Method for preparing glucose oxidase electrode based on carbon nanomaterials

Abstract

The application discloses a method for preparing a glucose oxidase electrode based on carbon nanomaterials and relates to the technical field of glucose oxidase electrodes, and comprises the following steps: including the following steps: (1) pretreating the carbon nanomaterials; (2) obtaining a saturated mixed solution; (2) obtaining a composite solution; (3) obtaining a covering solution; (4) obtaining a glucose oxidase electrode based on mesoporous materials; the method only needs one step to simultaneously cover the electron mediator ferrocene and the glucose oxidase on the electrode surface, and the operation is simple. The electrode surface prepared by the method contains the carbon nanomaterials, the effective electrode surface is greatly increased, the electron transfer rate of the electrode surface is accelerated, and then the electrode reaction is accelerated, so that the enhancement of the electrode response signal and sensitivity is realized.

Description

Technical Field

[0001] This invention belongs to the field of glucose oxidase electrode technology, and in particular to a method for preparing glucose oxidase electrodes based on carbon nanomaterials. Background Technology

[0002] In existing technologies, there are three methods for detecting glucose concentration using glucose oxidase electrodes. The first method detects glucose concentration by measuring the electrons transferred during the redox reaction of hydrogen peroxide, a product of the glucose oxidase enzymatic reaction, on the electrode surface. The second method detects glucose concentration by using electron mediators such as potassium ferricyanide and ferrocene to transfer electrons transferred during the enzymatic reaction to the electrode surface. The third method detects blood glucose concentration by using materials such as metal nanoparticles, graphene, and carbon nanotubes to achieve direct electron transfer between the enzyme and the electrode.

[0003] The method of rapid glucose concentration detection using glucose oxidase electrodes is widely used in the pharmaceutical and food industries. One of the most representative applications is the preparation of test strips for home blood glucose meters. The main principle of this method is to use screen printing technology to generate an electric current response to blood glucose concentration on the glucose oxidase electrode test strip. Electronic equipment detects the current intensity and determines the blood glucose concentration based on the current intensity, thus achieving rapid detection of blood glucose concentration. Among many electron mediators, ferrocene is commonly used in the fabrication of glucose oxidase electrodes due to its good redox reversibility and low soluble-insoluble nature in water. The glucose oxidase electrode is a highly selective electrochemical glucose sensor based on the redox reaction catalyzed by glucose oxidase. Ferrocene, also known as cyclopentadienyl iron, is a sandwich compound, insoluble in water but soluble in organic solvents such as ethanol and acetone. Because the compound contains iron, it is often used as an electron mediator in glucose oxidase electrodes. Because ferrocene is insoluble in water while glucose oxidase is soluble, the construction of glucose oxidase electrodes is generally carried out using a stepwise method. First, an ethanol solution of ferrocene is applied to the electrode surface, and after the solution evaporates, a ferrocene deposition layer is formed. Then, a glucose oxidase layer is further modified on top of the ferrocene layer. This method requires two steps, is cumbersome, and limits the contact area between ferrocene and glucose oxidase, hindering electron transfer between them and affecting the electrode's response. Furthermore, the ferrocene and glucose oxidase directly applied to the electrode surface will gradually detach over prolonged use, affecting the electrode's stability and sensitivity.

[0004] Due to the unique physicochemical properties of carbon nanomaterials, carbon nanomaterials of different dimensions have attracted increasing attention and research interest in fields such as electrochemistry, energy, and biomedicine. Because of their large specific surface area, good conductivity, low toxicity, low cost, ease of large-scale production, and ease of surface modification, carbon nanomaterials have become star materials for improving electrode performance in the field of electrochemistry and are widely used in the treatment and diagnosis of diseases. This invention introduces carbon nanomaterials into a glucose oxidase electrode to further improve the sensitivity and stability of the glucose oxidase electrode. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing glucose oxidase electrodes based on carbon nanomaterials, in order to overcome the shortcomings of the prior art.

[0006] The technical solution adopted in this invention is as follows:

[0007] A method for preparing glucose oxidase electrodes based on carbon nanomaterials includes the following steps:

[0008] (1) Pretreatment of carbon nanomaterials:

[0009] First, mix 3-4g of propyltriethoxysilane isocyanate with 5-7g of ethyl glycolate, then add 0.04-0.05mL of catalyst. Under nitrogen atmosphere protection, adjust the temperature to 45℃ and stir for 2 hours to obtain the pretreated solution.

[0010] Take 2-3g of carbon nanoparticles and disperse them evenly in 200-210mL of anhydrous ethanol solution to obtain a carbon nanoparticle dispersion.

[0011] The pretreated liquid prepared above was added dropwise to the carbon nanoparticle dispersion and mixed for 10 min. Then the pH was adjusted to 9.8, and the mixture was stirred and reacted for 5 hours under a water bath at 72-75℃. After that, the mixture was filtered, washed, and dried to obtain the pretreated carbon nanoparticles.

[0012] (2) Add excess ferrocene to ethanol and stir and mix for 10 min at room temperature to obtain a mixed solution;

[0013] The resulting mixed solution is then subjected to high-speed centrifugation.

[0014] The centrifugation speed for high-speed centrifugation is 5500 r / min;

[0015] Then let it stand for 30 minutes, take the supernatant solution, and obtain a saturated ethanol solution of ferrocene;

[0016] Take 0.1-0.2g of pretreated carbon nanomaterials and add them to a saturated ethanol solution. Perform ultrasonic dispersion treatment for 5min to obtain a saturated mixed solution.

[0017] (2) Take 120-130 μL of saturated mixed solution and add it to 880-950 μL of acetic acid solution. Stir and mix at 155 r / min for 30 min. After mixing evenly, stop stirring to obtain a composite solution.

[0018] (3) Then add 0.8-1.5 mg of glucose oxidase and 15-35 mg of composite modified mesoporous material to the composite solution obtained above, stir and mix evenly at 120 r / min, and then sonicate in a 30℃ water bath for 3-6 h to obtain the covering liquid.

[0019] (4) The coating liquid prepared above is applied to the electrode surface. After the coating liquid evaporates, a 5% Nafion solution is coated on the electrode surface. After drying, a glucose oxidase electrode based on mesoporous material is obtained.

[0020] As a further technical solution: the carbon nanomaterial mentioned in step (1) is any one of fullerene, graphene, and carbon nanotube;

[0021] The catalyst is dibutyltin dilaurate.

[0022] As a further technical solution: the acetic acid solution in step (3) has a mass fraction of 2%.

[0023] As a further technical solution: the method for preparing the composite modified mesoporous material in step (4) is as follows:

[0024] Add 0.2-0.3g of nano-bentonite, 8-10mg of hexadecyltrimethylamine bromide, and 130-150mL of isopropanol to 100mL of ammonia solution. Stir at 200r / min for 10min, then sonicate for 5min. Add 0.6-1.0mL of tetraethoxysilane, adjust the temperature to 40℃, and keep the mixture stirred for 10 hours. Dry by rotary evaporation, then wash and dry again.

[0025] As a further technical solution: the montmorillonite content in the nano-bentonite is 97.36%.

[0026] As a further technical solution: the thickness of the nano-bentonite wafer is ≤30nm.

[0027] As a further technical solution: the ammonia solution is a saturated ammonia solution.

[0028] As a further technical solution: the frequency of the ultrasonic treatment in step (4) is 40kHz.

[0029] As a further technical solution: in step (5), the covering liquid is applied to the electrode surface by immersion.

[0030] Specifically, the electrode is fully inserted into the covering liquid for 28-34 seconds, and then removed.

[0031] As a further technical solution: step (5) involves coating the electrode surface with Nafion solution, wherein the mass ratio of the electrode to the Nafion solution is 1:22-30.

[0032] The composite modified mesoporous material prepared by this invention can effectively adsorb and bind glucose oxidase without the need for excessive organic chemical reagents. Furthermore, it can be reused and causes very little damage to the active site of glucose oxidase, thus effectively ensuring the high activity of glucose oxidase.

[0033] By introducing pretreated carbon nanomaterials, during the adsorption of glucose oxidase onto the electrode, due to its nanoscale topology and size comparable to that of glucose oxidase molecules, the adsorption of glucose oxidase does not alter its biological structure and function. At the same time, the pretreated carbon nanomaterials can contact the active site of glucose oxidase, i.e., be within its electron tunnel range.

[0034] The carbon nanomaterials prepared in this invention can not only maintain biological activity when used to prepare glucose oxidase electrodes, but also have good biocatalytic activity in glucose solutions.

[0035] Beneficial effects:

[0036] This invention provides a method for preparing glucose oxidase electrodes based on carbon nanomaterials, with the following advantages:

[0037] 1. The method of the present invention can simultaneously coat the electron mediator ferrocene and glucose oxidase on the electrode surface in only one step, which is simple to operate.

[0038] 2. The electrode surface prepared by the method of the present invention contains carbon nanomaterials (fullerene, graphene, carbon nanotubes, etc.), which greatly increases the effective electrode surface area, accelerates the electron transfer rate on the electrode surface, and thus accelerates the electrode reaction, thereby enhancing the electrode response signal and sensitivity.

[0039] 3. The method of the present invention simultaneously immobilizes the electron mediator ferrocene and glucose oxidase in the pores of the composite modified mesoporous material, thereby enhancing the stability of the electron mediator ferrocene and glucose oxidase, and thus enhancing the stability of the enzyme electrode.

[0040] 4. The method of the present invention can uniformly mix carbon nanomaterials (fullerene, graphene, carbon nanotubes, etc.), electron mediator ferrocene, and glucose oxidase in only one step. The electron mediator ferrocene, carbon nanomaterials, and glucose oxidase are uniformly distributed in the film layer, with a large contact area, which greatly increases the effective electrode surface and the contact area between ferrocene and glucose oxidase, thereby accelerating the electrode reaction and improving the sensitivity of the glucose oxidase electrode. Attached Figure Description

[0041] Figure 1 The above are bar charts showing the current response time of the glucose oxidase electrode in the examples and comparative examples. Detailed Implementation

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Example 1

[0044] A method for preparing glucose oxidase electrodes based on carbon nanomaterials includes the following steps:

[0045] (1) Pretreatment of carbon nanomaterials:

[0046] First, 3g of propyltriethoxysilane isocyanate was mixed with 5g of ethyl glycolate, and then 0.04mL of catalyst was added. Under nitrogen atmosphere protection, the temperature was adjusted to 45℃ and stirred for 2 hours to obtain the pretreated solution.

[0047] Take 2g of carbon nanoparticles and disperse them evenly in 200mL of anhydrous ethanol solution to obtain a carbon nanoparticle dispersion.

[0048] The pretreated liquid prepared above was added dropwise to the carbon nanoparticle dispersion and mixed for 10 min. Then the pH was adjusted to 9.8, and the mixture was stirred and reacted for 5 hours under a water bath at 72°C. After that, the mixture was filtered, washed, and dried to obtain the pretreated carbon nanomaterial. The carbon nanomaterial was graphene.

[0049] The catalyst is dibutyltin dilaurate.

[0050] (2) Add excess ferrocene to ethanol and stir and mix for 10 min at room temperature to obtain a mixed solution;

[0051] The resulting mixed solution is then subjected to high-speed centrifugation.

[0052] The centrifugation speed for high-speed centrifugation is 5500 r / min;

[0053] Then let it stand for 30 minutes, take the supernatant solution, and obtain a saturated ethanol solution of ferrocene;

[0054] Take 0.1g of pretreated carbon nanomaterials and add them to a saturated ethanol solution. Perform ultrasonic dispersion treatment for 5min to obtain a saturated mixed solution.

[0055] (2) Take 120 μL of saturated mixed solution and add it to 880 μL of acetic acid solution. Stir and mix at 155 r / min for 30 min. After mixing evenly, stop stirring to obtain a composite solution.

[0056] (3) Subsequently, 0.8 mg of glucose oxidase and 15 mg of composite modified mesoporous material were added to the above-obtained composite solution, and the mixture was stirred at 120 r / min until homogeneous. Then, the solution was ultrasonically treated in a 30℃ water bath for 3 h to obtain a covering solution; the acetic acid solution had a mass fraction of 2%; the preparation method of the composite modified mesoporous material was as follows:

[0057] 0.2 g of nano-bentonite, 8 mg of hexadecyltrimethylamine bromide, and 130 mL of isopropanol were added to 100 mL of ammonia solution. The mixture was stirred at 200 r / min for 10 min, followed by ultrasonic treatment for 5 min. Then, 0.6 mL of tetraethoxysilane was added, the temperature was adjusted to 40 °C, and the mixture was kept at this temperature and stirred for 10 hours. The mixture was then dried by rotary evaporation, washed, and dried again. The montmorillonite content in the nano-bentonite was 97.36%. The wafer thickness of the nano-bentonite was ≤30 nm. The ammonia solution was a saturated ammonia solution. The ultrasonic treatment frequency was 40 kHz.

[0058] (4) The coating solution prepared above is applied to the electrode surface. After the coating solution evaporates, a 5% Nafion solution is coated onto the electrode surface. After drying, a glucose oxidase electrode based on mesoporous materials is obtained. The coating solution is applied to the electrode surface by impregnation.

[0059] Specifically, the electrode is fully inserted into the covering solution for 28 seconds and then removed; the mass ratio of the electrode to the Nafion solution is 1:22.

[0060] Example 2

[0061] A method for preparing glucose oxidase electrodes based on carbon nanomaterials includes the following steps:

[0062] (1) Pretreatment of carbon nanomaterials:

[0063] First, 3.2 g of propyltriethoxysilane isocyanate was mixed with 5.3 g of ethyl glycolate, and then 0.041 mL of catalyst was added. Under nitrogen atmosphere protection, the temperature was adjusted to 45 °C and stirred for 2 hours to obtain the pretreated solution.

[0064] Take 2.3g of carbon nanoparticles and disperse them evenly in 204mL of anhydrous ethanol solution to obtain a carbon nanoparticle dispersion.

[0065] The pretreated liquid prepared above was added dropwise to the carbon nanoparticle dispersion and mixed for 10 min. Then the pH was adjusted to 9.8, and the mixture was stirred and reacted for 5 hours under a water bath at 73°C. After that, the mixture was filtered, washed, and dried to obtain the pretreated carbon nanomaterials. The carbon nanomaterials were carbon nanotubes.

[0066] The catalyst is dibutyltin dilaurate.

[0067] (2) Add excess ferrocene to ethanol and stir and mix for 10 min at room temperature to obtain a mixed solution;

[0068] The resulting mixed solution is then subjected to high-speed centrifugation.

[0069] The centrifugation speed for high-speed centrifugation is 5500 r / min;

[0070] Then let it stand for 30 minutes, take the supernatant solution, and obtain a saturated ethanol solution of ferrocene;

[0071] Take 0.15g of pretreated carbon nanomaterials and add them to a saturated ethanol solution. Perform ultrasonic dispersion treatment for 5min to obtain a saturated mixed solution.

[0072] (2) Take 125 μL of saturated mixed solution and add it to 890 μL of acetic acid solution. Stir and mix at 155 r / min for 30 min. After mixing evenly, stop stirring to obtain a composite solution.

[0073] (3) Subsequently, 1.0 mg of glucose oxidase and 20 mg of composite modified mesoporous material were added to the above-obtained composite solution, and the mixture was stirred at 120 r / min until homogeneous. Then, the solution was ultrasonically treated in a 30℃ water bath for 4 h to obtain a covering solution; the acetic acid solution had a mass fraction of 2%; the preparation method of the composite modified mesoporous material was as follows:

[0074] 0.26 g of nano-bentonite, 8.8 mg of hexadecyltrimethylamine bromide, and 135 mL of isopropanol were added to 100 mL of ammonia solution. The mixture was stirred at 200 r / min for 10 min, followed by ultrasonic treatment for 5 min. Then, 0.8 mL of tetraethoxysilane was added, the temperature was adjusted to 40 °C, and the mixture was kept at this temperature and stirred for 10 hours. The mixture was then dried by rotary evaporation, washed, and dried again. The montmorillonite content in the nano-bentonite was 97.36%. The wafer thickness of the nano-bentonite was ≤30 nm. The ammonia solution was a saturated ammonia solution. The ultrasonic treatment frequency was 40 kHz.

[0075] (4) The coating solution prepared above is applied to the electrode surface. After the coating solution evaporates, a 5% Nafion solution is coated onto the electrode surface. After drying, a glucose oxidase electrode based on mesoporous materials is obtained. The coating solution is applied to the electrode surface by impregnation.

[0076] Specifically, the electrode is fully inserted into the covering solution for 30 seconds and then removed; the mass ratio of the electrode to the Nafion solution is 1:25.

[0077] Example 3

[0078] A method for preparing glucose oxidase electrodes based on carbon nanomaterials includes the following steps:

[0079] (1) Pretreatment of carbon nanomaterials:

[0080] First, 3.6 g of propyltriethoxysilane isocyanate was mixed with 6 g of ethyl glycolate, and then 0.045 mL of catalyst was added. Under nitrogen atmosphere protection, the temperature was adjusted to 45 °C and stirred for 2 hours to obtain the pretreated solution.

[0081] Take 2.8g of carbon nanoparticles and disperse them evenly in 204mL of anhydrous ethanol solution to obtain a carbon nanoparticle dispersion.

[0082] The pretreated liquid prepared above was added dropwise to the carbon nanoparticle dispersion and mixed for 10 min. Then the pH was adjusted to 9.8, and the mixture was stirred and reacted for 5 hours under a water bath at 73°C. After that, the mixture was filtered, washed, and dried to obtain the pretreated carbon nanomaterial. The carbon nanomaterial was graphene.

[0083] The catalyst is dibutyltin dilaurate.

[0084] (2) Add excess ferrocene to ethanol and stir and mix for 10 min at room temperature to obtain a mixed solution;

[0085] The resulting mixed solution is then subjected to high-speed centrifugation.

[0086] The centrifugation speed for high-speed centrifugation is 5500 r / min;

[0087] Then let it stand for 30 minutes, take the supernatant solution, and obtain a saturated ethanol solution of ferrocene;

[0088] Take 0.16g of pretreated carbon nanomaterials and add them to a saturated ethanol solution. Perform ultrasonic dispersion treatment for 5min to obtain a saturated mixed solution.

[0089] (2) Take 124 μL of saturated mixed solution and add it to 882 μL of acetic acid solution. Stir and mix at 155 r / min for 30 min. After mixing evenly, stop stirring to obtain a composite solution.

[0090] (3) Subsequently, 1.2 mg of glucose oxidase and 25 mg of composite modified mesoporous material were added to the above-obtained composite solution, and the mixture was stirred at 120 r / min until homogeneous. Then, the solution was ultrasonically treated in a 30℃ water bath for 4 h to obtain a covering solution; the acetic acid solution had a mass fraction of 2%; the preparation method of the composite modified mesoporous material was as follows:

[0091] 0.26 g of nano-bentonite, 9 mg of hexadecyltrimethylamine bromide, and 140 mL of isopropanol were added to 100 mL of ammonia solution. The mixture was stirred at 200 r / min for 10 min, followed by ultrasonic treatment for 5 min. Then, 0.8 mL of tetraethoxysilane was added, the temperature was adjusted to 40 °C, and the mixture was kept at this temperature and stirred for 10 hours. The mixture was then dried by rotary evaporation, washed, and dried again. The montmorillonite content in the nano-bentonite was 97.36%. The wafer thickness of the nano-bentonite was ≤30 nm. The ammonia solution was a saturated ammonia solution. The ultrasonic treatment frequency was 40 kHz.

[0092] (4) The coating solution prepared above is applied to the electrode surface. After the coating solution evaporates, a 5% Nafion solution is coated onto the electrode surface. After drying, a glucose oxidase electrode based on mesoporous materials is obtained. The coating solution is applied to the electrode surface by impregnation.

[0093] Specifically, the electrode is fully inserted into the covering solution for 31 seconds and then removed; the mass ratio of the electrode to the Nafion solution is 1:26.

[0094] The glucose oxidase content on the electrode surface was 23.1 μg / cm□;

[0095] Example 4

[0096] A method for preparing glucose oxidase electrodes based on carbon nanomaterials includes the following steps:

[0097] (1) Pretreatment of carbon nanomaterials:

[0098] First, 4g of propyltriethoxysilane isocyanate was mixed with 7g of ethyl glycolate, and then 0.04mL of catalyst was added. Under nitrogen atmosphere protection, the temperature was adjusted to 45℃ and stirred for 2 hours to obtain the pretreated solution.

[0099] Take 2.6g of carbon nanoparticles and disperse them evenly in 205mL of anhydrous ethanol solution to obtain a carbon nanoparticle dispersion.

[0100] The pretreated liquid prepared above was added dropwise to the carbon nanoparticle dispersion and mixed for 10 min. Then the pH was adjusted to 9.8, and the mixture was stirred and reacted for 5 hours under a water bath at 73°C. After that, the mixture was filtered, washed, and dried to obtain the pretreated carbon nanomaterial. The carbon nanomaterial is any one of fullerene, graphene, or carbon nanotube.

[0101] The catalyst is dibutyltin dilaurate.

[0102] (2) Add excess ferrocene to ethanol and stir and mix for 10 min at room temperature to obtain a mixed solution;

[0103] The resulting mixed solution is then subjected to high-speed centrifugation.

[0104] The centrifugation speed for high-speed centrifugation is 5500 r / min;

[0105] Then let it stand for 30 minutes, take the supernatant solution, and obtain a saturated ethanol solution of ferrocene;

[0106] Take 0.15g of pretreated carbon nanomaterials and add them to a saturated ethanol solution. Perform ultrasonic dispersion treatment for 5min to obtain a saturated mixed solution.

[0107] (2) Take 120 μL of saturated mixed solution and add it to 920 μL of acetic acid solution. Stir and mix at 155 r / min for 30 min. After mixing evenly, stop stirring to obtain a composite solution.

[0108] (3) Subsequently, 1.2 mg of glucose oxidase and 30 mg of composite modified mesoporous material were added to the above-obtained composite solution, and the mixture was stirred at 120 r / min until homogeneous. Then, the solution was ultrasonically treated in a 30℃ water bath for 4 h to obtain a covering solution; the acetic acid solution had a mass fraction of 2%; the preparation method of the composite modified mesoporous material was as follows:

[0109] 0.25 g of nano-bentonite, 9 mg of hexadecyltrimethylamine bromide, and 138 mL of isopropanol were added to 100 mL of ammonia solution. The mixture was stirred at 200 r / min for 10 min, followed by ultrasonic treatment for 5 min. Then, 0.8 mL of tetraethoxysilane was added, the temperature was adjusted to 40 °C, and the mixture was kept at this temperature and stirred for 10 hours. The mixture was then dried by rotary evaporation, washed, and dried again. The montmorillonite content in the nano-bentonite was 97.36%. The wafer thickness of the nano-bentonite was ≤30 nm. The ammonia solution was a saturated ammonia solution. The ultrasonic treatment frequency was 40 kHz.

[0110] (4) The coating solution prepared above is applied to the electrode surface. After the coating solution evaporates, a 5% Nafion solution is coated onto the electrode surface. After drying, a glucose oxidase electrode based on mesoporous materials is obtained. The coating solution is applied to the electrode surface by impregnation.

[0111] Specifically, the electrode is fully inserted into the covering solution for 32 seconds and then removed; the mass ratio of the electrode to the Nafion solution is 1:28.

[0112] Example 5

[0113] A method for preparing glucose oxidase electrodes based on carbon nanomaterials includes the following steps:

[0114] (1) Pretreatment of carbon nanomaterials:

[0115] First, 4g of propyltriethoxysilane isocyanate was mixed with 7g of ethyl glycolate, and then 0.05mL of catalyst was added. Under nitrogen atmosphere protection, the temperature was adjusted to 45℃ and stirred for 2 hours to obtain the pretreated solution.

[0116] Take 3g of carbon nanoparticles and disperse them evenly in 210mL of anhydrous ethanol solution to obtain a carbon nanoparticle dispersion.

[0117] The pretreated liquid prepared above was added dropwise to the carbon nanoparticle dispersion and mixed for 10 min. Then the pH was adjusted to 9.8, and the mixture was stirred and reacted for 5 hours under a water bath at 75°C. After that, the mixture was filtered, washed, and dried to obtain the pretreated carbon nanomaterial. The carbon nanomaterial was graphene.

[0118] The catalyst is dibutyltin dilaurate.

[0119] (2) Add excess ferrocene to ethanol and stir and mix for 10 min at room temperature to obtain a mixed solution;

[0120] The resulting mixed solution is then subjected to high-speed centrifugation.

[0121] The centrifugation speed for high-speed centrifugation is 5500 r / min;

[0122] Then let it stand for 30 minutes, take the supernatant solution, and obtain a saturated ethanol solution of ferrocene;

[0123] Take 0.2g of pretreated carbon nanomaterials and add them to a saturated ethanol solution. Perform ultrasonic dispersion treatment for 5min to obtain a saturated mixed solution.

[0124] (2) Take 130 μL of saturated mixed solution and add it to 950 μL of acetic acid solution. Stir and mix at 155 r / min for 30 min. After mixing evenly, stop stirring to obtain a composite solution.

[0125] (3) Subsequently, 1.5 mg of glucose oxidase and 35 mg of composite modified mesoporous material were added to the above-obtained composite solution, and the mixture was stirred at 120 r / min until homogeneous. Then, the solution was ultrasonically treated in a 30℃ water bath for 6 h to obtain a covering solution; the acetic acid solution had a mass fraction of 2%; the preparation method of the composite modified mesoporous material was as follows:

[0126] 0.3g of nano-bentonite, 10mg of hexadecyltrimethylamine bromide, and 150mL of isopropanol were added to 100mL of ammonia solution. The mixture was stirred at 200r / min for 10min, followed by ultrasonic treatment for 5min. Then, 1.0mL of tetraethoxysilane was added, the temperature was adjusted to 40℃, and the mixture was kept at this temperature and stirred for 10 hours. The mixture was then dried by rotary evaporation, washed, and dried again. The montmorillonite content in the nano-bentonite was 97.36%. The wafer thickness of the nano-bentonite was ≤30nm. The ammonia solution was a saturated ammonia solution. The ultrasonic treatment frequency was 40kHz.

[0127] (4) The coating solution prepared above is applied to the electrode surface. After the coating solution evaporates, a 5% Nafion solution is coated onto the electrode surface. After drying, a glucose oxidase electrode based on mesoporous materials is obtained. The coating solution is applied to the electrode surface by impregnation.

[0128] Specifically, the electrode is fully inserted into the covering solution for 34 seconds and then removed; the mass ratio of the electrode to the Nafion solution is 1:30.

[0129] Comparative Example 1:

[0130] Based on Example 1, the pre-treated carbon nanomaterial in step (2) is replaced with water, and the remaining steps are the same as the technical solution of Example 1.

[0131] Comparative Example 2:

[0132] Based on Example 1, the nano-bentonite was not added during the preparation of the composite modified mesoporous material in step (3), while the remaining steps were the same as those in Example 1.

[0133] test

[0134] The experiment was conducted using platinum sheets as electrodes:

[0135] Sensitivity test:

[0136] In a three-electrode system consisting of a glucose oxidase electrode, a saturated calomel electrode, and a platinum electrode, using PBS buffer (pH 5.5) as the electrolyte, under the same current-carrying conditions, when the current tended to stabilize, a low concentration of glucose solution was added to the electrolyte system. When the current changed, and the change was greater than three times the fluctuation of the current baseline (i.e., the signal-to-noise ratio was greater than 3), the glucose oxidase electrode was considered to have responded to that concentration of glucose. By sequentially detecting different concentrations of glucose, the lowest glucose concentration that elicited a response was obtained, which is the limit of detection, as shown in Table 1.

[0137] Table 1

[0138]

[0139]

[0140] As can be seen from Table 1, the glucose oxidase electrode prepared by the method of the present invention has a lower detection limit.

[0141] The current response time of the glucose oxidase electrode in the examples and the comparative examples was recorded and compared, wherein the glucose solution concentration was 1.0 mmol / mL.

[0142] Table 2

[0143] Example 1 2.06 Example 2 2.03 Example 3 2.00 Example 4 2.02 Example 5 1.98 Comparative Example 1 9.63 Comparative Example 2 6.82

[0144] As can be seen from Table 2, the glucose oxidase electrode prepared by this invention has a shorter current response time.

[0145] The sensitivity of the glucose oxidase sensors prepared from the examples and comparative samples was tested:

[0146] Table 3

[0147] Example 1 0.192 Example 2 0.201 Example 3 0.198 Example 4 0.203 Example 5 0.199 Comparative Example 1 0.116 Comparative Example 2 0.163

[0148] As can be seen from Table 3, the glucose oxidase sensor prepared by the carbon nanomaterials of this invention has higher sensitivity.

[0149] Using Example 1 as the base sample, the effect of adding different masses of pretreated carbon nanoparticles to an ethanol saturated solution on the current response time of the glucose oxidase electrode was compared.

[0150] The above description is only a preferred embodiment of the present invention, but the present invention is not limited to the scope of implementation shown. Any changes made in accordance with the concept of the present invention, or equivalent embodiments modified to have equivalent changes, shall be within the protection scope of the present invention as long as they do not exceed the spirit covered by the specification.

Claims

1. A method for preparing glucose oxidase electrodes based on carbon nanomaterials, characterized in that: Includes the following steps: (1) Pretreatment of carbon nanomaterials: First, 3-4g of propyltriethoxysilane isocyanate and 5-7g of ethyl glycolate are mixed, and then 0.04-0.05mL of catalyst is added. Under nitrogen atmosphere protection, the temperature is adjusted to 45℃ and stirred for 2 hours to obtain a pretreated solution; the catalyst is dibutyltin dilaurate. Take 2-3g of carbon nanoparticles and disperse them evenly in 200-210mL of anhydrous ethanol solution to obtain a carbon nanoparticle dispersion. The pretreated liquid prepared above was added dropwise to the carbon nanoparticle dispersion and mixed for 10 min. Then the pH was adjusted to 9.8, and the mixture was stirred and reacted for 5 hours under a water bath at 72-75℃. Then the mixture was filtered, washed, and dried to obtain the pretreated carbon nanoparticles. (2) Add excess ferrocene to ethanol and stir and mix for 10 min at room temperature to obtain a mixed solution; The resulting mixed solution is then subjected to high-speed centrifugation. The centrifugation speed for high-speed centrifugation is 5500 r / min; Then let it stand for 30 minutes, take the supernatant solution, and obtain a saturated ethanol solution of ferrocene; Take 0.1-0.2g of pretreated carbon nanomaterials and add them to a saturated ethanol solution. Perform ultrasonic dispersion treatment for 5min to obtain a saturated mixed solution. (3) Take 120-130µL of saturated mixed solution and add it to 880-950µL of acetic acid solution. Stir and mix at 155r / min for 30min. After mixing evenly, stop stirring to obtain a composite solution. (4) Then add 0.8-1.5 mg of glucose oxidase and 15-35 mg of composite modified mesoporous material to the composite solution obtained above, stir and mix evenly at 120 r / min, and then sonicate in a 30℃ water bath for 3-6 h to obtain the covering liquid. The method for preparing the composite modified mesoporous material is as follows: Add 0.2-0.3g of nano-bentonite, 8-10mg of hexadecyltrimethylamine bromide, and 130-150mL of isopropanol to 100mL of ammonia solution, stir at 200r / min for 10min, then sonicate for 5min, then add 0.6-1.0mL of tetraethoxysilane, adjust the temperature to 40℃, keep warm and stir for 10 hours, dry by rotary evaporation, then wash, and dry again. (5) The coating liquid prepared above is applied to the electrode surface. After the coating liquid evaporates, a 5% Nafion solution is applied to the electrode surface. After drying, a glucose oxidase electrode based on mesoporous material is obtained.

2. The method for preparing a glucose oxidase electrode based on carbon nanomaterials according to claim 1, characterized in that: The carbon nanomaterial mentioned in step (1) is any one of fullerene, graphene, and carbon nanotube.

3. The method for preparing a glucose oxidase electrode based on carbon nanomaterials according to claim 1, characterized in that: The acetic acid solution in step (3) has a mass fraction of 2%.

4. The method for preparing a glucose oxidase electrode based on carbon nanomaterials according to claim 1, characterized in that: The nano-bentonite contains 97.36% montmorillonite.

5. The method for preparing a glucose oxidase electrode based on carbon nanomaterials according to claim 4, characterized in that: The thickness of the nano-bentonite wafers is ≤30nm.

6. The method for preparing a glucose oxidase electrode based on carbon nanomaterials according to claim 1, characterized in that: The ammonia solution is a saturated ammonia solution.

7. The method for preparing a glucose oxidase electrode based on carbon nanomaterials according to claim 1, characterized in that: The frequency of the ultrasonic treatment in step (4) is 40 kHz.

8. The method for preparing a glucose oxidase electrode based on carbon nanomaterials according to claim 1, characterized in that: In step (5), the covering liquid is applied to the electrode surface by immersion: Specifically, the electrode is fully inserted into the covering liquid for 28-34 seconds, and then removed.

9. The method for preparing a glucose oxidase electrode based on carbon nanomaterials according to claim 1, characterized in that: Step (5) involves coating the electrode surface with Nafion solution, wherein the mass ratio of electrode to Nafion solution is 1:22-30.

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