Glucose oxidase nano capsule sensor and preparation and application thereof
阅读说明：本技术 一种葡萄糖氧化酶纳米胶囊传感器及其制备和应用 (Glucose oxidase nano capsule sensor and preparation and application thereof ) 是由 卢宪波 郸嘉 陈吉平 于 2019-02-28 设计创作，主要内容包括：一种葡萄糖氧化酶纳米胶囊传感器及其制备和应用,酶基生物传感器在生物医疗、生命健康、食品安全、环境安全、食品发酵等领域具有广阔的应用前景,目前限制其商品化的主要因素是酶传感器的稳定性有待进一步提高。本发明提供了一种高稳定性酶传感器的制备方法及应用,即基于酶分子纳米胶囊制备酶传感器的方法。该酶纳米胶囊传感器的制备主要包括以下步骤：首先,利用原位自由基聚合法制备酶单分子纳米胶囊(或酶多分子纳米胶囊)；然后将上述酶分子纳米胶囊用于制备酶纳米胶囊传感器。该酶传感器的优势在于其热稳定性、有机溶剂耐受性、存储和使用寿命等得到了明显提高。本发明所得到的酶纳米胶囊传感器在人体可穿戴设备、生物医疗、分析检测、食品发酵等多个领域具有巨大市场应用潜力。(A glucose oxidase nano-capsule sensor and preparation and application thereof are provided, the enzyme-based biosensor has wide application prospect in the fields of biological medical treatment, life health, food safety, environmental safety, food fermentation and the like, and the main factor for limiting the commercialization of the enzyme-based biosensor at present is that the stability of the enzyme sensor needs to be further improved. The invention provides a preparation method and application of a high-stability enzyme sensor, namely a method for preparing the enzyme sensor based on an enzyme molecule nanocapsule. The preparation method of the enzyme nanocapsule sensor mainly comprises the following steps: firstly, preparing an enzyme monomolecular nanocapsule (or an enzyme polymolecular nanocapsule) by using an in-situ free radical polymerization method; and then the enzyme molecule nanocapsule is used for preparing the enzyme nanocapsule sensor. The enzyme sensor has the advantages that the thermal stability, organic solvent tolerance, storage and service life and the like are obviously improved. The enzyme nanocapsule sensor obtained by the invention has huge market application potential in a plurality of fields such as human body wearable equipment, biomedical treatment, analysis and detection, food fermentation and the like.)
1. A biosensor, wherein the biosensor uses an enzyme nanocapsule as an enzyme electrode biorecognition molecule or as part of an enzyme electrode biorecognition element.
2. The biosensor of claim 1, wherein the enzyme nanocapsule is an enzyme monomolecular nanocapsule or an enzyme polymolecular nanocapsule.
3. The biosensor of claim 1, wherein the enzyme nanocapsules are less than 100nm in size; the enzyme nanocapsule comprises at least one enzyme molecule as or as part of an inner core; a layer of reticular polymer film is wrapped outside the inner core to be used as a shell; the thickness of the polymer film is 0.1 nm-100 nm, and the polymer film is formed by monomers or polymers in a covalent mode; the monomer or polymer contains at least one of carboxyl, amino, positive charge, negative charge or double bond groups.
4. The biosensor according to any one of claims 1 to 3, wherein the enzyme nanocapsule is a glucose oxidase nanocapsule.
5. A method for preparing the sensor of claim 4, wherein the method for preparing the biosensor comprises two steps:
the method comprises the following steps: mixing an electrode sensitization material and an electrode film forming material with ultrapure water to obtain a mixed solution, then dropwise adding, pouring or printing the mixed solution onto the surface of an electrode, and drying to obtain a sensor precursor; finally, dripping, printing or pouring a glucose oxidase nanocapsule (nGOx) solution on the sensor precursor, and drying to obtain the biosensor; the concentrations of the electrode sensitization material and the electrode film forming material in the mixed solution are both 0.05-20 mg mL-1Preferably 2mg mL-1(ii) a The dropping amount of the mixed solution is 0.1-3 mu L/mm2(ii) a The concentration of the glucose oxidase nano capsule solution is 0.1-30 mg mL-1Preferably 10mg mL-1(ii) a The dripping, printing or pouring amount of the nGOx solution on the sensor precursor is 0.1-3 mu L/mm2；
The second method comprises the following steps: mixing an electrode sensitization material, an electrode film forming material, a glucose oxidase nanocapsule (nGOx) and ultrapure water to obtain a mixed solution, and then dropwise adding, printing or pouring the mixed solutionInjecting the solution onto the surface of the electrode, and drying to obtain the biosensor; in the mixed solution, the concentrations of the electrode sensitization material and the electrode film forming material are both 0.0-20 mgmL-1Preferably 2mg mL-1(ii) a The concentration of nGOx in the mixed solution is 0.1-30 mg mL-1Preferably 5mg mL-1(ii) a The dropping amount of the mixed solution is 0.1-3 mu L/mm2；
The electrode sensitization material is a carbon nano material, graphite powder, an inert metal nano material, a transition metal oxide nano material or a composite material containing any one of the materials;
the electrode film-forming material is chitosan, gelatin, fibroin, sodium alginate, Nafion emulsion, and silica sol-gel or conductive adhesive.
6. The preparation method according to claim 5, wherein the mixed solution in the first and second methods further comprises a cosolvent, and the volume ratio of the cosolvent to water in the mixed solution is 0: 100-100: 0; the cosolvent is dimethylformamide, dimethyl sulfoxide, ethanol, a hydrophilic surfactant or hydrophilic ionic liquid.
7. The production method according to claim 5, wherein the electrode surface to which the mixed solution is dropped is subjected to polishing treatment and/or cleaning treatment, and then dried to obtain a clean electrode surface for standby.
8. The method of claim 5, wherein the electrode is a Glassy Carbon Electrode (GCE), an inert metal electrode, a conductive glass electrode, a graphite electrode, a screen-printed electrode, a test paper electrode, a flexible electrode; the metal electrode is a metal gold, platinum or silver electrode.
9. The method according to claim 5, wherein the carbon nanomaterial is a carbon nanotube, graphene, mesoporous carbon, carbon dot, or heteroatom-doped carbon nanomaterial; the hetero atoms are nitrogen, phosphorus, boron, oxygen and hydrogenA hydroxyl, carboxyl or quinonyl group; the inert metal in the inert metal nano material is gold, silver or platinum; the transition metal oxide in the transition metal oxide nano material is ZnO and Fe3O4、Co3O4、NiO、TiO2、MnO2、ZrO2。
10. Use of the sensor according to claim 4 for detecting glucose concentration in an aqueous solution, organic solvent or mixed solution, and for detecting glucose concentration in blood glucose, urine glucose or sweat glucose, body fluid glucose, food; the application is the application of the sensor in the fields of blood glucose test paper, wearable equipment, implantable equipment, flexible electrodes, food fermentation, enzyme chemical engineering and analysis and detection.
Technical Field
The invention belongs to the field of biosensors and analysis and detection, and particularly relates to a sensor based on enzyme nanocapsules and glucose oxidase nanocapsules, a preparation method of the sensor, and application of the sensor in the fields of glucose detection, human body wearable equipment and the like.
Background
Blood glucose testing is the basis for the diagnosis, treatment and management of diabetes. At present, the number of diabetes patients is more than 4.2 hundred million worldwide, and the number of diabetes patients in China only reaches 1.09 hundred million in 2015. The global market capacity for glucometers and glucose strips (i.e., enzyme-based electrochemical glucose sensors) is as high as $ 200 and billions. There are two main types of blood glucose test strips available on the market: one is the glucose oxidase (GOx) electrode measurement, which is dominant, and indicates blood glucose concentration by detecting changes in the concentration of oxygen in blood on a blood glucose test strip, and has advantages of specific selectivity for glucose, poor storage stability (generally within 3 months, the test strip is used within 5 minutes after being taken out of a container), and poor measurement reproducibility (about 15% error). The other method is glucose dehydrogenase electrode measurement method, which has the advantages of easy storage and good stability, and has the disadvantage that xylose, maltose, galactose and the like in blood can generate interference. During the year 2009 1997-2009, the U.S. Food and Drug Administration (FDA) received at least 13 fatal reports, and the U.S. FDA was once 3 consecutive times warning of defects, and blood glucose meters or dipsticks using glucose dehydrogenase (GDH-PQQ) technology may cause abnormal hypoglycemia, coma, and even death during treatment. To solve the problems of storage stability, measurement accuracy and repeatability of the blood glucose test strip (glucose sensor), many domestic and foreign scientists including many international companies are still making continuous efforts.
The problems with blood glucose strips are also some of the common problems with enzyme-based biosensors. From the domestic and foreign aspects, a main focus of enzyme-based biosensor research is focused on the key scientific problems of how to further improve the long-term stability, the measurement accuracy, the repeatability and the like of the sensor, and the key common problem of limiting the development of the enzyme-based biosensor is also provided. On a traditional enzyme electrode (including blood glucose test paper), enzymes are generally fixed on the enzyme electrode in a physical embedding or chemical crosslinking mode by using biocompatible polymers or nano materials, and the current situation that the enzymes as bioactive molecules are easy to inactivate is not effectively changed by a traditional enzyme electrode preparation method, so that how to further improve the stability and the service life of the enzyme molecules and the sensors is a first core problem to be solved, and the pursuit of researchers is always kept. In particular, with the rise of wearable devices in recent years, higher requirements are being placed on the stability of enzyme sensors. The traditional blood glucose test paper is disposable and disposable, and cannot meet the requirements of human body wearable equipment on the stability and reusability of an enzyme sensor. The stability of the enzyme sensor is improved, the problems of the traditional blood glucose test paper are solved, the requirements of human body wearable equipment on the stability and other performances of the sensor are met, and the method is urgent.
Organisms contain complex subcellular units, in eukaryotic cells, most enzymes do not diffuse freely in the cytosol, but are confined spatially to subcellular (organelle) cells, or together with other enzymes, into enzyme complexes. These enzyme molecules and enzyme complexes, which are spatially confined within subcellular and organelles, have higher activity than the free enzyme molecules (Wilner, o.i., et al., Nature nanotechnology 2009,
Compared with the traditional technology for preparing the sensor by using original enzyme (unmodified), the enzyme nanocapsule technology provides a favorable tool for maintaining the long-term stability, the thermal stability and the biological activity of biomolecules, is expected to greatly improve the key performances of the enzyme sensor, such as the thermal stability, the long-term storage stability, the service life, the measurement accuracy and the like, solves the key common scientific problem of limiting the development of the enzyme sensor field, and promotes the commercialization of more kinds of enzyme sensors. The literature reports a technology of immobilizing enzyme in porous inorganic nano materials such as mesoporous carbon, mesoporous silicon and the like to improve enzyme stability, and since the macroscopic scales of the two inorganic nano materials are large (the pores are nano-scale and micron-scale, but the three-dimensional size of the materials is micron or millimeter-scale), the water phase dispersibility of the enzyme immobilized by the inorganic nano materials is relatively poor, and the diffusion resistance of an enzyme substrate is also large. According to the enzyme nanocapsule technology, a thin-layer reticular polymer is formed on the surface of an enzyme, so that the enzyme nanocapsule technology has better dispersibility in a water phase, smaller diffusion resistance of an enzyme substrate and more importantly, the polymer has better biocompatibility to enzyme molecules than an inorganic nano material, and therefore the performances of the enzyme nanocapsule, such as thermal stability, organic solvent tolerance and the like, are remarkably superior to those reported in the previous literature (ChemSusChem,2012,5, 1918-1925; Chinese patent ZL 201110377608.2). The enzyme nanocapsule sensor obtained by the invention has great market application potential in a plurality of fields such as human body wearable equipment related to glucose detection, life health, analysis and detection, food and beverage fermentation and the like.
We searched for relevant documents and patents at home and abroad, and found no relevant document report and patent application of enzyme molecule nanocapsules, particularly glucose oxidase nanocapsules, for an enzyme sensor, although there are a single-protein nanocapsule for protein delivery having a long-term effect (patent application No. 201080020405.1), a patent application No. 201280043352.4 for oral delivery of an enzyme using nanocapsules to achieve targeted metabolism of alcohol or toxic metabolites, an antioxidant enzyme nanocapsule aerosol and application thereof in eliminating smoke radicals (application publication No. CN 104549085A).
Disclosure of Invention
The invention mainly aims to provide a preparation method and application of an enzyme sensor with higher stability for glucose detection. The method uses glucose oxidase nanocapsule (nGOx) as a biological recognition element of an enzyme sensor or a part of a biological recognition element, and the developed sensor can be used for detecting the concentration of glucose in an aqueous solution or an organic solvent and the detection of blood sugar, urine sugar, sweat glucose, body fluid glucose and the like. Compared with the traditional sensor based on original (native) glucolase molecules, the enzyme sensor based on the glucose oxidase nanocapsule has advantages in the aspects of thermal stability, storage life, service life, organic solvent tolerance and the like, and has outstanding advantages in the fields of traditional blood glucose detection and the like, particularly in the fields of human body wearable equipment and the like. The invention adopts the following technical scheme:
an aspect of the present invention provides a biosensor using an enzyme nanocapsule as an enzyme electrode biorecognition molecule or as a part of an enzyme electrode biorecognition element, the enzyme nanocapsule including a protein nanocapsule. Based on the technical scheme, the enzyme nanocapsule is an enzyme monomolecular nanocapsule or an enzyme polymolecular capsule.
Based on the technical scheme, preferably, the size of the enzyme nanocapsule is less than 100 nm; the enzyme nanocapsule comprises at least one enzyme molecule as or as part of an inner core; a layer of reticular polymer film is wrapped outside the inner core to be used as a shell; the thickness of the polymer film is 0.1 nm-100 nm, and the polymer film is formed by monomers or polymers in a covalent mode; the monomer or polymer contains at least one of carboxyl, amino, positive charge, negative charge or double bond groups.
Based on the technical scheme, the enzyme types in the enzyme nanocapsule comprise oxidoreductases, hydrolases, isomerases, synthetases (polymerases), transferases and lyases. After the enzymes are formed into nano capsules, the stability of the enzymes is obviously improved, and the enzymes can be used as enzyme electrode biological recognition molecules or as a part of an enzyme electrode biological recognition element to improve the performance of a sensor. The scheme emphasizes glucose detection, so that the following scheme takes glucose oxidase nanocapsules as an example for detailed description, and other enzymes are not described again. It is to be noted that other enzymes are based on the similar schemes and the like described belowAll in one SubstitutionAnd the obvious effect of improving the performances of the sensor such as stability and the like can be obtained. Based on the technical scheme, preferably, the enzyme nanocapsule is a glucose oxidase nanocapsule.
Based on the technical scheme, preferably, the size of the glucose oxidase nanocapsule is less than 100 nm; the glucose oxidase nanocapsule comprises at least one glucose oxidase molecule as an inner core; a layer of reticular polymer film is wrapped outside the inner core to be used as a shell; the thickness of the polymer film is generally 0.1-100 nm, preferably 0.1-20 nm; the polymer film thickness is preferably chosen such that the transport of substrate molecules which do not significantly affect the enzyme and the signal exchange of the biosensor are not significantly affected; the polymer film is a monomer or a polymer, forms a polymer film shell in a covalent mode and is anchored on the surface of the inner core (taking a glucose oxidase molecule as a core); the monomer or polymer is at least one of carboxyl, amino, positive charge, negative charge or double bond group. The polymer film shell can only wrap glucose oxidase molecules, and can also wrap one or more of coenzyme, redox mediator, nano material and enzyme stabilizer while wrapping the enzyme molecules.
The preparation method of the enzyme nanocapsule is various, and the exemplified typical preparation process is divided into two processes, firstly fixing organic small molecules (such as N-propenyl succinimide) with double bonds (alkenyl) on the surface of enzyme molecules by means of adsorption, covalent crosslinking and the like in a buffer saline solution or an organic solvent of enzyme with a proper concentration to enable the enzyme to be enzyme single molecules with polymerizable groups (double bonds) (at a proper concentration, the enzyme wrapped by the small molecules is generally in single molecule distribution in the solution), then adding at least one organic monomer molecule with double bonds (such as acrylamide and the like) and/or an organic monomer molecule crosslinking agent with two double bonds (such as N, N' -methylene bisacrylamide), finally adding initiators such as ammonium persulfate, tetramethylethylenediamine and the like to initiate an in-situ (controllable) free radical polymerization reaction at a proper reaction temperature (room temperature or low temperature and the like), the polymer generated by the reaction forms a thin-layer reticular polymer on the surface of the enzyme molecule to form the enzyme monomolecular (or enzyme polymolecular) nanocapsule.
According to the glucose oxidase nanocapsule, a thin polymer film is formed on the surface of a single molecule (or multiple molecules) of glucose oxidase by a physical or chemical method, and the film can play a role in protecting enzyme molecules and improving enzyme stability, and meanwhile, material transmission and signal (electron, ion and the like) transmission between the enzyme and substrate molecules cannot be obviously influenced. The sensor uses the glucose oxidase nanocapsule as a biological recognition molecule, and the developed glucose oxidase nanocapsule sensor can be used for detecting the concentration of glucose.
In another aspect, the present invention provides a method for preparing the sensor, including the following steps:
(1) preparation of electrodes: the method for cleaning the surface of the electrode to obtain a clean electrode for standby application mainly comprises the following steps: polishing the surface of the electrode by physical, chemical or electrochemical methods, or (and) cleaning with organic solvent such as ethanol and water, and drying under nitrogen flow or naturally drying to obtain clean electrode surface for later use. Taking a glassy carbon electrode as an example, before the glassy carbon electrode for preparing a biosensor is used, the surface of the glassy carbon electrode is firstly washed by deionized water, and is properly polished on a polishing cloth by using one or more of alumina powders of 1.0 micron, 0.3 micron and 0.05 micron in sequence, the polished electrode is respectively subjected to ultrasonic treatment in ethanol and water to remove any attached alumina powder, and then, the glassy carbon electrode is washed by the deionized water and dried under nitrogen flow to obtain a clean polished glassy carbon mirror surface for later use. The polishing and cleaning aims to obtain a clean electrode surface, eliminate the influence of impurities on the electrode surface on the preparation of the sensor and ensure that the repeatability of the sensors prepared in different batches is good.
(2) Preparing an electrode sensitization material solution: dissolving an electrode sensitization material in water or a cosolvent, and preparing an electrode sensitization material solution by ultrasonic dispersion; (3) preparing an electrode film-forming material solution: dissolving an electrode film-forming material in a buffer salt solution or purified water to obtain an electrode film-forming material solution; the buffer salt is preferably acetate or phosphate; the concentration of the buffer salt solution is 0.05-1.0 mg mL-1Preferably 0.1mg mL-1(ii) a (4) Preparation of glucose oxidase nanocapsule (nGOx) solution: dissolving a glucose oxidase nanocapsule (nGOx) in a buffered salt solution to obtain a glucose oxidase nanocapsule solution, wherein the pH of the buffered salt solution is 5-9, and a phosphate buffer solution is preferred; the concentration of the glucose oxidase nano capsule solution is 0.1-30 mg mL-1Preferably 10mg mL-1；
(5) Preparing a sensor:
the method comprises the following steps: mixing the electrode sensitization material solution and the electrode film forming solution with ultrapure water to obtain a mixed solution, then dropwise adding, pouring or printing the mixed solution onto a polished mirror surface of an electrode, and drying to obtain a sensor precursor; finally, dripping the nGOx solution on the sensor precursor, and drying to obtain the biosensor; in the mixed solution, the concentration of the electrode sensitization material and the electrode film forming material is 0.05-20 mg mL-1Preferably 2mg mL-1(ii) a The dropping amount of the mixed solution is 0.1-3 microliter per square millimeter of the electrode surface; the dropwise adding amount of the nGOx solution is 0.1-3 microliter per square millimeter of the electrode surface;
the second method comprises the following steps: mixing the electrode sensitization material solution, the electrode film forming material solution, the nGOx solution and ultrapure water to obtain a mixed solution, then dropwise adding, printing or pouring the mixed solution onto the surface of the electrode, and drying to obtain the biosensor; electrodes in the mixed solutionThe concentration of the sensitizing material and the concentration of the electrode film forming material are respectively 0.0-20.0 mgmL-1Preferably 2mg mL-1(ii) a The concentration of the nGOx solution in the mixed solution is 0.1-30 mg mL respectively-1Preferably 5.0mg mL-1(ii) a The dropping amount of the mixed solution is 0.1-3 mu L/mm2. In the preparation method of the sensor, the electrode sensitivity enhancing material, the electrode film forming material and the glucose oxidase nanocapsule can be mixed with a solvent to form a mixed solution, or the electrode sensitivity enhancing material, the electrode film forming material and the glucose oxidase nanocapsule can be prepared into solutions respectively, and then the solutions are mixed to form the mixed solution, and only the concentrations of the electrode sensitivity enhancing material, the electrode film forming material and the glucose oxidase nanocapsule in the mixed solution need to be ensured.
Based on the above technical scheme, preferably, the mixed solution in the step (5) may further include a cosolvent, and the volume ratio of the cosolvent to water in the mixed solution is 0: 100-100: 0; the cosolvent is a solvent or a dissolving-assisting reagent which can be mutually dissolved with water, such as dimethylformamide, dimethyl sulfoxide, ethanol, a hydrophilic surfactant or hydrophilic ionic liquid and the like; in the mixed solution, the volume ratio of the cosolvent to water is 0: 100-100: 0, and preferably 20: 80.
Based on the above technical solution, preferably, the electrode for preparing the biosensor may be a Glassy Carbon Electrode (GCE), a metal electrode, a conductive glass electrode, a graphite electrode, a screen printing electrode, a test paper electrode, a flexible electrode, and the like.
Based on the technical scheme, preferably, the electrode sensitization material is a micro-nano scale material with good conductivity, such as graphite powder, a carbon nano material and the like, or a nano material or a conventional material with conductivity or semiconductor properties, such as an inert metal nano material, a metal oxide nano material, a metal-organic framework compound and the like, or a composite material containing any one of the nano materials.
Based on the above technical scheme, it is further preferable that the carbon nanomaterial is a carbon nanotube, graphene, mesoporous carbon, or heteroatom-doped carbon nanomaterial; the heteroatom is nitrogen, phosphorus, boron, oxygen, hydrogen,Hydroxy, carboxy or quinonyl; the inert metal in the inert metal nano material is gold, silver or platinum; the transition metal oxide in the transition metal oxide nano material is ZnO and Fe3O4、Co3O4、TiO2、MnO2、NiO、ZrO2(ii) a More preferably, the electrode sensitization material is nitrogen-doped carbon nanotubes (N-CNTs), and the electrode sensitization material can be replaced by a material which is equivalent to the N-CNTs or has a better sensitization effect
Based on the above technical scheme, preferably, the electrode film forming material may be a polymer material solution or a hydrogel solution with good film forming performance, such as chitosan (containing oligosaccharide), gelatin, fibroin, alginic acid (salt), Nafion emulsion, conductive adhesive (containing carbon powder or silver powder) or silica sol-gel; the film-forming material can be replaced by a material which is equivalent to chitosan or has a better film-forming effect.
The sensor of the present invention is not limited to the above-described production method, and other methods may be employed. The materials for preparing the sensor are not limited to the above materials, and other materials which are beneficial to improving the performance can be used for preparing the sensor, or similar effects can be achieved without the materials.
In another aspect, the invention provides a use of the sensor described above.
Based on the above technical solution, preferably, the application is to use the sensor to detect the glucose concentration in an aqueous solution, an organic solvent or a mixed solution and to detect the glucose concentration in blood sugar, urine sugar or sweat, body fluid glucose or food.
Based on the technical scheme, the sensor is preferably applied to the fields of blood glucose test paper, wearable equipment, implantable equipment, food fermentation, enzyme chemical industry, analysis and detection and the like.
Advantageous effects
The insufficient stability of the conventional biosensor is a major obstacle limiting its commercialization and large-scale application. Compared with the traditional technology of preparing the sensor by using the original enzyme (unmodified), the glucose oxidase nanocapsule sensor has the advantages of higher thermal stability, longer storage life and service life, better organic solvent tolerance and the like, and the adaptability and the tolerance of the biosensor used in various environments (high temperature, organic solvent and the like) are greatly improved. The literature reports a technology of immobilizing enzyme in porous inorganic nano materials such as mesoporous carbon, mesoporous silicon and the like to improve enzyme stability, and since the macroscopic scales of the two inorganic nano materials are large (the pores are nano-scale and micron-scale, but the three-dimensional size of the materials is micron or millimeter-scale), the water phase dispersibility of the enzyme immobilized by the inorganic nano materials is relatively poor, and the diffusion resistance of an enzyme substrate is also large. According to the enzyme nanocapsule technology, a thin-layer reticular polymer is formed on the surface of the enzyme, so that the enzyme nanocapsule has better dispersibility in a water phase and smaller diffusion resistance of an enzyme substrate, and more importantly, the polymer has better biocompatibility to enzyme molecules than an inorganic nano material, so that the performances of the enzyme nanocapsule such as thermal stability, organic solvent tolerance and the like are remarkably superior to those reported in the previous literature. The nano-capsule sensor based on the glucose oxidase has obvious advantages and huge market application potential in a plurality of fields of human body wearable equipment, life health, analysis and detection, food and beverage fermentation, enzyme chemical industry and the like, and especially has obvious advantages on prolonging the service life of the sensor and improving the accuracy and reliability of measurement in the human body wearable equipment.
Drawings
The invention is further illustrated with reference to the following figures and examples.
FIG. 1 is a schematic diagram of a typical process for preparing a monomolecular enzyme nanocapsule.
FIG. 2 is a schematic diagram of a typical process for preparing a sensor based on glucose oxidase nanocapsules.
FIG. 3 is a graph showing (A) the particle size distribution of native glucose oxidase molecules (GOx) and glucose oxidase nanocapsule molecules (nGOx) prepared in example 1, (B) a transmission electron micrograph of the glucose oxidase nanocapsule molecules prepared in example 1, (C) UV-visible spectra of the native glucose oxidase molecules (GOx) and the glucose oxidase nanocapsule molecules (nGOx) prepared in example 1, and (D) a transmission electron micrograph of N-CNTs used in the preparation of nGOx/N-CNTs-Chi/GCE biosensors.
FIG. 4 is (A) a cyclic voltammogram (sweep rate: 0.1V/s) of various modified electrodes in 0.1M phosphate buffer saturated (deoxygenated) with nitrogen; (B) and (3) a cyclic voltammogram of a glucose oxidase nanocapsule modified electrode (nGOx/N-CNTs-Chi/GCE).
FIG. 5 is (A) a cyclic voltammogram of a glucose oxidase nanocapsule modified electrode (nGOx/N-CNTs-Chi/GCE); (B) calibration curve graph of relative sweep rate of cathode and anode peak currents of glucose oxidase nanocapsule modified electrode (nGOx/N-CNTs-Chi/GCE).
FIG. 6 is (A) a cyclic voltammogram of a glucose oxidase nanocapsule-modified electrode (nGOx/N-CNTs-Chi/GCE) before and after addition of 1000. mu.M glucose; (B) and (3) a cyclic voltammetry response graph of a glucose oxidase nanocapsule modified electrode (nGOx/N-CNTs-Chi/GCE) to glucose with different concentrations. Sweeping speed: 100mV s-1。
FIG. 7 is the relative sensitivity of glucose oxidase nanocapsule modified electrode (nGOx/N-CNTs-Chi/GCE) and native glucose oxidase modified electrode (GOx/N-CNTs-Chi/GCE) retained after incubation for 1 hour at different temperatures: (A) ampere-type current-time response curve (i-t) graph: (B) cyclic Voltammogram (CV).
FIG. 8 is the relative sensitivity of glucose oxidase nanocapsule modified electrode (nGOx/N-CNTs-Chi/GCE) and native glucose oxidase modified electrode (GOx/N-CNTs-Chi/GCE) retained after incubation at 65 ℃ for 1, 2, 3, 4 hours: (A) ampere-type current-time response curve (i-t) graph: (B) cyclic Voltammogram (CV).
FIG. 9 is a comparison of the relative sensitivities of glucose oxidase nanocapsule-modified electrodes (nGOx/N-CNTs-Chi/GCE) and native glucose oxidase-modified electrodes (GOx/N-CNTs-Chi/GCE) retained after incubation for 1 hour in a mixed system of organic solvent and buffer solution (50: 50): (A) ampere-type current-time response curve (i-t) graph: (B) cyclic Voltammogram (CV).
FIG. 10 is a Cyclic Voltammogram (CV) of a glucose oxidase nanocapsule-modified electrode (nGOx/N-CNTs-Chi/GCE).
FIG. 11 is a graph of the current-time response of glucose oxidase nanocapsule modified electrode (nGOx/N-CNTs-Chi/GCE) to glucose solution (A), and the corresponding calibration graph of response current and glucose concentration (B).
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The following are some of the instruments and equipment used in the examples of the invention, other experimental conditions not specifically noted, according to conventional or equipment manufacturer's suggested conditions. The electrochemical amperometric detection was performed using an apparatus known as chenhua electrochemical workstation CHI440, and the electrochemical impedance detection was performed using PGSTAT 302N (Autolab, switzerland). The electrochemical detection adopts a three-electrode system, a prepared Glassy Carbon (GC) modified electrode is used as a working electrode, Ag/AgCl (3M KCl) is used as a reference electrode, and a platinum electrode is used as a counter electrode. The glassy carbon electrode for preparing the modified electrode needs to be pretreated before use: respectively polishing the surface of the electrode by using 1.0, 0.3 and 0.05 mu m of aluminum oxide powder, then repeatedly ultrasonically cleaning the electrode in absolute ethyl alcohol and deionized water for 3 times, wherein each time lasts for 1.5 minutes, and then blowing the glassy carbon electrode by using high-purity nitrogen for later use. The three-electrode system used in the experiment is only convenient for verifying the principle and the experimental conclusion, and in practical application, the working electrode (modified electrode) can be prepared and integrated into the blood glucose test paper, and can also be prepared into wearable equipment for human bodies, and is not limited to an enzyme modified electrode based on a glassy carbon electrode matrix and is not limited to the three-electrode system. The modification materials (film-forming materials such as nanomaterials and chitosan) used in the examples are also merely illustrative and not intended to be limiting.
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