Biocompatible membrane for biosensor and glucose sensor
By using a biocompatible membrane made of cofactor grafted polymer in biosensors, the problem of cofactor loss and high glucose concentration affecting detection accuracy is solved, and the stability and durability of the biosensor is achieved, ensuring that Accurate detection of glucose concentration and long-term continuous detection.
Patent Information
- Application Number
- CN202411376132.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2025-01-07
AI Technical Summary
In biosensors, cofactor loss and high glucose concentration lead to a decrease in detection accuracy, making it difficult to achieve long-term continuous detection.
A biocompatible film made of cofactor grafted polymer is used to wrap it on the outside of the electrode sensing layer through a coating process to prevent cofactor loss and regulate glucose penetration, expanding the detection linear range.
It improves the stability and durability of the biosensor, realizes accurate detection of glucose concentration, extends the detection time, and maintains the linearity and sensitivity of the electrical signal.
Smart Images
Figure CN119264305A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biosensors, and in particular to a biocompatible membrane and a glucose sensor for biosensors. 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 biocompatible membrane and a glucose sensor for biosensors to overcome the above problems.
[0005] In order to achieve the above purpose, this application adopts the following technical solutions:
[0006] On one hand, the present application discloses a biocompatible membrane for a biosensor, wherein the biocompatible membrane is prepared by coating a membrane solution through a coating process;
[0007] The membrane solution comprises a cofactor grafted polymer, an organic solvent and a buffer; the cofactor is grafted to the end of the side chain of the cofactor grafted polymer;
[0008] The main chain of the cofactor grafted polymer is one or more of polyurethane, polyvinyl alcohol, polyvinyl alcohol derivatives, polyvinyl pyrrolidone, polyvinyl pyrrolidone derivatives, polyvinyl pyridine and polyvinyl pyridine derivatives; the side chain of the cofactor grafted polymer is one or more of acetic acid, butyric acid, ethyl butyrate, hexanoic acid and ethyl hexanoate.
[0009] On the other hand, the present application discloses a glucose sensor, which includes an electrode, and the outer side of the sensing layer of the electrode is wrapped with the above-mentioned biocompatible film for the biosensor.
[0010] In summary, the beneficial effects of this application are:
[0011] The biocompatible membrane in the present application is made highly biocompatible by selecting a cofactor grafted polymer composed of a cofactor, a main chain and a side chain as a raw material, and by selecting the specific types of the main chain and the side chain. The biocompatible membrane can be applied to biosensors, which can not only achieve precise regulation of the penetration of chemical substances and expand the linear range of the biosensor for chemical substance detection, but also provide the cofactors required for the biosensor to react during the chemical substance detection process, maintain the content of the cofactor in the detection system, improve the stability and durability of the biosensor, and enable the biosensor to perform multiple consecutive detections. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 The structural formula of the cofactor grafted polymer in which the cofactor is NADP in one embodiment of the present application;
[0013] 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;
[0014] Figure 3 The titration curves of the glucose sensor covered with a biocompatible membrane at different glucose concentrations (0-30 mM);
[0015] Figure 4 A graph showing the change of current detected by the glucose sensor covered with a biocompatible film over time when detecting glucose concentration;
[0016] Figure 5 The titration curves of the glucose sensor covered with a biocompatible film at different glucose concentrations (0-20 mM) from Day 1 to Day 35;
[0017] Figure 6 The graph is a graph showing the change in current over time when the glucose sensor covered with a biocompatible film detects glucose concentration from Day 1 to Day 35;
[0018] Figure 7 The graph is a graph showing the change of current detected by the glucose sensor covered with a biocompatible membrane over time when detecting glucose concentration. DETAILED DESCRIPTION
[0019] 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.
[0020] In one embodiment of the present application, a biocompatible membrane for a biosensor is provided. The biocompatible membrane can be applied to the biosensor and, when in use, is specifically wrapped around the outer side of the electrode sensing layer of the biosensor. The biocompatible membrane is prepared by coating a membrane solution through a coating process; wherein the coating process is one of dip coating, spot coating, pull coating and vapor deposition, preferably dip pulling.
[0021] The membrane solution includes a cofactor-grafted polymer, an organic solvent, and a buffer; e.g. Figure 1 As shown, the cofactor grafted polymer consists of a main chain and side chains, a plurality of side chains of the cofactor grafted polymer are grafted onto the main chain of the cofactor grafted polymer, and the ends of the side chains of the cofactor grafted polymer are grafted with cofactors, so that the cofactors are fixed on the biocompatible membrane to prevent them from being lost.
[0022] Specifically, the main chain of the cofactor grafted polymer is one or more of polyurethane, polyvinyl alcohol, polyvinyl alcohol derivatives, polyvinyl pyrrolidone, polyvinyl pyrrolidone derivatives, polyvinyl pyridine and polyvinyl pyridine derivatives; preferably, the main chain of the cofactor grafted polymer is one or more of polyurethane, polyvinyl alcohol, polyvinyl pyrrolidone and polyvinyl pyridine. The side chain of the cofactor grafted polymer is one or more of acetic acid, butyric acid, ethyl butyrate, caproic acid and ethyl caproate. The main chain of the cofactor grafted polymer of the above type and the side chain of the cofactor grafted polymer are highly biocompatible materials, so that the biocompatible membrane has high biocompatibility.
[0023] The following tests were performed on a glucose sensor covered with a biocompatible membrane:
[0024] The electrode surface of the glucose sensor is covered with a sensing layer, which is formed by depositing an enzyme mixture containing NAD(P), glucose dehydrogenase (GDH), and diaphorase. 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.
[0025]
[0026] Table 1
[0027] Cofactor grafted polymer 100 mg Anhydrous ethanol 0.95 mL HEPES (10 mM, pH = 8) 0.05 mL
[0028] Table 2
[0029] The glucose sensor was tested in SBF buffer containing different concentrations of glucose. The test results are shown below:
[0030] from Figure 2 It can be seen that when the electrode of the glucose sensor 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 the permeation of glucose in equal proportion, and can accurately and sensitively indicate the glucose concentration through the 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.
[0031] Figure 3 The titration curves of the glucose sensor covered with a biocompatible film at different glucose concentrations (0-30 mM) are shown. It can be seen that the glucose sensor can reflect a linear relationship to glucose, and the linear relationship responds continuously; Figure 4 This is a graph showing the change in current over time when the glucose sensor covered with a biocompatible membrane detects glucose concentration. From the graph, it can be seen that the slope of the calibration curve, linear strength 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 a high detection sensitivity.
[0032] Figure 5 and Figure 6 The linear titration data of glucose concentration of the glucose sensor electrode on the 1st, 7th, 14th, 20th, 30th and 35th days are shown. The glucose concentration-electrical signal of the glucose sensor has a linear relationship for 35 days, and the signal size remains almost unchanged. The 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. Table 3 shows the linear relationship and sensitivity of the sensor, proving that the glucose sensor has good stability under long-term operation, and the linearity and sensitivity of the glucose concentration response remain almost unchanged.
[0033] Slope <![CDATA[R 2 ]]> Sensitivity Day 1 2.07 0.9994 2.065 Day 7 1.93 0.9992 1.978 Day 14 2.25 0.9961 2.205 Day 20 1.92 0.9963 1.953 Day 30 1.91 0.9996 1.892 Day 35 1.77 0.9996 1.757
[0034] Table 3
[0035] Figure 7This is a graph showing the change in the current of the glucose sensor in an SBF buffer solution containing glucose over 35 days. It can be seen that when the biosensor is covered with a biocompatible membrane, the current hardly changes over 35 days and can stably maintain the same level (change range <7%), indicating that the biocompatible membrane has good stability.
[0036] The preparation method of the biocompatible membrane in this embodiment is as follows:
[0037] Step 01, polymerization of main chain monomers; the main chain monomers and initiators are mixed in a solvent in a certain proportion, and reacted in an oxygen-free environment at 30°C to 80°C for 6h to 18h, and then the reaction products are separated and purified to obtain the first intermediate product, i.e., the cofactor grafted polymer is composed of a main chain. Among them, the main chain monomer is one or more of urethane monomers, vinyl alcohol monomers, vinyl pyrrolidone monomers and vinyl pyridine monomers, preferably vinyl pyridine monomers; the initiator is one or more of azobisisobutyronitrile, azobisisoheptanenitrile or benzoyl peroxide; the mass ratio of the main chain monomer to the initiator is 50 to 500:1.
[0038] Step 02, side chain modification: the first intermediate product and the side chain modifier are mixed in a solvent, and reacted in an oxygen-free environment at 40 to 90°C for 12 to 24 hours, and then the reaction product is separated and purified to obtain a second intermediate product. The side chain modifier is one or more of dibromoacetic acid, bromobutyric acid, ethyl bromobutyrate, bromohexanoic acid and ethyl bromohexanoate, preferably bromohexanoic acid; the mass ratio of the first intermediate product to the side chain modifier is 0.5 to 2:1.
[0039] Step 03, side chain coenzyme modification: the second intermediate product and the side chain modified coenzyme are mixed in a third solvent, and reacted in an anaerobic environment at 30-80°C for 6h-18h, and then the reaction product is separated and purified to obtain a cofactor grafted polymer. 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; the mass ratio of the second intermediate product and the side chain modified coenzyme is 2-10:1.
[0040] Step 04, coating to form a membrane: uniformly mix the cofactor grafted polymer, organic solvent and buffer 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.
[0041] In summary, the biocompatible membrane in this embodiment has high biocompatibility by selecting a cofactor grafted polymer composed of a cofactor, a main chain and a side chain as a raw material, and by selecting the specific types of the main chain and the side chain. The compatible membrane can be applied to biosensors, which can not only achieve precise regulation of the penetration of chemical substances, but also expand the linear range of chemical substance detection by the biosensor, that is, it can limit the sensitivity concentration of 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 substance, so that the biosensor can accurately and sensitively detect the concentration of the chemical substance, and can also provide the cofactors required for the biosensor to react in the process of chemical substance detection, maintain the content of the cofactor in the detection system, improve the stability and durability of the biosensor, and enable the biosensor to perform multiple consecutive detections.
[0042] In this embodiment, the grafting rate of the cofactor on the cofactor grafted polymer is 5%-30%. On the one hand, the magnitude of the response current on the biosensor electrode will increase with the increase in the percentage of the cofactor on the cofactor grafted polymer. A too high response current will affect the activity and stability of the enzyme, and a too low response current is not suitable for the biosensor to receive signals; on the other hand, the percentage of the cofactor in the biocompatible membrane will affect the stability of the membrane structure. If the value is too high, the cofactor in the membrane layer will participate in the reaction more strongly, causing the membrane structure to be unstable, resulting in the enzyme layer in the membrane directly contacting the high concentration of glucose, thereby accelerating the inactivation of the enzyme. Therefore, the cofactor with the above grafting rate can not only accurately control the magnitude of the response current, so that the magnitude of the response current is within a reasonable range, but also ensure the stability of the membrane structure.
[0043] 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. The mass concentration of the cofactor grafted polymer makes the membrane quality better.
[0044] In addition, the organic solvent is one or more of ethanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide, preferably ethanol. The buffer is PBS buffer, HEPES buffer, TAPS buffer or MES buffer. The pH range of the buffer is 5.5 to 9. 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 organic solvents and buffers of the above types and volume ratios enable the cofactors to dissolve the grafted polymer evenly, thereby making the film-forming effect of the membrane solution better.
[0045] Furthermore, the environment in which the membrane solution is coated to form a biocompatible membrane is a solvent vapor atmosphere and a 100,000-level clean constant temperature and humidity environment, thereby making the quality of the 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 a film need to be carried out in this environment.
[0046] In this embodiment, the cofactor is one or more of NADP, NAD, NADH and NADPH, preferably NADP.
[0047] In addition, 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.
[0048] In another embodiment of the present application, a glucose sensor is provided, comprising an electrode, wherein the outer side of the sensing layer of the electrode is wrapped with the above-mentioned biocompatible film for biosensor. The glucose sensor has the advantages of high detection accuracy and good durability.
[0049] The thickness of the biocompatible membrane is 5 μm to 60 μm, and the biocompatible membrane with this thickness has the best restriction effect on glucose permeation.
[0050] 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 biocompatible membrane for 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 cofactor is grafted to the end of the side chain of the cofactor grafted polymer; The main chain of the cofactor grafted polymer is one or more of polyurethane, polyvinyl alcohol, polyvinyl alcohol derivatives, polyvinyl pyrrolidone, polyvinyl pyrrolidone derivatives, polyvinyl pyridine and polyvinyl pyridine derivatives; the side chain of the cofactor grafted polymer is one or more of acetic acid, butyric acid, ethyl butyrate, hexanoic acid and ethyl hexanoate.
2. The biocompatible film 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, polyvinyl pyrrolidone and polyvinyl pyridine.
3. The biocompatible film 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 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.
5. The biocompatible film for a biosensor according to claim 1, characterized in that: The organic solvent is one or more of ethanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide.
6. The biocompatible film for a biosensor according to claim 1, characterized in that: The buffer is PBS buffer, HEPES buffer, TAPS buffer or MES buffer.
7. The biocompatible film for a biosensor according to claim 1, characterized in that: The volume proportion of the organic solvent in the total volume of the organic solvent and the buffer solution is 50% to 97%; the volume proportion of the buffer solution in the total volume of the organic solvent and the buffer solution is 3% to 50%.
8. The 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.
9. The biocompatible film for a biosensor according to claim 8, characterized in that: The environment in which the membrane solution is coated to form the biocompatible membrane is a solvent vapor atmosphere and a 100,000-grade clean constant temperature and humidity environment.
10. The biocompatible film for a biosensor according to claim 1, characterized in that: 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 film for biosensor as claimed in claim 10.
12. The glucose sensor according to claim 11, characterized in that: The biocompatible film has a thickness of 5 μm to 60 μm.