A chitosan-modified glucose oxidase sensor
The glucose oxidase sensor modified with chitosan solves the problem of activity loss caused by contact between glucose oxidase and electrode, improves the sensitivity and stability of the sensor, and achieves higher detection accuracy and range.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENZHEN COFOE BIOTECHNOLOGY CO LTD
- Filing Date
- 2023-10-24
- Publication Date
- 2026-07-24
AI Technical Summary
When glucose oxidase comes into contact with the bare electrode surface, it can cause changes in protein structure and function, resulting in loss of biological activity and affecting the detection accuracy of the sensor.
A chitosan-modified glucose oxidase sensor underwent multiple treatments, including pretreatment of carbon nanotubes, mixing of chitosan and cellulose, and forming a Nafion protective film on the electrode surface, which improved electron transfer efficiency and enzyme immobilization.
It significantly improves the current response sensitivity and detection accuracy of the glucose oxidase sensor, broadens the detection range, and enhances the sensor's stability and long-term storage performance.
Smart Images
Figure CN117517420B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of glucose oxidase sensor processing technology, and in particular to a glucose oxidase sensor based on chitosan modification. Background Technology
[0002] Glucose is an important carbohydrate in plants and animals, serving as an energy source for living cells and a metabolic intermediate.
[0003] Blood sugar is very important for human health, but high blood sugar can cause diabetes, which in turn can induce various diseases such as cardiovascular disease, high blood pressure, and neurological disorders, endangering human life and health.
[0004] Acute hypoglycemia can be life-threatening, while hyperglycemia can lead to various complications and severe damage to many organs. Furthermore, diabetes has a high incidence rate in my country. Diabetes is a serious chronic disease and one of the most pressing health problems globally. Proper blood glucose management is crucial for patients and is essential for guiding insulin therapy. However, traditional finger-prick blood glucose testing is accompanied by intense pain, significantly reducing patient compliance and leading to inadequate blood glucose management, thus hindering effective treatment.
[0005] The rapidly increasing number of people with diabetes worldwide requires daily glucose testing to control their blood sugar, leading to a surge in demand for glucose testing.
[0006] In most glucose sensors, glucose oxidase is often used for glucose detection because it has good selectivity and high specificity for glucose molecules.
[0007] Glucose oxidase (GOD) is a dimer molecule composed of two identical polypeptide chains. It is an oxidoreductase with a molecular weight of approximately 150–185 kDa. Each of the two polypeptide chains contains an identical flavin adenine dinucleotide (FAD).
[0008] However, direct contact between glucose oxidase and the bare electrode surface usually causes changes in the structure and function of the protein and causes it to lose its biological activity, thus inhibiting electron transfer between the protein and the electrode. Moreover, the active site of the protein is embedded in a double helix structure and cannot approach the electrode surface, making it difficult to achieve direct electron transfer between the protein and the electrode, resulting in a significant decrease in the detection accuracy of the sensor.
[0009] Therefore, further improvements to existing technologies are needed. Summary of the Invention
[0010] The purpose of this invention is to provide a chitosan-modified glucose oxidase sensor to address the shortcomings of existing technologies.
[0011] The technical solution adopted in this invention is as follows:
[0012] A chitosan-modified glucose oxidase sensor includes the following steps:
[0013] (1) Preparation of the modifier:
[0014] First, the carbon nanotubes are modified to obtain pretreated carbon nanotubes.
[0015] Chitosan was added to an acetic acid solution and stirred until the chitosan dissolved to obtain a chitosan solution.
[0016] Add cellulose to water, then add sulfuric acid solution dropwise while stirring. Once the cellulose is completely dissolved, continue stirring for 30-40 minutes and adjust the pH to neutral to obtain a cellulose mixture.
[0017] Pretreated nanotubes were added to the chitosan solution and stirred for 10-15 minutes. Then, the solution was ultrasonically treated for 10 minutes at room temperature to obtain the first dispersion.
[0018] Add the first dispersion to the cellulose mixture, stir for 10-15 min, then adjust the temperature to 60-68℃ and sonicate for 12 min to obtain the modifier;
[0019] (2) First, immerse the screen-printed carbon electrode in a 5% sodium hydroxide solution for 30 minutes at a temperature of 70°C. Then, remove it, rinse it with water, and dry it.
[0020] The screen-printed carbon electrode, after being soaked in sodium hydroxide solution, was placed in a mixed solution of cobalt chloride and cerium chloride. A cyclic voltammetric scan was performed for 20 cycles in the range of 0.8V-1.5V at a scan rate of 100-120mV / s to obtain the pretreated electrode.
[0021] (3) Use a micropipette to pick up the modifier and then uniformly drop it onto the surface of the pretreated electrode obtained above. The mass ratio of the modifier to the surface of the pretreated electrode is 30:1-1.5. Then place it directly under the ultraviolet mercury lamp and irradiate it uniformly for 1-1.5 hours. Then place it in a drying oven at 30°C for 2 hours to dry it and obtain the modified electrode.
[0022] (4) The modified electrode was soaked in glucose oxidase solution for 20-24 hours, then removed and rinsed with PBS phosphate buffer. After drying, Nafion solution was added to the surface. The mass ratio of Nafion solution to modified electrode was 1:20. After drying, the enzyme was immobilized on the electrode surface to obtain a glucose oxidase sensor.
[0023] As a further technical solution: the method for preparing pretreated carbon nanotubes in step (1) is as follows:
[0024] First, 30g of carbon nanotubes were added to 200mL of nitric acid solution, the temperature was adjusted to 75℃, and the mixture was stirred for 40min. Then, the mixture was filtered, washed with water until neutral, and dried to obtain the treated carbon nanotubes.
[0025] The prepared carbon nanotubes were added to 150 mL of n-octanol, followed by 10 mL of sulfuric acid solution. The temperature was adjusted to 80 °C, and the mixture was stirred for 2 hours. The mixture was then filtered, washed with water until neutral, and dried under vacuum.
[0026] As a further technical solution: the vacuum drying temperature is 55℃ and the vacuum drying time is 2 hours.
[0027] As a further technical solution: the acetic acid solution in step (1) has a mass fraction of 3-4%;
[0028] The chitosan solution has a mass fraction of 2.2-2.8%.
[0029] As a further technical solution: the mass fraction of the cellulose mixture in step (1) is 1.5-1.8%;
[0030] The sulfuric acid solution has a mass fraction of 10%.
[0031] As a further technical solution: the mixing ratio of the pretreated carbon nanotubes and chitosan solution in step (1) is 3-5g:120mL;
[0032] The frequency of the ultrasonic treatment is 35kHz.
[0033] The volume ratio of the first dispersion to the cellulose mixture is 3:1.
[0034] As a further technical solution: the concentration of cobalt chloride in the cobalt chloride and cerium chloride mixed solution in step (2) is 1.5-2 mmol / L;
[0035] The concentration of cerium chloride is 0.001-0.002 mmol / L.
[0036] As a further technical solution: in step (3), the wavelength of the ultraviolet lamp is 254nm, the optical power is 0.8mW, and the irradiation distance is 12cm.
[0037] As a further technical solution: the enzyme activity in the glucose oxidase solution in step (4) is 500-5000 U / mL.
[0038] As a further technical solution: the mass fraction of the Nafion solution in step (4) is 0.5%.
[0039] The prepared glucose oxidase sensor was stored at 4°C for future use.
[0040] First, the glucose oxidase sensor prepared by this invention can effectively avoid the phenomenon that direct contact between glucose oxidase and the bare electrode surface usually causes changes in the structure and function of proteins. Thus, it can effectively protect the biological activity of glucose oxidase and ensure that electron transfer of proteins on the electrode is not affected.
[0041] The glucose oxidase sensor prepared in this invention has a linear range of 6.5 × 10⁻⁶ for detecting glucose. -6 -1.0×10 -4 mol / L.
[0042] This invention modifies the glucose oxidase electrode through multiple treatments, specifically by performing multiple modifications at the microscopic level. This significantly improves the efficiency of direct electron transfer between the active site of glucose oxidase and the electrode. Furthermore, the pretreated carbon nanotube particles and Co particles synergistically act as electron transfer conductors.
[0043] Carbon nanotubes possess good electrical conductivity, biocompatibility, and catalytic activity, which can improve the electrochemical reaction rate of electrodes, increase electrode current density, reduce electrode polarization, and enhance electrode stability. This invention pretreats the carbon nanotubes, improving their dispersibility and preventing aggregation. Simultaneously, they can bind to chitosan, nanocellulose, and other materials onto the electrode surface, effectively improving the reversibility of biomolecular redox reactions. Furthermore, due to their electrical conductivity, carbon nanotubes can act as molecular conductors.
[0044] This invention, through the combination of multiple processing steps, can effectively improve the function of immobilized biological enzyme molecules, thereby broadening the detection range of the sensor.
[0045] Chitosan and glucose oxidase can form stable ion pairs, and chitosan has good film-forming properties, enabling biological enzymes to be well immobilized on the protective membrane of Nafion.
[0046] By adding Nafion solution at the end, a protective film of Nafion can be formed, which can significantly improve the selectivity and stability of the glucose oxidase sensor.
[0047] Beneficial effects:
[0048] This invention provides a glucose oxidase sensor based on chitosan modification. Through the combination of multiple processing methods of this invention, the current response sensitivity of the glucose oxidase sensor can be significantly improved. The improved current response sensitivity can further improve the detection accuracy, and the detection range and detection limit are significantly improved.
[0049] The glucose oxidase sensor prepared by this invention has higher stability and its detection stability decreases only slightly after long-term storage, indicating that the sensor prepared by this invention has excellent long-term stability.
[0050] This invention, through multiple superposition treatments of the electrodes, not only better immobilizes glucose oxidase but also maintains high enzyme activity of glucose oxidase, thereby improving detection stability and sensitivity. Attached Figure Description
[0051] Figure 1 This is a graph showing the effect of placement time on the percentage decrease in the sensor's subsequent sensitivity compared to the initial detection value. Detailed Implementation
[0052] 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.
[0053] Example 1
[0054] A chitosan-modified glucose oxidase sensor includes the following steps:
[0055] (1) Preparation of the modifier:
[0056] First, the carbon nanotubes are modified to obtain pretreated carbon nanotubes.
[0057] Chitosan was added to an acetic acid solution and stirred until the chitosan dissolved to obtain a chitosan solution.
[0058] Cellulose is added to water, and then sulfuric acid solution is added dropwise while stirring. After the cellulose is completely dissolved, stirring is continued for 30 minutes. The pH is then adjusted to neutral to obtain a cellulose mixture.
[0059] Pretreated nanotubes were added to the chitosan solution and stirred for 10 minutes. Then, the solution was ultrasonically treated at room temperature for 10 minutes to obtain the first dispersion.
[0060] The first dispersion was added to the cellulose mixture and stirred for 10 min. The temperature was then adjusted to 60℃ and ultrasonically treated for 12 min to obtain the modifier. The pretreated carbon nanotubes were prepared as follows:
[0061] First, 30g of carbon nanotubes were added to 200mL of nitric acid solution, the temperature was adjusted to 75℃, and the mixture was stirred for 40min. Then, the mixture was filtered, washed with water until neutral, and dried to obtain the treated carbon nanotubes.
[0062] The prepared carbon nanotubes were added to 150 mL of n-octanol, followed by 10 mL of sulfuric acid solution. The temperature was adjusted to 80 °C, and the mixture was stirred for 2 hours. The mixture was then filtered, washed with water until neutral, and dried under vacuum.
[0063] The vacuum drying temperature is 55°C and the vacuum drying time is 2 hours.
[0064] The acetic acid solution has a mass fraction of 3%.
[0065] The chitosan solution has a mass fraction of 2.2%.
[0066] The cellulose mixture has a mass fraction of 1.5%.
[0067] The sulfuric acid solution has a mass fraction of 10%.
[0068] The mixing ratio of pretreated carbon nanotubes to chitosan solution was 3g:120mL.
[0069] The frequency of the ultrasonic treatment is 35kHz.
[0070] The volume ratio of the first dispersion to the cellulose mixture is 3:1;
[0071] (2) First, immerse the screen-printed carbon electrode in a 5% sodium hydroxide solution for 30 minutes at a temperature of 70°C. Then, remove it, rinse it with water, and dry it.
[0072] The screen-printed carbon electrode, after being soaked in sodium hydroxide solution, was placed in a mixed solution of cobalt chloride and cerium chloride. Cyclic voltammetry was performed for 20 cycles within the range of 0.8V at a scan rate of 100mV / s to obtain the pretreated electrode. The concentration of cobalt chloride in the mixed solution of cobalt chloride and cerium chloride was 1.5mmol / L.
[0073] The concentration of cerium chloride was 0.001 mmol / L;
[0074] (3) Use a micropipette to pick up the modifier and then uniformly drop it onto the surface of the pretreated electrode obtained above. The mass ratio of the modifier to the surface of the pretreated electrode is 30:1. Then place it directly under a UV mercury lamp and irradiate it uniformly for 1 hour. Then place it in a drying oven at 30°C for 2 hours to dry it and obtain the modified electrode. The wavelength of the UV lamp is 254nm, the light power is 0.8mW, and the irradiation distance is 12cm.
[0075] (4) The modified electrode was soaked in glucose oxidase solution for 20 h, then removed, rinsed with PBS phosphate buffer, dried, and then Nafion solution was added to the surface. The mass ratio of Nafion solution to modified electrode was 1:20. After drying, the enzyme was fixed to the electrode surface to obtain a glucose oxidase sensor. The enzyme activity in the glucose oxidase solution was 2000 U / mL.
[0076] The Nafion solution has a mass fraction of 0.5%.
[0077] Example 2
[0078] A chitosan-modified glucose oxidase sensor includes the following steps:
[0079] (1) Preparation of the modifier:
[0080] First, the carbon nanotubes are modified to obtain pretreated carbon nanotubes.
[0081] Chitosan was added to an acetic acid solution and stirred until the chitosan dissolved to obtain a chitosan solution.
[0082] Cellulose is added to water, and then sulfuric acid solution is added dropwise while stirring. After the cellulose is completely dissolved, stirring is continued for 35 minutes. The pH is then adjusted to neutral to obtain a cellulose mixture.
[0083] Pretreated nanotubes were added to the chitosan solution and stirred for 12 minutes. Then, the solution was ultrasonically treated at room temperature for 10 minutes to obtain the first dispersion.
[0084] The first dispersion was added to the cellulose mixture and stirred for 12 minutes. The temperature was then adjusted to 65°C and ultrasonically treated for 12 minutes to obtain the modifier. The pretreated carbon nanotubes were prepared as follows:
[0085] First, 30g of carbon nanotubes were added to 200mL of nitric acid solution, the temperature was adjusted to 75℃, and the mixture was stirred for 40min. Then, the mixture was filtered, washed with water until neutral, and dried to obtain the treated carbon nanotubes.
[0086] The prepared carbon nanotubes were added to 150 mL of n-octanol, followed by 10 mL of sulfuric acid solution. The temperature was adjusted to 80 °C, and the mixture was stirred for 2 hours. The mixture was then filtered, washed with water until neutral, and dried under vacuum.
[0087] The vacuum drying temperature is 55°C and the vacuum drying time is 2 hours.
[0088] The acetic acid solution has a mass fraction of 3.3%.
[0089] The chitosan solution has a mass fraction of 2.5%.
[0090] The cellulose mixture has a mass fraction of 1.6%.
[0091] The sulfuric acid solution has a mass fraction of 10%.
[0092] The mixing ratio of pretreated carbon nanotubes to chitosan solution was 3.5g:120mL.
[0093] The frequency of the ultrasonic treatment is 35kHz.
[0094] The volume ratio of the first dispersion to the cellulose mixture is 3:1;
[0095] (2) First, immerse the screen-printed carbon electrode in a 5% sodium hydroxide solution for 30 minutes at a temperature of 70°C. Then, remove it, rinse it with water, and dry it.
[0096] The screen-printed carbon electrode, after being soaked in sodium hydroxide solution, was placed in a mixed solution of cobalt chloride and cerium chloride. Cyclic voltammetry was performed for 20 cycles at a scan rate of 105 mV / s within a range of 1.0 V to obtain the pretreated electrode. The concentration of cobalt chloride in the mixed solution of cobalt chloride and cerium chloride was 1.8 mmol / L.
[0097] The concentration of cerium chloride was 0.0012 mmol / L;
[0098] (3) Use a micropipette to pick up the modifier and then uniformly drop it onto the surface of the pretreated electrode obtained above. The mass ratio of the modifier to the pretreated electrode surface is 30:1.2. Then place it directly under a UV mercury lamp and irradiate it uniformly for 1.1 hours. Then place it in a drying oven at 30°C for 2 hours to dry it and obtain the modified electrode. The wavelength of the UV lamp is 254nm, the light power is 0.8mW, and the irradiation distance is 12cm.
[0099] (4) The modified electrode was soaked in glucose oxidase solution for 22 hours, then removed, rinsed with PBS phosphate buffer, dried, and then Nafion solution was added to the surface. The mass ratio of Nafion solution to modified electrode was 1:20. After drying, the enzyme was fixed on the electrode surface to obtain a glucose oxidase sensor. The enzyme activity in the glucose oxidase solution was 2000 U / mL.
[0100] The Nafion solution has a mass fraction of 0.5%.
[0101] Example 3
[0102] A chitosan-modified glucose oxidase sensor includes the following steps:
[0103] (1) Preparation of the modifier:
[0104] First, the carbon nanotubes are modified to obtain pretreated carbon nanotubes.
[0105] Chitosan was added to an acetic acid solution and stirred until the chitosan dissolved to obtain a chitosan solution.
[0106] Cellulose is added to water, and then sulfuric acid solution is added dropwise while stirring. After the cellulose is completely dissolved, stirring is continued for 38 minutes. The pH is then adjusted to neutral to obtain a cellulose mixture.
[0107] Pretreated nanotubes were added to the chitosan solution and stirred for 13 minutes. Then, the solution was ultrasonically treated at room temperature for 10 minutes to obtain the first dispersion.
[0108] The first dispersion was added to the cellulose mixture and stirred for 12 min. The temperature was then adjusted to 63℃ and ultrasonically treated for 12 min to obtain the modifier. The pretreated carbon nanotubes were prepared as follows:
[0109] First, 30g of carbon nanotubes were added to 200mL of nitric acid solution, the temperature was adjusted to 75℃, and the mixture was stirred for 40min. Then, the mixture was filtered, washed with water until neutral, and dried to obtain the treated carbon nanotubes.
[0110] The prepared carbon nanotubes were added to 150 mL of n-octanol, followed by 10 mL of sulfuric acid solution. The temperature was adjusted to 80 °C, and the mixture was stirred for 2 hours. The mixture was then filtered, washed with water until neutral, and dried under vacuum.
[0111] The vacuum drying temperature is 55°C and the vacuum drying time is 2 hours.
[0112] The acetic acid solution has a mass fraction of 3.8%.
[0113] The chitosan solution has a mass fraction of 2.6%.
[0114] The cellulose mixture has a mass fraction of 1.6%.
[0115] The sulfuric acid solution has a mass fraction of 10%.
[0116] The mixing ratio of pretreated carbon nanotubes to chitosan solution was 4g:120mL.
[0117] The frequency of the ultrasonic treatment is 35kHz.
[0118] The volume ratio of the first dispersion to the cellulose mixture is 3:1;
[0119] (2) First, immerse the screen-printed carbon electrode in a 5% sodium hydroxide solution for 30 minutes at a temperature of 70°C. Then, remove it, rinse it with water, and dry it.
[0120] The screen-printed carbon electrode, after being soaked in sodium hydroxide solution, was placed in a mixed solution of cobalt chloride and cerium chloride. Cyclic voltammetry was performed for 20 cycles within a range of 1.2V at a scan rate of 110mV / s to obtain the pretreated electrode. The concentration of cobalt chloride in the mixed solution of cobalt chloride and cerium chloride was 1.8mmol / L.
[0121] The concentration of cerium chloride was 0.0015 mmol / L;
[0122] (3) Use a micropipette to pick up the modifier and then uniformly drop it onto the surface of the pretreated electrode obtained above. The mass ratio of the modifier to the pretreated electrode surface is 30:1.2. Then place it directly under a UV mercury lamp and irradiate it uniformly for 1.2 hours. Then place it in a drying oven at 30°C for 2 hours to dry it and obtain the modified electrode. The wavelength of the UV lamp is 254nm, the light power is 0.8mW, and the irradiation distance is 12cm.
[0123] (4) The modified electrode was soaked in glucose oxidase solution for 22 hours, then removed, rinsed with PBS phosphate buffer, dried, and then Nafion solution was added to the surface. The mass ratio of Nafion solution to modified electrode was 1:20. After drying, the enzyme was fixed on the electrode surface to obtain a glucose oxidase sensor. The enzyme activity in the glucose oxidase solution was 2000 U / mL.
[0124] The Nafion solution has a mass fraction of 0.5%.
[0125] Example 4
[0126] A chitosan-modified glucose oxidase sensor includes the following steps:
[0127] (1) Preparation of the modifier:
[0128] First, the carbon nanotubes are modified to obtain pretreated carbon nanotubes.
[0129] Chitosan was added to an acetic acid solution and stirred until the chitosan dissolved to obtain a chitosan solution.
[0130] Cellulose is added to water, and then sulfuric acid solution is added dropwise while stirring. After the cellulose is completely dissolved, stirring is continued for 40 minutes. The pH is then adjusted to neutral to obtain a cellulose mixture.
[0131] Pretreated nanotubes were added to the chitosan solution and stirred for 15 minutes. Then, the solution was ultrasonically treated at room temperature for 10 minutes to obtain the first dispersion.
[0132] The first dispersion was added to the cellulose mixture and stirred for 15 minutes. The temperature was then adjusted to 68°C and ultrasonically treated for 12 minutes to obtain the modifier. The pretreated carbon nanotubes were prepared as follows:
[0133] First, 30g of carbon nanotubes were added to 200mL of nitric acid solution, the temperature was adjusted to 75℃, and the mixture was stirred for 40min. Then, the mixture was filtered, washed with water until neutral, and dried to obtain the treated carbon nanotubes.
[0134] The prepared carbon nanotubes were added to 150 mL of n-octanol, followed by 10 mL of sulfuric acid solution. The temperature was adjusted to 80 °C, and the mixture was stirred for 2 hours. The mixture was then filtered, washed with water until neutral, and dried under vacuum.
[0135] The vacuum drying temperature is 55°C and the vacuum drying time is 2 hours.
[0136] The acetic acid solution has a mass fraction of 4%.
[0137] The chitosan solution has a mass fraction of 2.8%.
[0138] The cellulose mixture has a mass fraction of 1.8%.
[0139] The sulfuric acid solution has a mass fraction of 10%.
[0140] The mixing ratio of pretreated carbon nanotubes to chitosan solution was 5g:120mL.
[0141] The frequency of the ultrasonic treatment is 35kHz.
[0142] The volume ratio of the first dispersion to the cellulose mixture is 3:1;
[0143] (2) First, immerse the screen-printed carbon electrode in a 5% sodium hydroxide solution for 30 minutes at a temperature of 70°C. Then, remove it, rinse it with water, and dry it.
[0144] The screen-printed carbon electrode, after being soaked in sodium hydroxide solution, was placed in a mixed solution of cobalt chloride and cerium chloride. Cyclic voltammetry was performed for 20 cycles within a range of 1.5V at a scan rate of 120mV / s to obtain the pretreated electrode. The concentration of cobalt chloride in the mixed solution of cobalt chloride and cerium chloride was 1.5-2 mmol / L.
[0145] The concentration of cerium chloride was 0.002 mmol / L;
[0146] (3) Use a micropipette to pick up the modifier and then uniformly drop it onto the surface of the pretreated electrode obtained above. The mass ratio of the modifier to the pretreated electrode surface is 30:1.5. Then place it directly under a UV mercury lamp and irradiate it uniformly for 1.5 hours. Then place it in a drying oven at 30°C for 2 hours to dry it and obtain the modified electrode. The wavelength of the UV lamp is 254nm, the light power is 0.8mW, and the irradiation distance is 12cm.
[0147] (4) The modified electrode was soaked in glucose oxidase solution for 24 hours, then removed, rinsed with PBS phosphate buffer, dried, and then Nafion solution was added to the surface. The mass ratio of Nafion solution to modified electrode was 1:20. After drying, the enzyme was fixed to the electrode surface to obtain a glucose oxidase sensor. The enzyme activity in the glucose oxidase solution was 2000 U / mL.
[0148] The Nafion solution has a mass fraction of 0.5%.
[0149] Comparative Example 1:
[0150] Based on Example 1, step (1) is adjusted so that carbon nanotubes are not pretreated, while the remaining steps are the same as the technical solution of Example 1.
[0151] Comparative Example 2:
[0152] Based on Example 1, the process of screen printing carbon electrode in step (2) is omitted, while the remaining steps are the same as those in Example 1.
[0153] Comparative Example 3:
[0154] Based on Example 1, step (3) is adjusted so that ultraviolet mercury lamp irradiation is not performed, while the remaining steps are the same as the technical solution of Example 1.
[0155] Viscosity test:
[0156] Experiments were conducted using the examples and comparative samples in a 0.2 mol / L pH 7.4 phosphate buffer solution. The working potential was fixed at 0.5 V, and the reaction cell temperature was kept constant at 25 °C. A certain concentration of glucose sample was injected while continuously stirring, and the current response was recorded using a chronoamperometric method.
[0157] Table 1
[0158] Example 1 33.5 Example 2 33.9 Example 3 34.5 Example 4 34.2 Comparative Example 1 29.8 Comparative Example 2 24.1 Comparative Example 3 26.3
[0159] As can be seen from Table 1, the glucose oxidase sensor prepared in this invention has high sensor sensitivity.
[0160] Using Example 3 as the base sample, the effects of different glucose oxidase activities on the sensitivity of the prepared glucose oxidase sensor were compared:
[0161] Table 2
[0162] 500 29.3 1000 32.0 2000 34.5 3000 34.3 4000 34.1 5000 34.2
[0163] As can be seen from Table 2, in this invention, the sensor sensitivity increases significantly with the increase of glucose oxidase activity. However, with further increases in glucose oxidase activity, the sensor sensitivity no longer increases and tends to stabilize.
[0164] Using Example 3 as the base sample, the effect of different soaking times in glucose oxidase solution on sensor sensitivity was compared.
[0165] Table 3
[0166] 14 30.4 18 32.0 22 34.5 26 34.3 30 33.8 34 33.1
[0167] As shown in Table 3, the sensor sensitivity increases with the extension of the soaking time in the glucose oxidase solution. However, further extending the soaking time will cause a slight decrease in the sensor sensitivity.
[0168] Using Example 3 as the base sample, at a temperature of 4°C, the effect of prolonged placement time on the percentage decrease in subsequent sensor sensitivity compared to the initial detection value was compared. Figure 1 .
[0169] 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 chitosan-modified glucose oxidase sensor, characterized in that: Includes the following steps: (1) Preparation of the modifier: First, the carbon nanotubes are modified to obtain pretreated carbon nanotubes. Chitosan was added to an acetic acid solution and stirred until the chitosan dissolved to obtain a chitosan solution. Add cellulose to water, then add sulfuric acid solution dropwise while stirring. Once the cellulose is completely dissolved, continue stirring for 30-40 minutes and adjust the pH to neutral to obtain a cellulose mixture. Pretreated nanotubes were added to the chitosan solution and stirred for 10-15 minutes. Then, the solution was ultrasonically treated for 10 minutes at room temperature to obtain the first dispersion. Add the first dispersion to the cellulose mixture, stir for 10-15 min, then adjust the temperature to 60-68℃ and sonicate for 12 min to obtain the modifier; (2) First, immerse the screen-printed carbon electrode in a 5% sodium hydroxide solution for 30 minutes at a temperature of 70°C. Then, remove it, rinse it with water, and dry it. The screen-printed carbon electrode, after being soaked in sodium hydroxide solution, was placed in a mixed solution of cobalt chloride and cerium chloride. A cyclic voltammetric scan was performed for 20 cycles in the range of 0.8V-1.5V at a scan rate of 100-120mV / s to obtain the pretreated electrode. (3) Use a micropipette to pick up the modifier and then uniformly drop it onto the surface of the pretreated electrode obtained above. The mass ratio of the modifier to the surface of the pretreated electrode is 30:1-1.
5. Then place it directly under the ultraviolet mercury lamp and irradiate it uniformly for 1-1.5 hours. Then place it in a drying oven at 30°C for 2 hours to dry it and obtain the modified electrode. (4) The modified electrode was soaked in glucose oxidase solution for 20-24 hours, then removed and rinsed with PBS phosphate buffer. After drying, Nafion solution was added to the surface. The mass ratio of Nafion solution to modified electrode was 1:
20. After drying, the enzyme was immobilized on the electrode surface to obtain a glucose oxidase sensor.
2. The chitosan-modified glucose oxidase sensor according to claim 1, characterized in that: The method for preparing pretreated carbon nanotubes in step (1) is as follows: First, 30g of carbon nanotubes were added to 200mL of nitric acid solution, the temperature was adjusted to 75℃, and the mixture was stirred for 40min. Then, the mixture was filtered, washed with water until neutral, and dried to obtain the treated carbon nanotubes. The prepared carbon nanotubes were added to 150 mL of n-octanol, followed by 10 mL of sulfuric acid solution. The temperature was adjusted to 80 °C, and the mixture was stirred for 2 hours. The mixture was then filtered, washed with water until neutral, and dried under vacuum.
3. The chitosan-modified glucose oxidase sensor according to claim 2, characterized in that: The vacuum drying temperature is 55℃, and the vacuum drying time is 2 hours.
4. The chitosan-modified glucose oxidase sensor according to claim 1, characterized in that: The acetic acid solution in step (1) has a mass fraction of 3-4%. The chitosan solution has a mass fraction of 2.2-2.8%.
5. The chitosan-modified glucose oxidase sensor according to claim 1, characterized in that: The cellulose mixture in step (1) has a mass fraction of 1.5-1.8%; The sulfuric acid solution has a mass fraction of 10%.
6. The chitosan-modified glucose oxidase sensor according to claim 1, characterized in that: The mixing ratio of the pretreated carbon nanotubes and chitosan solution in step (1) is 3-5g:120mL; The frequency of the ultrasonic treatment is 35kHz. The volume ratio of the first dispersion to the cellulose mixture is 3:
1.
7. The chitosan-modified glucose oxidase sensor according to claim 1, characterized in that: The concentration of cobalt chloride in the cobalt chloride and cerium chloride mixed solution in step (2) is 1.5-2 mmol / L; The concentration of cerium chloride is 0.001-0.002 mmol / L.
8. The chitosan-modified glucose oxidase sensor according to claim 1, characterized in that: In step (3), the wavelength of the ultraviolet lamp is 254nm, the optical power is 0.8mW, and the irradiation distance is 12cm.
9. A chitosan-modified glucose oxidase sensor according to claim 1, characterized in that: The enzyme activity in the glucose oxidase solution described in step (4) is 500-5000 U / mL.
10. A chitosan-modified glucose oxidase sensor according to claim 1, characterized in that: The Nafion solution in step (4) has a mass fraction of 0.5%.