Biosensor, biosensor outer membrane as well as preparation method and application of biosensor outer membrane

Abstract

The invention relates to a biosensor, an outer membrane of the biosensor and a preparation method and application of the outer membrane. The outer membrane of the biosensor comprises a polymer molecule with a thiourea group, and the polymer molecule is obtained by reacting and polymerizing a polymer containing a pyridine group and a monomer containing the thiourea group, that is, the thiourea group of the monomer is combined with the pyridine group in the polymer through the action of a chemical bond; the outer membrane of the biosensor is provided with a thiourea group, so that the thiourea group is utilized to generate an adsorption effect on silver ions, seepage of the silver ions in the reference electrode is prevented, the biocompatibility of the biosensor is improved, and the toxicity is reduced.

Description

Technical Field

[0001] The present invention relates to the field of sensors, and in particular to a biosensor, an outer membrane of the biosensor, and a preparation method and application thereof. Background Art

[0002] Biosensors are a field of technology that has developed rapidly and has been actively researched in recent years. Sensors include enzymes, Microorganisms, animal and plant tissues, organelles, antigens, antibodies and other biologically active materials, after the substance to be detected enters the sensor, the molecules recognize and then undergo a biological reaction and generate information, which is converted into sound, light, electricity and other signals by chemical transducers or physical transducers. The instrument outputs the signal and can test the concentration of the substance to be detected. Biosensors are playing an increasingly important role in the medical field due to their specificity, high sensitivity and fast response, providing a new and fast method for basic medical research and clinical diagnosis.

[0003] As a biosensor that collects physiological information of the human body, it must contact the human body or even enter the human body, so it must have good biocompatibility and specific detection of the target substance. Glucose sensor is a biosensor that has been studied a lot, developed more maturely and commercialized. At present, the service life of most sensors is 14 days. In order to further increase the service life of the sensor, it is necessary to improve the biocompatibility layer of the sensor. For the structure of the sensor and the analysis of the results of the combined cytotoxicity test, an important factor affecting the biocompatibility of the sensor is the leakage of silver ions in the reference electrode. Excessive silver ions have certain biological toxicity. Summary of the invention

[0004] Based on this, it is necessary to provide a biosensor with improved biocompatibility and reduced toxicity, an outer membrane of the biosensor and a preparation method thereof.

[0005] In addition, an application of a biosensor is also provided.

[0006] The outer membrane of a biosensor comprises polymer molecules with thiourea groups, wherein the polymer molecules are obtained by polymerizing a polymer containing a pyridine group and a monomer containing a thiourea group.

[0007] In one embodiment, the polymer containing pyridine groups is selected from one or more of poly(4-vinylpyridine-co-styrene), poly(4-vinylpyridine-co-butyl methacrylate) and poly(4-vinylpyridine); and the monomer is selected from monothiocarbazide and / or bisthiocarbazide.

[0008] In one embodiment, the monomer is selected from one or more of thiourea, 1-propylthiourea, 1-butylthiourea, N-benzylthiourea, anilinothiourea, phenylthiourea, cyclopentylthiourea, methylthiourea, guanylthiourea, ethylthiourea, isopropylthiourea, allylthiourea, semicarbazide and tert-butylthiourea.

[0009] In one embodiment, the molar ratio of the monomer containing thiourea groups to the pyridine groups in the polymer containing pyridine groups is 1:100-30:100.

[0010] A method for preparing a biosensor outer membrane comprises the following steps: Dissolving a polymer containing a pyridine group using an amide solvent to obtain a polymer solution; Adding a bromine-terminated carboxylic acid to the polymer solution to modify the polymer so that the polymer contains a carboxyl group; Adding a monomer containing a thiourea group to a modified polymer solution to carry out a grafting reaction, purifying and drying the product obtained after the grafting reaction, and dissolving the dried product in an ethanol aqueous solution to obtain a grafted polymer solution; A cross-linking agent with epoxy ether end-capping having a functionality greater than two is added to the grafted polymer solution, and the implanted part of the sensor is dip-coated in the solution to form an outer membrane of the biosensor.

[0011] In one embodiment, the polymer containing pyridine groups is selected from one or more of poly(4-vinylpyridine-co-styrene), poly(4-vinylpyridine-co-butyl methacrylate) and poly(4-vinylpyridine); the monomer is selected from monothiocarbazide and / or bisthiocarbazide; the bromine-terminated carboxylic acid is selected from one or more of 3-bromopropionic acid, 4-bromobutyric acid, 5-bromovaleric acid, 6-bromohexanoic acid, 8-bromooctanoic acid and 9-bromononanoic acid; the epoxy ether-terminated cross-linking agent with a functionality greater than two is selected from one or more of hydroxymethylpropane glycidyl ether, pentaerythritol glycidyl ether, glycerol glycol glycidyl ether and polyethylene glycol glycidyl ether.

[0012] In one embodiment, the amide solvent is N,N-dimethylformamide and / or N,N-dimethylacetamide; The conditions for modifying the polymer include: a reaction temperature of 60-120° C. and a reaction time of 12-72 hours.

[0013] In one embodiment, the grafting reaction satisfies any one or more of the following conditions: (1) The temperature of the grafting reaction is 20-50°C; (2) The grafting reaction time is 12-72 hours.

[0014] In one embodiment, the coating process is: dip coating with a solution having a solid content of 30-200 mg / ml, dipping 4-12 times, each dipping time being 2-20 seconds, and curing for 24-72 hours at a temperature of 37°C and a humidity of 20%-90% after dipping; wherein the solid content refers to the total mass of the cross-linking agent and the dried product in each milliliter of solution being 30-200 mg.

[0015] A method for preparing a biosensor outer membrane comprises the following steps: Mixing a cross-linking agent terminated with an epoxy ether having a functionality greater than three and a monomer containing a thiourea group to obtain a mixed solution; dissolving a polymer containing a pyridine group to obtain a polymer solution; After the mixed solution is added to the polymer solution, dip coating is performed to form the biosensor outer membrane.

[0016] In one embodiment, the polymer containing pyridine groups is selected from one or more of poly(4-vinylpyridine-co-styrene), poly(4-vinylpyridine-co-butyl methacrylate) and poly(4-vinylpyridine); the monomer is selected from monothiocarbazide and / or bisthiocarbazide; the epoxy ether-terminated cross-linking agent with a functionality greater than three is selected from one or more of hydroxymethylpropane glycidyl ether, pentaerythritol glycidyl ether, glycerol glycol glycidyl ether and polyethylene glycol glycidyl ether.

[0017] In one embodiment, the step of mixing a cross-linking agent having a functionality greater than trifunctional epoxy ether and a monomer containing a thiourea group to obtain a mixed solution comprises: The epoxy ether-terminated crosslinking agent with a functionality greater than three and the monomer containing a thiourea group are dissolved in a first solvent and a second solvent respectively, and the mixture is stirred and mixed for 8-24 hours to obtain the mixed solution; wherein the first solvent and the second solvent are both ethanol solutions, and the volume ratio of water to ethanol in the ethanol solution is 1:3.

[0018] In one embodiment, the coating process is: dip coating with a solution having a solid content of 30-200 mg / ml, dipping 4-12 times, each dipping time being 2-20 seconds, and curing for 24-72 hours at a temperature of 37°C and a humidity of 20%-90% after dipping; wherein the solid content refers to the total mass of the cross-linking agent and the dried product in each milliliter of solution being 30-200 mg.

[0019] A biosensor comprises the outer membrane as described above, or comprises the outer membrane prepared by the above preparation method.

[0020] The above-mentioned biosensor is used in detecting glucose, lactic acid, ketone bodies, alcohol or uric acid.

[0021] The outer membrane of the above-mentioned biosensor includes a polymer molecule with a thiourea group, which is obtained by polymerizing a polymer containing a pyridine group and a monomer containing a thiourea group, that is, the thiourea group of the monomer is combined with the pyridine group in the polymer through the action of a chemical bond, so that the outer membrane of the biosensor carries a thiourea group, thereby utilizing the thiourea group to produce an adsorption effect on silver ions, preventing the leakage of silver ions in the reference electrode, thereby improving the biocompatibility of the biosensor and reducing toxicity.

[0022] The above-mentioned preparation method of the outer membrane of the biosensor makes the outer membrane of the biosensor carry thiourea groups by cross-linking or grafting, so that the thiourea groups can be used to adsorb silver ions, prevent the silver ions in the reference electrode from leaking out, and improve the biocompatibility of the biosensor. In addition, the preparation method is simple and easy to operate, and can be applied to different types of outer membrane layers, thereby providing the biocompatibility of various types of biosensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a process flow chart of a method for preparing an outer membrane of a biosensor according to an embodiment; Figure 2 The present invention is a process flow chart of a method for preparing an outer membrane of a biosensor according to another embodiment. DETAILED DESCRIPTION

[0024] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below in conjunction with the specific embodiments. Preferred embodiments of the present invention are provided in the specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0026] In the present invention, "one or several" refers to any one, any two or any two or more of the listed items. Among them, "several" refers to any two or any two or more.

[0027] In the present invention, the percentage concentration involved, unless otherwise specified, refers to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0028] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0029] When a numerical range is disclosed in the present invention, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0030] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0031] The terms "including" and "having" and any variations thereof in the embodiments of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or device that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units that are not listed, or may optionally include other steps or components that are inherent to these processes, methods, products, or devices.

[0032] Reference to "embodiments" in the present invention means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described in the present invention may be combined with other embodiments.

[0033] See also Figure 1 A method for preparing a biosensor outer membrane according to an embodiment of the present invention comprises the following steps: Step S110: dissolving the polymer containing pyridine groups using an amide solvent to obtain a polymer solution.

[0034] Wherein, the polymer containing pyridine groups is selected from one or more of poly(4-vinylpyridine-co-styrene), poly(4-vinylpyridine-co-butyl methacrylate) and poly(4-vinylpyridine). The amide solvent is N,N-dimethylformamide and / or N,N-dimethylacetamide. The pyridine group is introduced to facilitate the subsequent introduction of the thiourea group. In this embodiment, the polymer containing pyridine groups is poly(4-vinylpyridine-co-styrene), and the dimethylamide is N,N-dimethylformamide. When using dimethylamide to dissolve poly(4-vinylpyridine-co-styrene), the dissolution can be carried out at room temperature.

[0035] Step S120: adding bromine-terminated carboxylic acid to the polymer solution to modify the polymer so that the polymer contains carboxyl groups.

[0036] Wherein, the bromine-terminated carboxylic acid is selected from one or more of 3-bromopropionic acid, 4-bromobutyric acid, 5-bromovaleric acid, 6-bromohexanoic acid, 8-bromooctanoic acid and 9-bromononanoic acid. In this embodiment, the bromine-terminated carboxylic acid is 4-bromobutyric acid. The conditions for modifying the polymer include: a reaction temperature of 60-120°C and a reaction time of 12-72 hours. Preferably, the reaction temperature is 80-100°C and the reaction time is 20-36 hours. The amount of the bromine-terminated carboxylic acid is a molar ratio of 1:1 to the subsequently added thiourea.

[0037] Specifically, after the polymer containing pyridine groups is completely dissolved in an amide solvent in step S110, a bromine-terminated carboxylic acid is added to the polymer solution, and the reaction is carried out at 60-120° C. for 12-72 hours, and a nucleophilic substitution reaction occurs, and the polymer containing pyridine groups is connected to the bromine-terminated carboxylic acid, so that the polymer containing pyridine groups has a carboxyl group. Among them, the nitrogen in the pyridine group and the bromine atom in the bromine-terminated carboxylic acid form a nitrogen cation and a bromine anion, respectively, and the two are bonded by an ionic bond, thereby achieving the connection of the polymer containing pyridine groups to the bromine-terminated carboxylic acid.

[0038] Step S130: adding a monomer containing a thiourea group to the modified polymer solution to carry out a grafting reaction, purifying and drying the product obtained after the grafting reaction, and dissolving the dried product in an ethanol aqueous solution to obtain a polymer solution.

[0039] Specifically, after the modification reaction in step S120, a monomer containing a thiourea group is added to the modified polymer solution, and the reaction is continued at 20-50° C. for 12-72 hours to perform a grafting reaction, and the amino group contained in the monomer undergoes a condensation reaction with the carboxyl group in the modified polymer, thereby grafting the monomer containing the thiourea group onto the side chain of the modified polymer. Next, the product after the grafting reaction is purified and dried to obtain a powder of the polymer after the grafting reaction, and then the powder is dissolved in an ethanol aqueous solution to obtain a polymer solution. Preferably, the volume ratio of water to ethanol in the ethanol aqueous solution is 1:3.

[0040] In one embodiment, the monomer containing the thiourea group is selected from monothiocarbazide and / or bisthiocarbazide. Preferably, the monomer containing the thiourea group is selected from one or more of thiourea, 1-propylthiourea, 1-butylthiourea, N-benzylthiourea, anilinothiourea, phenylthiourea, cyclopentylthiourea, methylthiourea, guanylthiourea, ethylthiourea, isopropylthiourea, allylthiourea, thiocarbazide and tert-butylthiourea.

[0041] Further, in one embodiment, the molar ratio of the monomer containing thiourea groups to the pyridine groups in the polymer containing pyridine groups is 1:100-30:100. Preferably, the molar ratio of the monomer containing thiourea groups to the pyridine groups in the polymer containing pyridine groups is 20:100.

[0042] Step S140: adding a cross-linking agent terminated with epoxy ether having a functionality greater than two into the purified solution, and dipping the implanted part of the sensor into the solution to form an outer membrane of the biosensor.

[0043] Specifically, a cross-linking agent terminated with epoxy ether having a functionality greater than two is added to the purified solution obtained in step S130 to obtain a coating solution, and then the implanted part of the sensor is dip-coated in the coating solution, thereby forming a biosensor outer film on the outer surface of the implanted part, so that the outer surface of the sensor has thiourea groups. The cross-linking agent terminated with epoxy ether having a functionality greater than two can be selected from one or more of hydroxymethylpropane glycidyl ether, pentaerythritol glycidyl ether, glycerol glycol glycidyl ether and polyethylene glycol glycidyl ether.

[0044] In one embodiment, the coating process is: dip coating with a solution having a solid content of 30-200 mg / ml, dipping 4-12 times, each dipping time being 2-20 seconds, and curing for 24-72 hours at a temperature of 37°C and a humidity of 20%-90% after dipping; wherein the solid content refers to the total mass of the cross-linking agent and the dried product in each milliliter of solution being 30-200 mg.

[0045] The above-mentioned method for preparing the outer membrane of the biosensor has at least the following advantages: (1) By grafting a monomer containing a thiourea group onto the side chain of a polymer containing a pyridine group, the outer membrane of the biosensor has a thiourea group. The adsorption of silver ions by the thiourea group is then utilized to prevent the silver ions from leaking out of the electrode, thereby improving the biocompatibility of the outer membrane of the biosensor.

[0046] See also Figure 2 Another embodiment of the method for preparing the outer membrane of the biosensor comprises the following steps: Step S210: Mixing a cross-linking agent terminated with an epoxy ether having a functionality greater than three and a monomer containing a thiourea group to obtain a mixed solution.

[0047] Optionally, a first solvent and a second solvent are used to dissolve a crosslinker with a functional group greater than three and a monomer containing a thiourea group, respectively, and the mixture is stirred for 8-24 hours to obtain a mixed solution; wherein the first solvent and the second solvent are both ethanol solutions, and the volume ratio of water to ethanol in the ethanol solution is 1:3. When the crosslinker is mixed with the monomer containing a thiourea group, the amino group on the monomer undergoes a ring-opening addition reaction with the epoxy group in the crosslinker.

[0048] It should be noted that the first solvent and the second solvent may also be ethyl acetate, etc., and the volume ratio of water to ethanol in the ethanol solution may also be 1:3, 1:5, etc. Wherein, the epoxy ether-terminated crosslinking agent having a functionality greater than three is selected from one or more of hydroxymethylpropane glycidyl ether, pentaerythritol glycidyl ether, and glycerol glycol glycidyl ether. The monomer containing the thiourea group is selected from one or more of monosemicarbazide and / or bis-semicarbazide. Preferably, the monomer containing the thiourea group is selected from one or more of thiourea, 1-propylthiourea, 1-butylthiourea, N-benzylthiourea, anilinothiourea, phenylthiourea, cyclopentylthiourea, methylthiourea, guanylthiourea, ethylthiourea, isopropylthiourea, allylthiourea, thiosemicarbazide and tert-butylthiourea.

[0049] S220: dissolving the polymer containing the pyridine group to obtain a polymer solution.

[0050] Wherein, the polymer containing pyridine groups is selected from one or more of poly (4-vinylpyridine-co-styrene), poly (4-vinylpyridine-co-butyl methacrylate) and poly (4-vinylpyridine). The solvent used for dissolution can be dimethylamide, and further, the dimethylamide can be N, N-dimethylformamide and / or N, N-dimethylacetamide. The pyridine group is introduced to facilitate the subsequent introduction of the thiourea group. In the present embodiment, the polymer containing pyridine groups is poly (4-vinylpyridine-co-styrene), and the dimethylamide is N, N-dimethylformamide. When using dimethylamide to dissolve poly (4-vinylpyridine-co-styrene), it can be dissolved at room temperature.

[0051] Further, in one embodiment, the molar ratio of the thiourea group-containing monomer to the pyridine group in the pyridine group-containing polymer is 1:100-30:100. Preferably, the molar ratio of the thiourea group in the thiourea group-containing monomer to the pyridine group in the pyridine group-containing polymer is 20:100.

[0052] S230: After adding the mixed solution to the polymer solution, dip coating is performed to form an outer membrane of the biosensor.

[0053] Specifically, the mixed solution obtained in step S210 is added to the polymer solution obtained in step S220 to form a new mixed solution, and then the implanted part of the sensor is dipped in the new mixed solution to form a biosensor outer film on the outer surface of the implanted part, so that the outer surface of the sensor has thiourea groups.

[0054] The above-mentioned method for preparing the outer membrane of the biosensor has at least the following advantages: By cross-linking, the outer membrane of the biosensor has thiourea groups, and then the adsorption of silver ions by the thiourea groups is used to prevent the silver ions from leaking out of the electrode, thereby improving the biocompatibility of the outer membrane of the biosensor. In addition, the cross-linking method is simple and easy to operate.

[0055] The present invention provides an outer membrane of a biosensor according to an embodiment, comprising: a polymer molecule having a thiourea group, wherein the polymer molecule is obtained by polymerization of a polymer containing a pyridine group and a monomer containing a thiourea group.

[0056] Wherein, the polymer containing pyridine groups is selected from one or more of poly(4-vinylpyridine-co-styrene), poly(4-vinylpyridine-co-butyl methacrylate) and poly(4-vinylpyridine). The monomer containing thiourea groups is selected from monosemicarbazide and / or bissemicarbazide. Preferably, the monomer containing thiourea groups is selected from one or more of thiourea, 1-propylthiourea, 1-butylthiourea, N-benzylthiourea, anilinothiourea, phenylthiourea, cyclopentylthiourea, methylthiourea, guanylthiourea, ethylthiourea, isopropylthiourea, allylthiourea, thiosemicarbazide and tert-butylthiourea. The molar ratio of the monomer containing thiourea groups to the pyridine groups in the polymer containing pyridine groups is 1:100-30:100. Preferably, the molar ratio of the monomer containing thiourea groups to the pyridine groups in the polymer containing pyridine groups is 20:100.

[0057] Specifically, the outer membrane of the biosensor can be prepared by the preparation method of the above embodiment, which will not be described in detail.

[0058] The outer membrane of the above-mentioned biosensor includes a polymer molecule with a thiourea group, which is obtained by polymerizing a polymer containing a pyridine group and a monomer containing a thiourea group, that is, the thiourea group of the monomer is combined with the pyridine group in the polymer through the action of a chemical bond, so that the outer membrane of the biosensor carries a thiourea group, thereby utilizing the thiourea group to produce an adsorption effect on silver ions, preventing the leakage of silver ions in the reference electrode, thereby improving the biocompatibility of the biosensor and reducing toxicity.

[0059] The present invention provides a biosensor according to an embodiment, comprising the outer membrane or the outer membrane obtained by the above preparation method. The biosensor has good biocompatibility.

[0060] The present invention also provides an application of a biosensor according to an embodiment of the present invention in detecting glucose, lactic acid, ketone bodies, alcohol or uric acid.

[0061] The above biosensor has good biocompatibility and can be used to detect substances such as glucose, lactic acid, ketone bodies, alcohol or uric acid by adjusting the types of biological enzymes in the biosensor.

[0062] In order to make the purpose and advantages of the present invention clearer, the biosensor of the present invention and its effects are further described in detail in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and shall not be used to limit the present invention. The following embodiments do not include other components except inevitable impurities unless otherwise specified. The drugs and instruments used in the embodiments are conventionally selected in the art unless otherwise specified. The experimental methods for which specific conditions are not specified in the embodiments are implemented according to conventional conditions, such as the conditions described in the literature, books, or methods recommended by the manufacturer.

[0063] Example 1 This embodiment provides an outer membrane of a biosensor, and the specific preparation process thereof is as follows: (1) Poly(4-vinylpyridine-co-styrene) is dissolved in N,N-dimethylformamide at room temperature, wherein the mass of poly(4-vinylpyridine-co-styrene) is 10 g and the mass of N,N-dimethylformamide is 100 ml.

[0064] (2) 0.28 g of 4-bromobutyric acid was added to the polymer solution obtained in step (1), and the mixture was reacted at 110° C. for 48 hours.

[0065] (3) Add 0.15 g of methylthiourea to the modified polymer solution obtained in step (2), react at 45° C. for 24 hours to carry out a grafting reaction, and use an ether solution to precipitate the polymer obtained after the grafting reaction. After removing small molecular impurities, place it in a vacuum oven at 50° C. and dry it for 24 hours. Finally, dissolve the dried product in an ethanol aqueous solution (the volume ratio of water to ethanol is 1:3) to obtain a polymer solution with a solid content of 100 mg / ml. The molar ratio of the monomer containing thiourea groups to the pyridine groups in the polymer containing pyridine groups is 1:100.

[0066] (4) Add the cross-linking agent polyethylene glycol glycidyl ether to an ethanol aqueous solution (the volume ratio of ethanol to water is 3:1) to prepare a solution with a solid content of 100 mg / ml, add it to the polymer solution prepared in step (3) to obtain a dip coating solution, immerse the implanted part of the sensor in the dip coating solution for 6 times, each time for 4 seconds. After the dip coating is completed, cure at a temperature of 37°C and a humidity of 50% for 24 hours.

[0067] Example 2 This embodiment provides an outer membrane of a biosensor, and the specific preparation process thereof is similar to that of Embodiment 1, except that the molar ratio of the monomer containing thiourea groups to the pyridine groups in the polymer containing pyridine groups is 5:100.

[0068] Example 3 This embodiment provides an outer membrane of a biosensor, and the specific preparation process thereof is similar to that of Embodiment 1, except that the molar ratio of the monomer containing thiourea groups to the pyridine groups in the polymer containing pyridine groups is 10:100.

[0069] Example 4 This embodiment provides an outer membrane of a biosensor, and the specific preparation process thereof is similar to that of Embodiment 1, except that the molar ratio of the monomer containing thiourea groups to the pyridine groups in the polymer containing pyridine groups is 20:100.

[0070] Example 5 This embodiment provides an outer membrane of a biosensor, and the specific preparation process thereof is similar to that of Embodiment 1, except that the molar ratio of the monomer containing thiourea groups to the pyridine groups in the polymer containing pyridine groups is 30:100.

[0071] Comparative Example 1 Comparative Example 1 provides a biosensor, and its specific preparation process is as follows: (1) Poly(4-vinylpyridine-co-styrene) was dissolved in an ethanol aqueous solution (the volume ratio of ethanol to water was 3:1) at room temperature, wherein the mass of poly(4-vinylpyridine-co-styrene) was 10 g and the volume of the ethanol aqueous solution was 100 ml.

[0072] (2) Add the crosslinking agent polyethylene glycol glycidyl ether to an ethanol aqueous solution (the volume ratio of ethanol to water is 3:1) to prepare a solution with a solid content of 100 mg / ml, add it to the solution prepared in step (1) to obtain a dip coating solution, immerse the implanted part of the sensor in the dip coating solution for 6 times, each time for 4 seconds. After the dip coating is completed, cure at a temperature of 37°C and a humidity of 50% for 24 hours.

[0073] Example 6 (1) Weigh 0.76 g of hydroxymethylpropane glycidyl ether and 0.1 g of thiourea and dissolve them in an ethanol aqueous solution (the volume ratio of water to ethanol is 1:3), and stir the two dissolved solutions for 8 hours to obtain a mixed solution with a solid content of 100 mg / ml.

[0074] (2) Poly(4-vinylpyridine-co-styrene) was dissolved in an ethanol aqueous solution (the volume ratio of water to ethanol was 1:3) at room temperature. Finally, the solid content of poly(4-vinylpyridine-co-styrene) was 100 mg / ml.

[0075] (3) Add the mixed solution obtained in step (1) to the polymer solution obtained in step (2) to obtain a dipping solution, and immerse the implanted part of the sensor in the dipping solution for 6 times, each time for 4 seconds. After the dipping is completed, cure at a temperature of 37° C. and a humidity of 50% for 24 hours. The final molar ratio of the monomer containing thiourea groups to the pyridine groups in the polymer containing pyridine groups is 5:100.

[0076] Example 7 This embodiment provides an outer membrane of a biosensor, and its specific preparation process is similar to that of Embodiment 5, except that the molar ratio of the monomer containing thiourea groups to the pyridine groups in the polymer containing pyridine groups is 10:100; and the dip coating is performed 4 times.

[0077] Example 8 This embodiment provides an outer membrane of a biosensor, and its specific preparation process is similar to that of Embodiment 5, except that the molar ratio of the monomer containing thiourea groups to the pyridine groups in the polymer containing pyridine groups is 10:100.

[0078] Example 9 This embodiment provides an outer membrane of a biosensor, and its specific preparation process is similar to that of Embodiment 5, except that the molar ratio of the monomer containing thiourea groups to the pyridine groups in the polymer containing pyridine groups is 20:100.

[0079] Comparative Example 2 (1) 1 g of a cross-linking agent, hydroxymethylpropane glycidyl ether, was dissolved in an ethanol aqueous solution (the volume ratio of water to ethanol was 1:3) to obtain a cross-linking agent solution having a solid content of 100 m / ml.

[0080] (2) Poly(4-vinylpyridine-co-styrene) was dissolved in an ethanol aqueous solution (the volume ratio of water to ethanol was 1:3) at room temperature. Finally, the solid content of poly(4-vinylpyridine-co-styrene) was 100 mg / ml.

[0081] (3) Add the solution obtained in step (1) to the polymer solution obtained in step (2) to obtain a dip coating solution, and immerse the implanted part of the sensor in the dip coating solution for 6 times, each time for 4 seconds. After the dip coating is completed, cure at a temperature of 37° C. and a humidity of 50% for 24 hours.

[0082] The biosensor outer membrane prepared in the above embodiment was tested, and the testing method was as follows: 1. Silver ion concentration test The prepared sensors were immersed in PBS (10 sensors were immersed in 10 ml solution) for 14 days (temperature 37 degrees Celsius, constant temperature air bath shaker 50 rpm / min). After the expiration, the sensors were taken out and the silver ion concentration was tested in the immersed solution. The instrument used was inductively coupled plasma mass spectrometry (ICP-MS). During the test, the silver ion standard solution was first used for linear calibration, and then a certain amount of the test solution was taken for testing.

[0083] 2. In vitro cytotoxicity test In vitro cytotoxicity assay was performed using the MTT assay.

[0084] The implanted parts of the sensors prepared in the above Examples 1-9 and Comparative Examples 1-2 were tested for silver ion concentration, and Example 4, Example 8 and Comparative Examples 1-2 were tested for in vitro cytotoxicity. The specific results are shown in Tables 1 and 2.

[0085] Table 1

[0086] Table 2

[0087] As shown in Table 1 and Table 2, by adopting the grafting or cross-linking method, the higher the amount of thiourea groups introduced, the more silver ions adsorbed, which has a good effect on preventing the leakage of silver ions from the electrode. From Comparative Examples 1 and 2, the thiourea groups were not introduced, and the silver ion concentration test showed that the concentration was high, indicating that the silver ion adsorption effect was not good, thus indicating that the introduction of thiourea groups played a good role in the adsorption of silver ions. In addition, from the in vitro cytotoxicity test results of Example 4 and Comparative Example 1, and Example 9 and Comparative Example 2, the toxicity obtained by the in vitro cell test of Example 4 and Example 9 was low, thus indicating that the introduction of thiourea groups improved the biocompatibility of the sensor.

[0088] Table 3

[0089] In the first column of Table 3, 0, 2.2, 11, 18, and 15 represent the concentrations of glucose, respectively. The data corresponding to the columns of Examples 1 to 9, and Comparative Examples 1 and 2 are the current values ​​measured at the corresponding glucose concentrations. R is the linear correlation coefficient, and the response value unit is nA / mmol.

[0090] As shown in Table 3, combined with the response values ​​and linear correlation coefficients R of Examples 1-5, as the thiourea content increases, the response value becomes higher and higher, but the R value decreases after reaching a certain value. When the thiourea content exceeds 20, the R value decreases instead. This is because after the thiourea content reaches a certain value, the resulting polymer has hydrophilicity, which leads to poor film-forming properties. From Example 5, when the thiourea grafting rate reaches 30%, the film-forming properties are poor, resulting in poor sensor linearity (please refer to the R values ​​of Examples 4 and 5), which in turn affects the sensor performance. In addition, for Examples 6-9, similarly, when the thiourea content reaches a certain value, the sensor linearity deteriorates.

[0091] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0092] The above-described embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solutions of the present invention in detail, but cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to interpret the contents of the claims.

Claims

1. An outer membrane of a biosensor, characterized in that: The invention comprises a polymer molecule having a thiourea group, wherein the polymer molecule is obtained by polymerizing a polymer containing a pyridine group and a monomer containing a thiourea group.

2. The outer film according to claim 1, characterized in that The polymer containing pyridine groups is selected from one or more of poly(4-vinylpyridine-co-styrene), poly(4-vinylpyridine-co-butyl methacrylate) and poly(4-vinylpyridine); and the monomer is selected from monosemicarbazide and / or bissemicarbazide.

3. The outer film according to claim 1, characterized in that The monomer is selected from one or more of thiourea, 1-propylthiourea, 1-butylthiourea, N-benzylthiourea, anilinothiourea, phenylthiourea, cyclopentylthiourea, methylthiourea, guanylthiourea, ethylthiourea, isopropylthiourea, allylthiourea, semicarbazide and tert-butylthiourea.

4. The outer film according to claim 1, characterized in that The molar ratio of the monomer containing thiourea groups to the pyridine groups in the polymer containing pyridine groups is 1:100-30:

100.

5. A method for preparing a biosensor outer membrane, characterized in that: The following steps are involved: Dissolving a polymer containing a pyridine group using an amide solvent to obtain a polymer solution; Adding a bromine-terminated carboxylic acid to the polymer solution to modify the polymer so that the polymer contains a carboxyl group; Adding a monomer containing a thiourea group to a modified polymer solution to carry out a grafting reaction, purifying and drying the product obtained after the grafting reaction, and dissolving the dried product in an ethanol aqueous solution to obtain a grafted polymer solution; A cross-linking agent with epoxy ether end-capping having a functionality greater than two is added to the grafted polymer solution, and the implanted part of the sensor is dip-coated in the solution to form an outer membrane of the biosensor.

6. The method for preparing the outer membrane of the biosensor according to claim 5, characterized in that: The polymer containing pyridine groups is selected from one or more of poly(4-vinylpyridine-co-styrene), poly(4-vinylpyridine-co-butyl methacrylate) and poly(4-vinylpyridine); the monomer is selected from monothiocarbazide and / or bisthiocarbazide; the bromine-terminated carboxylic acid is selected from one or more of 3-bromopropionic acid, 4-bromobutyric acid, 5-bromovaleric acid, 6-bromohexanoic acid, 8-bromooctanoic acid and 9-bromononanoic acid; the epoxy ether-terminated crosslinking agent with a functionality greater than two is selected from one or more of hydroxymethylpropane glycidyl ether, pentaerythritol glycidyl ether, glycerol glycol glycidyl ether and polyethylene glycol glycidyl ether.

7. The method for preparing the biosensor outer membrane according to claim 5, characterized in that: The amide solvent is N,N-dimethylformamide or N,N-dimethylacetamide; The conditions for modifying the polymer include: a reaction temperature of 60-120° C. and a reaction time of 12-72 hours.

8. The method for preparing the outer membrane of a biosensor according to claim 5, characterized in that: The grafting reaction satisfies any one or more of the following conditions: (1) The temperature of the grafting reaction is 20-50°C; (2) The grafting reaction time is 12-72 hours.

9. The method for preparing the outer membrane of a biosensor according to claim 5, characterized in that: The coating process is: dip coating with a solution having a solid content of 30-200 mg / ml, dipping 4-12 times, each dipping time of 2-20 seconds, and curing for 24-72 hours at a temperature of 37° C. and a humidity of 20%-90% after dipping; wherein the solid content refers to the total mass of the cross-linking agent and the dried product in each milliliter of solution being 30-200 mg.

10. A method for preparing a biosensor outer membrane, characterized in that: The following steps are involved: Mixing a cross-linking agent terminated with an epoxy ether having a functionality greater than three and a monomer containing a thiourea group to obtain a mixed solution; dissolving a polymer containing a pyridine group to obtain a polymer solution; After the mixed solution is added to the polymer solution, dip coating is performed to form the biosensor outer membrane.

11. The method for preparing the outer membrane of a biosensor according to claim 10, characterized in that: The polymer containing pyridine groups is selected from one or more of poly(4-vinylpyridine-co-styrene), poly(4-vinylpyridine-co-butyl methacrylate) and poly(4-vinylpyridine); the monomer is selected from monothiosemicarbazide and / or bisthiosemicarbazide; the epoxy ether-terminated cross-linking agent with a functionality greater than three is selected from one or more of hydroxymethylpropane glycidyl ether, pentaerythritol glycidyl ether, glycerol glycol glycidyl ether and polyethylene glycol glycidyl ether.

12. The method for preparing the outer membrane of a biosensor according to claim 10, characterized in that: The step of mixing a cross-linking agent having a functionality greater than three and a monomer containing a thiourea group to obtain a mixed solution comprises: The epoxy ether-terminated crosslinking agent with a functionality greater than three and the monomer containing a thiourea group are dissolved in a first solvent and a second solvent respectively, and the mixture is stirred and mixed for 8-24 hours to obtain the mixed solution; wherein the first solvent and the second solvent are both ethanol solutions, and the volume ratio of water to ethanol in the ethanol solution is 1:

3.

13. The method for preparing the outer membrane of a biosensor according to claim 9, characterized in that: The coating process is: dip coating with a solution having a solid content of 30-200 mg / ml, dipping 4-12 times, each dipping time of 2-20 seconds, and curing for 24-72 hours at a temperature of 37° C. and a humidity of 20%-90% after dipping; wherein the solid content refers to the total mass of the cross-linking agent and the dried product in each milliliter of solution being 30-200 mg.

14. A biosensor, comprising the outer membrane according to any one of claims 1 to 4, or the outer membrane prepared by the preparation method according to any one of claims 5 to 9, or the outer membrane prepared by the preparation method according to any one of claims 10 to 13.

15. Use of the biosensor according to claim 14 in detecting glucose, lactic acid, ketone bodies, alcohol or uric acid.

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