Biocompatible film for biosensor and glucose sensor

Abstract

Disclosed are a biocompatible film for a biosensor and a glucose sensor. The biocompatible film is prepared by coating a film solution by means of a coating process. The film solution comprises a cofactor-grafted polymer, an organic solvent, and a buffer solution. An end portion of each branch of the cofactor-grafted polymer has a cofactor grafted thereto. The backbone of the cofactor-grafted polymer is one or more of polyurethane, polyvinyl alcohol, a polyvinyl alcohol derivative, polyvinylpyrrolidone, a polyvinylpyrrolidone derivative, polyvinylpyridine, and a polyvinylpyridine derivative. The branches of the cofactor-grafted polymer are one or more of acetic acid, butyric acid, ethyl butyrate, caproic acid, and ethyl hexanoate. The biocompatible film can achieve precise regulation of chemical substance permeability, expand the linear detection range of the biosensor for chemical substances, and provide the cofactors required during chemical substance detection, thereby improving the stability and durability of the biosensor.

Description

A biocompatible membrane for a biosensor and a glucose sensor TECHNICAL FIELD

[0001] The present application relates to the technical field of biosensors, in particular to a biocompatible membrane for a biosensor and a glucose sensor. BACKGROUND

[0002] An electrochemical biosensor is an analytical device based on biological substances and their derivative materials or biomimetic materials, which can combine technologies such as electrochemistry, polymer chemistry, biochemistry and electronic circuits, and usually contains biological enzymes that can specifically recognize target substances. The biochemical reaction signal is converted into an electrical signal for detection, which is used for quantitative detection of chemical substances in the human body, such as glucose, uric acid, etc.

[0003] In an implantable continuous blood glucose detection sensor, the sensing layer of the working electrode directly contacts the chemical substance, and the enzyme on the electrode reacts with the target glucose and exchanges electrons with the electrode to convert the chemical reaction signal into an electrical signal. There is a certain linear relationship between the electrical signal and the concentration of the chemical substance, and the concentration of the chemical substance can be determined according to the strength of the electrical signal. In the enzyme electrode direct electron transfer blood glucose reaction system, the cofactor (coenzyme factor) contained in the sensing layer plays a key role, but the cofactor is an exogenous small molecule that is lost during multiple uses of the sensor, resulting in insufficient amount of cofactor and affecting the accuracy of 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 current value collected does not have a corresponding relationship with the chemical substance concentration. 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 to enable stable detection for multiple long times is a technical problem that needs to be solved by those skilled in the art.

[0004] SUMMARY

[0005] In view of the problems of loss of cofactor during multiple uses of the existing biosensor and influence of high concentration of chemical substance on the accuracy of sensor detection, the present application proposes a biocompatible membrane for a biosensor and a glucose sensor to overcome the above problems.

[0006] In order to achieve the above purpose, the present application adopts the following technical scheme:

[0007] The present application discloses a biocompatible membrane for a biosensor, which is prepared by coating a membrane solution through a coating process.

[0008] The film solution comprises a cofactor grafting polymer, an organic solvent and a buffer; the branched chain of the cofactor grafting polymer is grafted with a cofactor at the end of the branch;

[0009] The main chain of the cofactor grafting polymer is one or more of polyurethane, polyvinyl alcohol, polyvinyl alcohol derivative, polyvinyl pyrrolidone, polyvinyl pyrrolidone derivative, polyvinyl pyridine and polyvinyl pyridine derivative; the branched chain of the cofactor grafting polymer is one or more of acetic acid, butyric acid, ethyl butyrate, hexanoic acid and ethyl hexanoate.

[0010] Another aspect of the present application discloses a glucose sensor comprising an electrode, the outer side of the sensing layer of the electrode being wrapped with the above-mentioned biocompatible film for biosensors.

[0011] In summary, the beneficial effects of the present application are:

[0012] The biocompatible film in the present application uses a cofactor grafting polymer composed of a cofactor, a main chain and a branched chain as raw material, and selects specific types of main chain and branched chain, so that the biocompatible film has high biocompatibility and can be applied to biosensors. Not only can it realize precise regulation of the permeation of chemical substances, expand the linear range of chemical substance detection by biosensors, but also can provide the cofactor required for the reaction in the process of chemical substance detection by biosensors, maintain the content of cofactor in the detection system, and improve the stability and durability of biosensors, so that biosensors can be used for continuous multiple detections. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is a structural formula of a cofactor grafting polymer with NADP as the cofactor in an embodiment of the present application;

[0014] Figure 2 is a graph of the relative current detected by glucose sensors with and without biocompatible films versus the glucose concentration;

[0015] Figure 3 is a titration curve of glucose sensors with biocompatible films under different concentrations of glucose (0-30mM);

[0016] Figure 4 is a graph of the current detected by glucose sensors with biocompatible films versus time when detecting the glucose concentration;

[0017] Figure 5 is a titration curve of glucose sensors with biocompatible films under different concentrations of glucose (0-20mM) from Day 1 to Day 35;

[0018] Figure 6 is a graph of the current detected by glucose sensors with biocompatible films versus time when detecting the glucose concentration from Day 1 to Day 35.

[0019] Figure 7 is a graph of the current detected by a glucose sensor coated with a biocompatible membrane as a function of time when detecting glucose concentration. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present application clearer, the embodiments of the present application will be further described in detail below with reference to the drawings.

[0021] In one embodiment of the present application, a biocompatible membrane for a biosensor is provided, which is applicable to a biosensor and specifically wrapped outside the electrode sensing layer of the biosensor when in use. The biocompatible membrane is prepared by coating a membrane solution through a coating process; wherein the coating process is one of dip coating, dot coating, draw coating and vapor deposition, and preferably is dip coating.

[0022] The membrane solution comprises a cofactor grafted polymer, an organic solvent and a buffer; as shown in Figure 1, the cofactor grafted polymer is composed of a main chain and a branch chain, the main chain of the cofactor grafted polymer is grafted with a plurality of branch chains of the cofactor grafted polymer, and the end of the branch chain of the cofactor grafted polymer is grafted with a cofactor, so as to fix the cofactor in the biocompatible membrane and prevent it from being lost.

[0023] Specifically, the main chain of the cofactor grafted polymer is one or more of polyurethane, polyvinyl alcohol, polyvinyl alcohol derivative, polyvinylpyrrolidone, polyvinylpyrrolidone derivative, polyvinylpyridine and polyvinylpyridine derivative; preferably, the main chain of the cofactor grafted polymer is one or more of polyurethane, polyvinyl alcohol, polyvinylpyrrolidone and polyvinylpyridine. The branch chain of the cofactor grafted polymer is one or more of acetic acid, butyric acid, ethyl butyrate, hexanoic acid and ethyl hexanoate. The main chain of the cofactor grafted polymer and the branch chain of the cofactor grafted polymer of the above-mentioned types are materials with high biocompatibility, so as to make the biocompatible membrane have high biocompatibility.

[0024] The following tests are conducted on the glucose sensor coated with the biocompatible membrane:

[0025] The electrode surface of the glucose sensor is coated with a sensing layer formed by depositing an enzyme mixture containing NAD(P), glucose dehydrogenase (GDH) and diaphorase, and the formula of the enzyme mixture is shown in Table 1. The biocompatible membrane is prepared by using a cofactor grafted polymer with NADP as the cofactor, and the formula of the corresponding membrane solution is shown in Table 2.

[0026] Table 1

[0027] Table 2

[0028] The glucose sensor was tested in SBF buffer solution containing different concentrations of glucose, and the test results are shown as follows:

[0029] As can be seen from FIG. 2, when the electrode of the glucose sensor is covered with the biocompatible membrane, the relative value of the current decreases, and the linear range increases, which can meet the linear correspondence in the range of 0-30 mM glucose, thereby indicating that the biocompatible membrane can effectively limit the glucose permeation in proportion, and at the same time accurately and sensitively indicate the glucose concentration through the current. Moreover, by comparing the output currents of the glucose sensor covered with the biocompatible membrane and the glucose sensor not covered with the biocompatible membrane after sensing glucose, it can be found that the presence of the biocompatible membrane can make the electrode of the glucose sensor have a wider linear range, which is more suitable for the working environment of continuous blood glucose detection.

[0030] FIG. 3 shows the titration curve of the glucose sensor covered with the biocompatible membrane under different concentrations of glucose (0-30 mM), and it can be seen that the glucose sensor can reflect a linear relationship with glucose, and the linear relationship continues to respond; FIG. 4 is a curve diagram of the current detected by the glucose sensor covered with the biocompatible membrane when detecting the glucose concentration changes with time, and from the diagram, the correction curve slope, linear intensity and sensitivity of the glucose sensor are 1.72, 0.9995 and 2.586 respectively, indicating that the glucose sensor can accurately measure the glucose concentration, and has high detection sensitivity.

[0031] FIGS. 5 and 6 show the glucose concentration linear titration data of the electrode of the glucose sensor on the 1st day, the 7th day, the 14th day, the 20th day, the 30th day and the 35th day. The glucose concentration-electrical signal of the glucose sensor has a linear relationship within 35 days, and the signal size remains almost unchanged. The electrochemical test results show that the biocompatible membrane can long-term maintain the linearity, stability and sensitivity of the signal response of the electrode of the glucose sensor. Table 3 shows the linear relationship and sensitivity of the sensor, which proves that the glucose sensor has good stability under long-term work, and the linearity and sensitivity of the glucose concentration response are almost unchanged.

[0032] Table 3

[0033] FIG. 7 is a diagram of the change of the current of the glucose sensor in the SBF buffer solution containing glucose within 35 days, and it can be seen that when the biosensor is covered with the biocompatible membrane, the current almost does not change within 35 days, and can be stably maintained at the same level (change amplitude <7%), indicating that the biocompatible membrane has good stability.

[0034] The preparation method of the biocompatible membrane in the embodiment is as follows:

[0035] Step 01, main chain monomer polymerization; the main chain monomer and initiator are mixed in a solvent in a certain proportion, and reacted at 30-80°C in an anaerobic environment for 6-18h, and then the reaction product is separated and purified to obtain the first intermediate product, i.e. the coenzyme grafted polymer from the main chain. Among them, the main chain monomer is one or more of urethane monomer, vinyl alcohol monomer, vinyl pyrrolidone monomer and vinyl pyridine monomer, preferably vinyl pyridine monomer; the initiator is one or more of azobisisobutyronitrile, azobisisoheptyl nitrile or benzoyl peroxide; the mass ratio of main chain monomer to initiator is 50-500:1.

[0036] Step 02, side chain modification; the first intermediate product and the side chain modifier are mixed in a solvent, and reacted at 40-90°C in an anaerobic environment for 12-24h, and then the reaction product is separated and purified to obtain the second intermediate product. Among them, the side chain modifier is one or more of dibromoacetic acid, bromobutyric acid, bromobutyric acid ethyl ester, bromohexanoic acid and bromohexanoic acid ethyl ester, preferably bromohexanoic acid; the mass ratio of the first intermediate product to the side chain modifier is 0.5-2:1.

[0037] Step 03, side chain coenzyme modification; the second intermediate product and the side chain modification coenzyme are mixed in a third solvent, and reacted at 30-80°C in an anaerobic environment for 6-18h, and then the reaction product is separated and purified to obtain the coenzyme grafted polymer. Among them, the side chain modification 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; the mass ratio of the second intermediate product to the side chain modification coenzyme is 2-10:1.

[0038] Step 04, coating and film forming; the coenzyme grafted polymer, organic solvent and buffer are uniformly mixed to form a film solution, and then a coating process is used to coat the film solution to form a biocompatible film. Among them, the biocompatible film needs to be dried or left to dry after being formed.

[0039] In summary, the biocompatible membrane in the embodiment has high biocompatibility by selecting the co-factor grafting polymer composed of co-factors, main chains and side chains as raw materials, and by selecting specific types of main chains and side chains. The biocompatible membrane can be applied to a biosensor, can not only realize accurate regulation of the permeation of chemical substances, expand the linear range of the biosensor for detecting chemical substances, limit the sensing layer chemical substance sensing concentration on the biosensor, expand the linear range of the current value collected by the biosensor and the concentration of chemical substances, enable the biosensor to accurately and sensitively detect the concentration of chemical substances, but also can provide the co-factor required for the reaction in the process of detecting chemical substances by the biosensor, maintain the content of the co-factor in the detection system, and improve the stability and durability of the biosensor, so that the biosensor can be continuously detected for multiple times.

[0040] In the embodiment, the grafting rate of the co-factor on the co-factor grafting polymer is 5%-30%. On the one hand, the response current size on the electrode of the biosensor will increase with the increase of the percentage content of the co-factor on the co-factor grafting polymer. Too high response current will affect the activity and stability of the enzyme, and too low response current is not suitable for the biosensor to receive signals. On the other hand, the percentage content of the co-factor in the biocompatible membrane will affect the stability of the membrane structure. If the value is too high, the co-factor in the membrane layer will participate in the reaction more strongly, causing the instability of the membrane structure, leading to the direct contact of the enzyme layer in the membrane with high concentration of glucose, thereby accelerating the deactivation of the enzyme. Therefore, the co-factor with the above grafting rate can not only accurately control the response current size, so that the response current size is in a reasonable range, but also can ensure the stability of the membrane structure.

[0041] In addition, the mass concentration of the co-factor grafting polymer in the membrane solution is 15 mg / mL-150 mg / mL, preferably 35 mg / mL-70 mg / mL, and most preferably 60 mg / mL. The co-factor grafting polymer with the mass concentration can form better quality membranes.

[0042] In addition, the organic solvent is one or more of ethanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide, and preferably ethanol. The buffer is PBS buffer, HEPES buffer, TAPS buffer or MES buffer. The pH range of the buffer is 5.5-9. The volume ratio of the organic solvent in the total volume of the organic solvent and the buffer is 50%-97%, preferably 85%-95%; the volume ratio of the buffer in the total volume of the organic solvent and the buffer is 3%-50%, preferably 5%-15%. The above types and volume ratios of the organic solvent and the buffer can uniformly dissolve the co-factor grafting polymer, and further make the membrane solution have better membrane forming effect.

[0043] Further, the environment for coating the membrane solution to form the biocompatible membrane is a solvent vapor atmosphere, and a 100,000-level clean constant temperature and humidity environment, thereby making the quality of the formed membrane higher. 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 the membrane are both required to be performed in the environment.

[0044] In the present embodiment, the cofactor is one or more of NADP, NAD, NADH and NADPH, and is preferably NADP.

[0045] In addition, when the membrane is formed by dip coating, the pulling rate is 500 μm / s to 3000 μm / s, for example, it can be 1000 μm / s to 3000 μm / s, or 500 μm / s to 1000 μm / s. The dip coating and the interval process can be repeated multiple times to increase the thickness of the biocompatible membrane, so that the biocompatible membrane can be uniformly stacked to 5-20 layers, so that the thickness of the biocompatible membrane reaches 5-60 microns. When the dip coating is performed multiple times, the interval time between adjacent two dip coatings is 0.5 min to 30 min, for example, it can be 0.5 min to 3 min, or 10 min to 20 min. The standing and drying time after dip coating is 8 h to 24 h, for example, it can be 8 h to 12 h, or 20 h to 24 h, etc. The standing temperature can be 20℃ to 50℃, for example, it can be 25℃ to 45℃, or 30℃ to 40℃, and is preferably 20℃ to 25℃.

[0046] In another embodiment of the present application, a glucose sensor is provided, which comprises an electrode, and the outer side of the sensing layer of the electrode is wrapped with the biocompatible membrane for biosensors described above. The glucose sensor has the advantages of high detection accuracy and good durability.

[0047] The thickness of the biocompatible membrane is 5 μm to 60 μm, and the biocompatible membrane with the thickness has the best limiting effect on glucose permeation.

[0048] The above is only a specific embodiment of the present application, and those skilled in the art can make other improvements or modifications on the basis of the above embodiment under the above teaching of the present application. Those skilled in the art should understand that the above specific description is only to better explain the purpose of the present application, and the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A biocompatible membrane for use in a biosensor, characterized in that, The biocompatible membrane is prepared by coating a membrane solution through a coating process; The membrane solution comprises a cofactor grafted polymer, an organic solvent and a buffer; the end of a branch of the cofactor grafted polymer is grafted with a cofactor; The main chain of the cofactor grafted polymer is one or more of polyurethane, polyvinyl alcohol, polyvinyl alcohol derivative, polyvinylpyrrolidone, polyvinylpyrrolidone derivative, polyvinylpyridine and polyvinylpyridine derivative; the branch of the cofactor grafted polymer is one or more of acetic acid, butyric acid, ethyl butyrate, hexanoic acid and ethyl hexanoate.

2. The biocompatible membrane for a biosensor according to claim 1, characterized in that, The main chain of the cofactor grafted polymer is one or more of polyurethane, polyvinyl alcohol, polyvinylpyrrolidone and polyvinylpyridine.

3. The biocompatible membrane for a biosensor according to claim 1, characterized in that, The grafting rate of the cofactor on the cofactor grafted polymer is 5%-30%.

4. The biocompatible membrane 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-150 mg / mL.

5. The biocompatible membrane for a biosensor of claim 1, wherein, The organic solvent is one or more of ethanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide.

6. The biocompatible membrane for a biosensor of claim 1, wherein, The buffer is PBS buffer, HEPES buffer, TAPS buffer or MES buffer.

7. The biocompatible membrane for a biosensor of claim 1, wherein, The volume ratio of the organic solvent in the total volume of the organic solvent and the buffer is 50%-97%; the volume ratio of the buffer in the total volume of the organic solvent and the buffer is 3%-50%.

8. The biocompatible membrane for a biosensor of claim 1, wherein, The coating process is one of dip coating, dot coating, draw coating and vapor deposition.

9. The biocompatible membrane for a biosensor according to claim 8, characterized in that, The environment for coating the membrane solution to form the biocompatible membrane is a solvent vapor atmosphere, and a 100,000-level clean constant temperature and humidity environment.

10. The biocompatible membrane for a biosensor of claim 1, wherein, The cofactor is one or more of NADP, NAD, NADH and NADPH.

11. 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 the biocompatible membrane for biosensors described in claim 10.

12. The glucose sensor of claim 11, wherein, The thickness of the biocompatible membrane is 5 μm-60 μm.

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