solid after production, and then prepared into a membrane solution when it is needed to be coated and used. In this way, the stability and service life of the membrane solution are greatly improved, so that the stability and service life of the products of the same batch do not change significantly within one year, thereby ensuring the consistency of the biocompatible membrane, and further ensuring the high consistency of the prepared implantable continuous glucose detection system containing the biocompatible membrane.

[0025] In summary, the biocompatible membrane prepared in this embodiment through main chain monomer polymerization reaction, side chain modification reaction, side chain coenzyme modification reaction and coating film formation can be applied to biosensors, specifically wrapped on the outside of the biosensor electrode sensing layer. The biocompatible membrane can not only achieve precise regulation of the penetration of chemical substances, expand the linear range of the biosensor for chemical substance detection, that is, it can limit the sensitivity concentration of the chemical substances in the sensing layer of the biosensor, expand the linear range of the current value collected by the biosensor and the concentration of the chemical substances, so that the biosensor can accurately and sensitively detect the concentration of the chemical substances, but also provide cofactors for the biosensor, thereby increasing the stability and durability of the biosensor, so that the biosensor can perform multiple consecutive detections.

[0026] In the present embodiment, the main chain monomer is one or more of an urethane monomer, a vinyl alcohol monomer, a vinyl pyrrolidone monomer and a vinyl pyridine monomer, preferably a vinyl pyridine monomer. The side chain modifier is one or more of dibromoacetic acid, bromobutyric acid, ethyl bromobutyrate, bromohexanoic acid and ethyl bromohexanoate, preferably bromohexanoic acid. The biocompatible membrane prepared by using the above-mentioned main chain monomers and side chain modifiers as raw materials has a high degree of biocompatibility.

[0027] Furthermore, the initiator is one or more of azobisisobutyronitrile, azobisisoheptanenitrile and benzoyl peroxide.

[0028] In addition, the first solvent includes water, and one or more of ethanol, methanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide; the second solvent includes water, and one or more of ethanol, methanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide; the third solvent includes water, and one or more of ethanol, methanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide. The first solvent, the second solvent and the third solvent may be the same type of solvent or different types of solvents.

[0029] Furthermore, the mass concentration of the cofactor grafted polymer in the membrane solution is 15 mg / mL to 150 mg / mL, preferably 35 mg / mL to 70 mg / mL, and most preferably 60 mg / mL, so that a sufficient amount of cofactor can be provided for the biosensor.

[0030] In this embodiment, the fourth solvent includes water, an organic solvent and a buffer solution, and the water, the organic solvent and the buffer solution need to be uniformly mixed when used.

[0031] The organic solvent is one or more of ethanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide, preferably ethanol; the buffer is one of PBS buffer, HEPES buffer, TAPS buffer and MES buffer. The volume proportion of the organic solvent in the total volume of the organic solvent and the buffer is 50% to 97%, preferably 85% to 95%; the volume proportion of the buffer in the total volume of the organic solvent and the buffer is 3% to 50%, preferably 5% to 15%; the pH range of the buffer is 5.5 to 9. The fourth solvent of this component enables the cofactor grafted polymer to be dissolved evenly, thereby making the film-forming effect of the membrane solution better.

[0032] In addition, the coating process is one of dip coating, spot coating, pull coating and vapor deposition, and preferably immersion pull (dip coating).

[0033] When the film is formed by dipping and pulling, the pulling rate is 500μm / s to 3000μm / s, for example, it can be 1000μm / s to 3000μm / s, or it can be 500μm / s to 1000μm / s. The dipping and pulling and dipping interval processes can be repeated multiple times to increase the thickness of the biocompatible film, so that the biocompatible film can be evenly stacked to 5 to 20 layers, so that the thickness of the biocompatible film reaches 5 to 60 microns. When multiple dipping and pulling are performed, the interval time between two adjacent dippings can be 0.5min to 30min, for example, it can be 0.5min to 3min, or it can be 10min to 20min. The static drying time after pulling and dipping is 8h to 24h, for example, it can be 8h to 12h, or it can be 20h to 24h, etc. The standing temperature may be 20°C to 50°C, for example, 25°C to 45°C, or 30°C to 40°C, and preferably 20°C to 25°C.

[0034] In addition, in step 04, the coating environment is a solvent vapor atmosphere and a 100,000-level clean constant temperature and humidity environment, thereby making the film-forming quality higher. Among them, the solvent vapor can be ethanol vapor. It should be noted that the preparation of the membrane solution and the process of coating the membrane solution to form a film need to be carried out in this environment.

[0035] In this embodiment, the side chain modified coenzyme is one or more of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate, preferably nicotinamide adenine dinucleotide phosphate.

[0036] In another embodiment of the present application, a glucose sensor is provided, which includes an electrode, and the outer side of the sensing layer of the electrode is wrapped with a biocompatible film prepared by the preparation method of the biocompatible film for the biosensor in the above embodiment. The glucose sensor has the advantages of high detection accuracy and good durability.

[0037] Example 1

[0038] Synthesis of membrane materials: First, vinylpyridine and initiator were fully mixed and dissolved in N, N-dimethylformamide (DMF) at a mass ratio of 400:1, and heated in a water bath at 60°C for 10 hours. The reaction product was separated by precipitation with a methanol solution to obtain a first intermediate product. Then, the first intermediate product and bromoacetic acid were dissolved in N, N-dimethylformamide (DMF) at a mass ratio of 1:1, and heated in a water bath at 80°C for 18 hours. The reaction product was separated by ethyl acetate solution to obtain a second intermediate product. Finally, the second intermediate product and nicotinamide adenine dinucleotide phosphate were dissolved in N, N-dimethylformamide (DMF) at a mass ratio of 10:1, and heated in a water bath at 70°C for 5 hours. The reaction product was separated and purified by a methanol solution, and the cofactor grafted polymer was obtained after drying.

[0039] Film formation on the biosensor electrode: First, in a 100,000-class clean room and an environment containing saturated ethanol vapor, the cofactor grafted polymer was dissolved at a concentration of 100 mg / mL in 95% by volume of anhydrous ethanol and 5% by volume of HEPES buffer (10 mM, pH = 8) to form a membrane solution. Secondly, the membrane solution was evenly coated on the glucose sensor containing electrochemically activated glucose dehydrogenase by the immersion pulling method. The pulling rate was 500 μm / s. Then, it was allowed to stand for 30 minutes at a temperature of 25°C and a relative humidity of 50%, and the immersion pulling and standing process was repeated 8 times. Finally, it was dried for 18 hours under constant temperature conditions to obtain an implantable glucose sensor for testing.

[0040] Electrochemical test: The glucose sensor in this embodiment was immersed in a human body simulation fluid (SBF buffer) containing different glucose concentrations, and the chronoamperometry method was used for detection. The detection results are as follows: Figure 2 As shown, when the biosensor electrode is covered with a biocompatible film, the relative value of the current decreases and the linear range increases, which can meet the linear correspondence within the range of 0-30mM glucose, further indicating that the biocompatible film can effectively limit glucose penetration in equal proportion, and can accurately and sensitively indicate the glucose concentration through current. In addition, by comparing the output current of the glucose sensor covered with a biocompatible film and the glucose sensor not covered with a biocompatible film after sensing glucose, it can be found that the presence of the biocompatible film can make the electrode of the glucose sensor have a wider linear range that is more suitable for the working environment of continuous blood glucose detection.

[0041] Example 2

[0042] Synthesis of membrane materials: First, vinylpyridine and initiator were fully mixed and dissolved in N, N-dimethylformamide (DMF) at a mass ratio of 400:1, and heated in a water bath at 60°C for 10 hours. The reaction product was separated by precipitation with a methanol solution to obtain a first intermediate product. Then, the first intermediate product and bromoacetic acid were dissolved in N, N-dimethylformamide (DMF) at a mass ratio of 1:1, and heated in a water bath at 80°C for 18 hours. The reaction product was separated by precipitation with an ethyl acetate solution to obtain a second intermediate product. Finally, the second intermediate product and nicotinamide adenine dinucleotide phosphate were dissolved in N, N-dimethylformamide (DMF) at a mass ratio of 10:1, and heated in a water bath at 70°C for 5 hours. Then, the reaction product was separated and purified by a methanol solution, and the cofactor grafted polymer was obtained after drying.

[0043] Film formation on the biosensor electrode: First, in a 100,000-class clean room and an environment containing saturated ethanol vapor, the cofactor grafted polymer is dissolved at a concentration of 60 mg / mL in 95% by volume anhydrous ethanol and 5% by volume HEPES buffer (10 mM, pH = 8) to form a biocompatible membrane solution. Secondly, the biocompatible membrane solution is evenly coated on the glucose sensor containing electrochemically activated glucose dehydrogenase by the immersion pulling method. The pulling rate is 500 μm / s. Then, it is allowed to stand for 30 minutes at a temperature of 25°C and a relative humidity of 50%, and the immersion pulling and standing process is repeated 8 times. Finally, it is dried for 18 hours under constant temperature conditions to obtain an implantable glucose sensor for testing.

[0044] Electrochemical test: The glucose sensor in this example was immersed in a human body simulation fluid (SBF buffer) containing glucose, and the chronoamperometry was used to detect the change in stability (current) over a long period of time (35 days). Figure 3 This is a graph showing the change in current of the glucose sensor in Example 2 in an SBF buffer solution containing 10 mM glucose over 35 days. It can be seen that when the biosensor is covered with a biocompatible film, the current hardly changes over 35 days and can stably maintain the same level (change range <7%). Figure 4 The glucose concentration linear titration data of the glucose sensor electrode on the 1st, 7th, 14th, 20th, 30th and 35th days were displayed. During the 35 days, the glucose concentration-electrical signal of the glucose sensor has always had a linear relationship, and the signal size remains almost unchanged. The above electrochemical test results show that the biocompatible membrane can maintain the linearity, stability and sensitivity of the signal response of the glucose sensor electrode for a long time.

[0045] The above is only a specific embodiment of the present invention. Under the above teachings of the present invention, those skilled in the art can make other improvements or modifications on the basis of the above embodiments. Those skilled in the art should understand that the above specific description is only to better explain the purpose of the present invention, and the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for preparing a biocompatible membrane for a biosensor, characterized in that: The preparation method comprises the following steps: Step 01, polymerizing the main chain monomer; mixing the main chain monomer and the initiator in a first solvent according to a certain ratio, reacting under a first reaction condition, and then separating and purifying the reaction product to obtain a first intermediate product; Step 02, side chain modification: mixing the first intermediate product and the side chain modifier in a second solvent, reacting under second reaction conditions, and then separating and purifying the reaction product to obtain a second intermediate product; Step 03, side chain coenzyme modification; mixing the second intermediate product and the side chain modified coenzyme in a third solvent, reacting under third reaction conditions, and then separating and purifying the reaction product to obtain a cofactor grafted polymer; Step 04, coating to form a film; The cofactor grafted polymer and the fourth solvent are uniformly mixed to form a membrane solution, and then a coating process is used to coat the membrane solution to form the biocompatible membrane.

2. The method for preparing a biocompatible film for a biosensor according to claim 1, characterized in that: The main chain monomer is one or more of urethane monomer, vinyl alcohol monomer, vinyl pyrrolidone monomer and vinyl pyridine monomer.

3. The method for preparing a biocompatible film for a biosensor according to claim 1, characterized in that: The side chain modifier is one or more of dibromoacetic acid, bromobutyric acid, ethyl bromobutyrate, bromohexanoic acid and ethyl bromohexanoate.

4. The method for preparing a biocompatible film for a biosensor according to claim 1, characterized in that: The reaction under the first reaction condition is specifically: reacting in an oxygen-free environment at 30° C. to 80° C. for 6 h to 18 h.

5. The method for preparing a biocompatible film for a biosensor according to claim 1, characterized in that: The reaction under the second reaction condition is specifically: reacting in an oxygen-free environment at 40 to 90° C. for 12 to 24 hours.

6. The method for preparing a biocompatible film for a biosensor according to claim 1, characterized in that: The reaction under the third reaction condition is specifically: reacting in an anaerobic environment at 30-80° C. for 6 h to 18 h.

7. The method for preparing a biocompatible film for a biosensor according to claim 1, characterized in that: The initiator is one or more of azobisisobutyronitrile, azobisisoheptanenitrile and benzoyl peroxide.

8. The method for preparing a biocompatible film for a biosensor according to claim 7, characterized in that: The mass ratio of the main chain monomer to the initiator is 50 to 500:

1.

9. The method for preparing a biocompatible film for a biosensor according to claim 1, characterized in that: The mass ratio of the first intermediate product to the side chain modifier is c.5-2:

1.

10. The method for preparing a biocompatible film for a biosensor according to claim 1, characterized in that: The mass ratio of the second intermediate product to the side chain modified coenzyme mixture is 2 to 10:

1.

11. The method for preparing a biocompatible film for a biosensor according to claim 1, characterized in that: The first solvent includes water, and one or more of ethanol, methanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide; the second solvent includes water, and one or more of ethanol, methanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide; the third solvent includes water, and one or more of ethanol, methanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide.

12. The method for preparing a biocompatible film for a biosensor according to claim 1, characterized in that: The mass concentration of the cofactor grafted polymer in the membrane solution is 15 mg / mL to 150 mg / mL.

13. The method for preparing a biocompatible film for a biosensor according to claim 1, characterized in that: The fourth solvent includes water, an organic solvent and a buffer; The organic solvent is one or more of ethanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide; the buffer is one of PBS buffer, HEPES buffer, TAPS buffer and MES buffer.

14. The method for preparing a biocompatible film for a biosensor according to claim 13, characterized in that: The volume proportion of the buffer solution in the total volume of the organic solvent and the buffer solution is 3% to 50%; and the pH range of the buffer solution is 5.5 to 9.

15. The method for preparing a biocompatible film for a biosensor according to claim 1, characterized in that: The coating process is one of dip coating, spot coating, stretch coating and vapor deposition.

16. The method for preparing a biocompatible film for a biosensor according to claim 15, characterized in that: In the step 04, the coating environment is a solvent vapor atmosphere and a 100,000 class clean constant temperature and humidity environment.

17. The method for preparing a biocompatible film for a biosensor according to claim 1, characterized in that: The side chain modified coenzyme is one or more of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate.

18. A glucose sensor, characterized in that: The glucose sensor comprises an electrode, and the outer side of the sensing layer of the electrode is wrapped with a biocompatible film prepared by the method for preparing a biocompatible film for a biosensor as claimed in claim 17.

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