Preparation method of glucose oxidase electrode

Abstract

The invention belongs to the technical field of electrochemistry, and particularly relates to a preparation method of a glucose oxidase electrode, which comprises the following steps: S1, filtering a ferrocene ethanol saturated solution, taking a proper amount of filtrate and an acetic acid solution in proportion, and uniformly mixing in a mixing container to obtain a mixed solution; s2, adding chitosan powder into the mixed solution according to the dosage of 6g / L, and stirring to obtain ferrocene chitosan dispersion gel; s3, adding glucose oxidase into the ferrocene chitosan dispersion gel according to the dosage of 1.3 g / L, dripping on the surface of an electrode, drying for 13.5 hours under the condition of 5 DEG C, dripping a glutaraldehyde solution on the surface of the electrode, washing by using a PBS buffer solution after 17 minutes, and drying to obtain a glucose oxidase electrode; the contact area of ferrocene and glucose oxidase is increased, so that electron transfer in the glucose oxidase electrode is promoted, and the grape oxidase electrode with high sensitivity and wide detection range is prepared.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemistry, in particular to a method for preparing a glucose oxidase electrode. Background Art

[0002] Enzyme is a kind of macromolecular organic substance that is widely present in living organisms and has the characteristics of high efficiency and specificity. It is one of the indispensable substances for maintaining life. The enzyme is fixed on the electrode and the catalytic effect of the enzyme is used to catalyze the electrochemical reaction. Such a device is called an enzyme electrode. By fixing glucose oxidase on the electrode surface, an electric current response is generated to the blood glucose concentration, thereby realizing the rapid detection of glucose concentration in the blood. Therefore, it is necessary to prepare glucose oxidase electrodes for blood glucose detection.

[0003] A Chinese patent with publication number CN1563969A discloses a bio-enzyme electrode for a biosensor and a preparation method thereof. The bio-enzyme electrode is composed of a working electrode, a counter electrode, a carrier, an insulator covering the electrode surface and a reaction layer. The reaction layer is composed of an anti-interference layer, nanoparticles, a matrix-embedded enzyme layer, an oxidase and a diffusion-limiting membrane covering the electrode substrate at the lower end of the working electrode. On an electrode substrate made of an organic insulating material, i.e., a carrier, a conductive material is printed by a spraying method to form a substrate for the working electrode and the counter electrode. A layer of polycarbonate insulator is coated on the surface of their middle part, and a reaction layer formed by comprehensively applying a high molecular polymer accumulation film technology or a ferrocene covering technology, an enzyme embedding technology, a nanotechnology and a diffusion-limiting membrane technology is coated on the lower end of the working electrode substrate to form a bio-enzyme electrode.

[0004] In the current prior art, since ferrocene is insoluble in water while biological enzymes are soluble in water, it is necessary to first cover the electrode surface with an ethanol solution of ferrocene to form a ferrocene deposition layer, and then further modify the biological enzyme layer on the ferrocene layer; this method limits the contact area between ferrocene and the biological enzyme, which is not conducive to the electron transfer between ferrocene and the enzyme, and the ferrocene and biological enzyme directly dripped onto the electrode surface will gradually fall off during long-term use, thereby affecting the stability and sensitivity of the electrode.

[0005] To this end, the present invention provides a method for preparing a glucose oxidase electrode. Summary of the invention

[0006] In order to make up for the deficiencies of the prior art, at least one technical problem raised in the background technology is solved.

[0007] The technical solution adopted by the present invention to solve the technical problem is: a method for preparing a glucose oxidase electrode according to the present invention comprises the following steps:

[0008] S1: filtering the saturated ferrocene ethanol solution, taking out appropriate amounts of the filtrate and the acetic acid solution in proportion, and mixing them evenly in a mixing container to obtain a mixed solution;

[0009] S2: adding chitosan powder into the mixed solution in an amount of 5 to 7 g / L, stirring, and obtaining a ferrocene chitosan dispersion gel;

[0010] S3: Glucose oxidase is added to ferrocene chitosan dispersion gel at a dosage of 0.5-2 g / L, and dripped on the electrode surface, dried at 3-7°C for 12-15 h, glutaraldehyde solution is dripped on the electrode surface, rinsed with PBS buffer after 15-20 min, and dried to obtain a glucose oxidase electrode.

[0011] Preferably, the concentration of the saturated ferrocene ethanol solution in S1 is 16-22 g / L; the volume ratio of the filtrate to the acetic acid solution is 1:18-25, and the mass percentage of the acetic acid solution is 0.5%-1%.

[0012] Preferably, the deacetylation degree of the chitosan in S2 is 85% to 95%, the pH of the chitosan solution is 5.0 to 6.0, and the mass percentage of the chitosan is 0.5% to 2%.

[0013] Preferably, the concentration of glucose oxidase in S3 is 180-280 U / mg, the pH value of the PBS buffer is 7.3-7.5, and the concentration is 0.05-0.1 mol / L; the glutaraldehyde solution is composed of 45% glutaraldehyde by mass and PBS buffer in a volume ratio of 2:98; the electrode uses a Pt electrode as a base electrode.

[0014] Preferably, the mixing container includes a mixing barrel, the bottom of which is fixedly connected to a fixed seat; the bottom of the mixing barrel is connected and fixedly connected to a discharge pipe, the middle part of the discharge pipe is connected and fixedly connected to a spherical tube, and a discharge valve is installed on the top of the discharge pipe; two fixed plates are fixedly connected to the fixed seat, a bidirectional ball screw is rotatably connected between the two fixed plates via bearings, two movable plates are threadedly connected to the bidirectional ball screw, one side wall of the movable plate is rotatably connected to an extrusion roller via a rotating shaft, and the two extrusion rollers are symmetrically arranged on both sides of the spherical tube; a second motor is fixedly connected to one of the fixed plates, and the output shaft of the second motor is fixedly connected to one end of the reciprocating screw; two guide rods are fixedly connected between the two fixed plates, and the movable plates slide on the guide rods.

[0015] Preferably, a base plate is provided below the fixed seat, and a pressure sensor is provided between the base plate and the fixed seat; a conveyor is fixedly connected to the top of the base plate, and a drive motor is provided on the conveyor; two photoelectric sensors are symmetrically fixedly connected to the bottom of the discharge pipe through a connecting plate; and the photoelectric sensors are electrically connected to the drive motor.

[0016] Preferably, a first bracket and a second bracket are fixedly connected to the frame of the conveyor, and the first bracket and the second bracket are respectively arranged on both sides of the mixing barrel; a second lower hopper is fixedly connected to the second bracket, and the bottom end of the second lower hopper extends through the interior of the mixing barrel; a second solenoid valve is installed on the second lower hopper, and the second solenoid valve is electrically connected to the pressure sensor.

[0017] Preferably, a first lower hopper is fixedly connected to the first bracket, and the bottom end of the first lower hopper extends through and into the interior of the mixing barrel; a first solenoid valve is installed on the first lower hopper, a filter cartridge is inserted into the top of the first lower hopper, four fixing rods are fixedly connected to the top of the first lower hopper, the filter cartridge is inserted into the four fixing rods, the top of the first lower hopper is rotatably connected to four buckles via a rotating shaft, the buckles are respectively clamped with the fixing rods, and a torsion spring is installed on the buckles; the bottom of the filter cartridge is slidably connected to a limiting rod, the bottom of the limiting rod is fixedly connected to a floating ball, the top of the limiting rod is fixedly connected to a lifting plate, the lifting plate is fitted with the inner wall of the filter cartridge, a spring is provided between the lifting plate and the bottom of the filter cartridge, and the spring is sleeved on the outside of the limiting rod.

[0018] Preferably, a first motor is fixedly connected to the top of the mixing barrel, an output shaft of the first motor is fixedly connected to a rotating rod, a plurality of stirring paddles are fixedly connected to the rotating rod, two scraping rods are fixedly connected to the rotating rod, one side of the scraping rod is in contact with the inner wall of the mixing barrel, a water level gauge is installed on one of the scraping rods and the stirring paddle, and the water level gauge is electrically connected to the first solenoid valve.

[0019] Preferably, a collecting box is provided on the top of the bottom plate, a scraper is fixedly connected to the bottom end of the conveyor, the scraper is in close contact with the bottom of the conveyor, and the collecting box is arranged below the scraper.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. A method for preparing a glucose oxidase electrode according to the present invention comprises the following steps: by combining ethanol, ferrocene and chitosan, ferrocene is dispersed in chitosan gel with the aid of ethanol, glucose oxidase is dissolved in chitosan gel, and ferrocene is dispersed in chitosan gel containing glucose oxidase by utilizing the characteristics that ethanol has both polar hydroxyl groups and non-polar hydrocarbon groups and can volatilize during gel film formation, thereby preparing a glucose oxidase electrode with ferrocene dispersed inside the enzyme film, thereby increasing the contact area between ferrocene and glucose oxidase, thereby promoting the transfer of electrons in the glucose oxidase electrode, and preparing a glucose oxidase electrode with high sensitivity and a wide detection range.

[0022] 2. A method for preparing a glucose oxidase electrode described in the present invention comprises the following steps: through the cooperation of an extrusion roller, a spherical tube and a movable plate, the second motor is turned on, and the output shaft of the second motor drives the bidirectional ball screw to rotate, and the movable plate is driven to move in opposite directions by the bidirectional ball screw, and the movable plate drives the extrusion roller to move in opposite directions, and the extrusion roller squeezes the spherical tube when moving toward the middle and then releases it, and during the change of air pressure, the GOD / Fc-CS composite gel in the mixing barrel drips onto the electrode through the discharge pipe and the spherical tube. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be further described below in conjunction with the accompanying drawings.

[0024] Figure 1 is a flow chart of a preparation method according to an embodiment of the present invention;

[0025] Figure 2 is a stereogram of the present invention;

[0026] Figure 3 is a cross-sectional view of the present invention;

[0027] Figure 4 It is a schematic diagram of the structure of the mixing barrel and the squeezing ball in the present invention;

[0028] Figure 5 It is a schematic diagram of the structure of the squeezing roller in the present invention;

[0029] Figure 6 It is a schematic diagram of the structure of the filter cartridge in the present invention;

[0030] Figure 7 is a cross-sectional view of the filter cartridge of the present invention;

[0031] In the figure: 1. fixed seat; 11. mixing barrel; 12. first motor; 13. rotating rod; 14. stirring paddle; 15. scraping rod; 16. water level gauge; 17. feeding pipe; 171. spherical tube; 172. discharging valve; 173. squeezing roller; 174. fixed plate; 175. movable plate; 176. second motor; 177. guide rod; 178. bidirectional ball screw; 179. photoelectric sensor; 2. conveyor; 21. scraper; 22. collecting box; 23. first bracket; 231. first feeding hopper; 232. first solenoid valve; 233. filter cartridge; 234. fixed rod; 235. buckle; 236. lifting plate; 237. spring; 238. limit rod; 239. float; 24. second bracket; 241. second feeding hopper; 242. second solenoid valve; 3. bottom plate; 31. pressure sensor. DETAILED DESCRIPTION

[0032] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0033] Embodiment 1:

[0034] like Figure 1 As shown, a method for preparing a glucose oxidase electrode according to an embodiment of the present invention comprises the following steps:

[0035] S1: filtering the saturated ferrocene ethanol solution, taking out appropriate amounts of the filtrate and the acetic acid solution in proportion, and mixing them evenly in a mixing container to obtain a mixed solution;

[0036] S2: adding chitosan to the mixed solution at an amount of 5 g / L, stirring, and obtaining a ferrocene chitosan dispersion gel;

[0037] S3: Glucose was added to the ferrocene chitosan dispersion gel at a dosage of 0.5 g / L, and dropped on the electrode surface. It was dried at 3°C ​​for 15 h. Glutaraldehyde solution was added to the electrode surface. After 15 min, it was rinsed with PBS buffer and dried to obtain a glucose oxidase electrode.

[0038] The concentration of the saturated ferrocene ethanol solution in S1 is 16 g / L; the volume ratio of the filtrate to the acetic acid solution is 1:18, and the mass percentage of the acetic acid solution is 0.5%.

[0039] The deacetylation degree of the chitosan in S2 is 85%, the pH of the chitosan solution is 5.0, and the mass percentage of the chitosan is 0.5%.

[0040] The concentration of glucose oxidase in the S3 is 180 U / mg, the pH value of the PBS buffer is 7.3, and the concentration is 0.05 mol / L; the glutaraldehyde solution is composed of 45% glutaraldehyde by mass and PBS buffer in a volume ratio of 2:98; the electrode uses a Pt electrode as a base electrode.

[0041] Embodiment 2:

[0042] like Figure 1 As shown, a method for preparing a glucose oxidase electrode according to an embodiment of the present invention comprises the following steps:

[0043] S1: filtering the saturated ferrocene ethanol solution, taking out appropriate amounts of the filtrate and the acetic acid solution in proportion, and mixing them evenly in a mixing container to obtain a mixed solution;

[0044] S2: adding chitosan to the mixed solution at an amount of 6 g / L, stirring, and obtaining a ferrocene chitosan dispersion gel;

[0045] S3: Glucose was added to the ferrocene chitosan dispersion gel at a dosage of 1.3 g / L and dropped on the electrode surface. It was dried at 5°C for 13.5 h. Glutaraldehyde solution was added to the electrode surface. After 17 min, it was rinsed with PBS buffer and dried to obtain a glucose oxidase electrode.

[0046] The concentration of the saturated ferrocene ethanol solution in S1 is 19 g / L; the volume ratio of the filtrate to the acetic acid solution is 1:21, and the mass percentage of the acetic acid solution is 0.7%.

[0047] The deacetylation degree of the chitosan in S2 is 90%, the pH of the chitosan solution is 5.5, and the mass percentage of the chitosan is 1.3%.

[0048] The concentration of glucose oxidase in the S3 is 230 U / mg, the pH value of the PBS buffer is 7.4, and the concentration is 0.75 mol / L; the glutaraldehyde solution is composed of glutaraldehyde with a mass fraction of 45% and PBS buffer in a volume ratio of 2:98; the electrode uses a Pt electrode as a base electrode.

[0049] Embodiment three:

[0050] like Figure 1 As shown, a method for preparing a glucose oxidase electrode according to an embodiment of the present invention comprises the following steps:

[0051] S1: filtering the saturated ferrocene ethanol solution, taking out appropriate amounts of the filtrate and the acetic acid solution in proportion, and mixing them evenly in a mixing container to obtain a mixed solution;

[0052] S2: adding chitosan powder into the mixed solution at an amount of 7 g / L, stirring, and obtaining a ferrocene chitosan dispersion gel;

[0053] S3: Glucose oxidase was added to the ferrocene chitosan dispersion gel at a dosage of 2 g / L, and dropped on the electrode surface. It was dried at 7°C for 12 h, and glutaraldehyde solution was added to the electrode surface. After 20 min, it was rinsed with PBS buffer and dried to obtain a glucose oxidase electrode.

[0054] The concentration of the saturated ferrocene ethanol solution in S1 is 22 g / L; the volume ratio of the filtrate to the acetic acid solution is 1:25, and the mass percentage of the acetic acid solution is 1%.

[0055] The deacetylation degree of the chitosan in S2 is 95%, the pH of the chitosan solution is 6.0, and the mass percentage of the chitosan is 2%.

[0056] The concentration of glucose oxidase in the S3 is 280 U / mg, the pH value of the PBS buffer is 7.5, and the concentration is 0.1 mol / L; the glutaraldehyde solution is composed of 45% glutaraldehyde and PBS buffer in a volume ratio of 2:98; the electrode uses a Pt electrode as a base electrode.

[0057] The method for preparing a glucose oxidase electrode provided by the present invention comprises the following steps: when in use, ferrocene is dispersed in a chitosan gel by means of ethanol, and then glucose oxidase is dissolved in the chitosan gel. By utilizing the characteristics that ethanol has both polar hydroxyl groups and non-polar hydrocarbon groups and can volatilize in the gel film-forming process, ferrocene is dispersed in the chitosan gel containing glucose oxidase, and a glucose oxidase electrode with ferrocene dispersed inside the enzyme film is prepared, thereby increasing the contact area between ferrocene and glucose oxidase, thereby promoting the transfer of electrons in the glucose oxidase electrode, and preparing a glucose oxidase electrode with high sensitivity and a wide detection range.

[0058] like Figures 2 to 5As shown, the mixing container includes a mixing barrel 11, and a fixed seat 1 is fixedly connected to the bottom of the mixing barrel 11; a discharge pipe 17 is connected and fixedly connected to the bottom of the mixing barrel 11, and a spherical tube 171 is connected and fixedly connected to the middle part of the discharge pipe 17, and a discharge valve 172 is installed on the top of the discharge pipe 17; two fixed plates 174 are fixedly connected to the fixed seat 1, and a bidirectional ball screw 178 is rotatably connected between the two fixed plates 174 through a bearing, and two movable plates 175 are threadedly connected to the bidirectional ball screw 178, and a side wall of the movable plate 175 is rotatably connected to an extrusion roller 173 through a rotating shaft, and the two extrusion rollers 173 are symmetrically arranged on both sides of the spherical tube 171; a second motor 176 is fixedly connected to one of the fixed plates 174, and the output shaft of the second motor 176 is fixedly connected to one end of the reciprocating screw; two guide rods 177 are fixedly connected between the two fixed plates 174, and the movable plates 175 all slide on the guide rods 177.

[0059] When the squeezing roller 173 provided by the present invention is used, glucose oxidase is added to the prepared ferrocene chitosan dispersion gel to form a GOD / Fc-CS composite gel, which can be dripped onto the electrode surface. By opening the discharge valve 172, since the physical state of the GOD / Fc-CS composite gel is an elastic semi-solid state and has no fluidity, it is not easy to drip directly. By turning on the second motor 176, the output shaft of the second motor 176 drives the bidirectional ball screw 178 to rotate, and the bidirectional ball screw 178 is driven to rotate. The movable plate 175 is driven to move in the opposite direction, and the movable plate 175 drives the squeezing roller 173 to move in the opposite direction. The squeezing roller 173 squeezes the spherical tube 171 when moving toward the middle and then releases. During the air pressure change, the GOD / Fc-CS composite gel in the mixing barrel 11 will drip onto the electrode through the discharge pipe 17 and the spherical tube 171; during the movement of the movable plate 175, the movable plate 175 slides on the guide rod 177, and the guide rod 177 limits the movable plate 175 in the horizontal direction.

[0060] like Figure 3 and Figure 4 As shown, a base plate 3 is provided below the fixed seat 1, and a pressure sensor 31 is provided between the base plate 3 and the fixed seat 1; a conveyor 2 is fixedly connected to the top of the base plate 3, and a driving motor is provided on the conveyor 2; two photoelectric sensors 179 are symmetrically fixedly connected to the bottom of the discharge pipe 17 through a connecting plate; the photoelectric sensors 179 are electrically connected to the driving motor.

[0061] When the pressure sensor 31 and the photoelectric sensor 179 provided by the present invention are in use, by setting the pressure sensor 31, it is convenient to monitor the mass changes in the mixing barrel 11 in real time, so as to control the discharge amount of the material by controlling the pressure sensor 31; by setting the photoelectric sensor 179, it is convenient to stop the conveyor 2 at any time, so as to determine the dripping position of the GOD / Fc-CS composite gel.

[0062] like Figure 3 As shown, a first bracket 23 and a second bracket 24 are fixedly connected to the frame of the conveyor 2, and the first bracket 23 and the second bracket 24 are respectively arranged on both sides of the mixing barrel 11; a second lower hopper 241 is fixedly connected to the second bracket 24, and the bottom end of the second lower hopper 241 extends through the interior of the mixing barrel 11; a second solenoid valve 242 is installed on the second lower hopper 241, and the second solenoid valve 242 is electrically connected to the pressure sensor 31.

[0063] The second discharge hopper 241 provided by the present invention is used for discharging powder materials when in use. By opening the second solenoid valve 242, when the pressure sensor 31 reaches the set value, the second solenoid valve 242 will be closed to stop discharging. The number of second discharge hoppers 241 can be set according to actual working conditions.

[0064] like Figure 6 and Figure 7 As shown, a first lower hopper 231 is fixedly connected to the first bracket 23, and the bottom end of the first lower hopper 231 extends through the interior of the mixing barrel 11; a first solenoid valve 232 is installed on the first lower hopper 231, and a filter cartridge 233 is inserted at the top of the first lower hopper 231. Four fixing rods 234 are fixedly connected to the top of the first lower hopper 231, and the filter cartridge 233 is inserted on the four fixing rods 234. The top of the first lower hopper 231 is rotatably connected to four buckles via a rotating shaft. 235, the buckles 235 are respectively engaged with the fixing rods 234, and a torsion spring is installed on the buckles 235; the bottom of the filter cartridge 233 is slidably connected to a limiting rod 238, the bottom of the limiting rod 238 is fixedly connected with a float 239, the top of the limiting rod 238 is fixedly connected with a lifting plate 236, the lifting plate 236 is fitted with the inner wall of the filter cartridge 233, a spring 237 is provided between the lifting plate 236 and the bottom of the filter cartridge 233, and the spring 237 is sleeved on the outside of the limiting rod 238.

[0065] The filter cartridge 233 provided by the present invention is used to filter a saturated solution of ferrocene ethanol when in use. When the saturated solution of ferrocene ethanol is poured into the filter cartridge 233, the spring 237 will expand and contract under the action of gravity, driving the lifting plate 236 to move up and down; the liquid in the first lower hopper 231 increases, and under the action of the float 239, the float 239 drives the lifting plate 236 to move upward through the limit rod 238, thereby prompting the lifting plate 236 to include a lifting movement. During the movement, impurities on the inner wall of the filter cartridge 233 are scraped off to prevent impurities from clogging the filter cartridge 233. After a period of time, a large amount of impurities are collected in the filter cartridge 233, and the filter cartridge 233 needs to be removed by rotating the buckle 235. The buckle 235 releases the fixation of the fixing rod 234, and the filter cartridge 233 is cleaned.

[0066] like Figure 3 and Figure 7 As shown, a first motor 12 is fixedly connected to the top of the mixing barrel 11, and a rotating rod 13 is fixedly connected to the output shaft of the first motor 12. A plurality of stirring paddles 14 are fixedly connected to the rotating rod 13, and two scraping rods 15 are fixedly connected to the rotating rod 13. One side of the scraping rod 15 is in contact with the inner wall of the mixing barrel 11, and a water level gauge 16 is installed on one of the scraping rods 15 and the stirring paddle 14, and the water level gauge 16 is electrically connected to the first solenoid valve 232.

[0067] The water level meter 16 provided by the present invention is used to monitor the liquid position in the mixing barrel 11 when in use, and controls the output of the liquid in the first lower hopper 231 by the liquid level difference. When the specified liquid is reached, the first solenoid valve 232 is controlled to be closed. When working, by turning on the first motor 12, the first motor 12 drives the rotating rod 13 to rotate through the output shaft, and the rotating rod 13 drives the stirring paddle 14 and the scraping rod 15 to rotate, and the mixing is fully stirred and uniform. Under the action of the scraping rod 15, the GOD / Fc-CS composite gel is scraped to prevent the GOD / Fc-CS composite from adhering to the inner wall of the mixing barrel 11, and it is not easy to drip, resulting in waste of raw materials.

[0068] like Figure 2 As shown, a collecting box 22 is provided on the top of the bottom plate 3 , a scraper 21 is fixedly connected to the bottom end of the conveyor 2 , the scraper 21 is in close contact with the bottom of the conveyor 2 , and the collecting box 22 is provided below the scraper 21 .

[0069] The scraper 21 provided by the present invention is used to scrape the GOD / Fc-CS composite gel on the surface of the conveyor 2 when in use. The GOD / Fc-CS composite gel drips on the motor and is easily partially overflowed. If it is not cleaned in time, the working environment will be affected and the raw materials will be wasted. The scraper 21 is used to scrape the GOD / Fc-CS composite gel on the surface of the conveyor 2 into the collection box 22 to complete the recovery of the GOD / Fc-CS composite gel.

[0070] Working principle: When preparing the glucose oxidase electrode, ferrocene is dispersed in chitosan gel with the help of ethanol, and then glucose oxidase is dissolved in chitosan gel. Taking advantage of the characteristics of ethanol having both polar hydroxyl groups and non-polar hydrocarbon groups and being able to volatilize during the gel film formation process, ferrocene is dispersed in chitosan gel containing glucose oxidase, and a glucose oxidase electrode with ferrocene dispersed inside the enzyme membrane is prepared, thereby increasing the contact area between ferrocene and glucose oxidase, thereby promoting the transfer of electrons in the glucose oxidase electrode, and preparing a glucose oxidase electrode with high sensitivity and wide detection range; adding oxidase to the prepared ferrocene chitosan dispersed gel will form GOD / Fc-CS The composite gel can be dripped onto the electrode surface by opening the discharge valve 172. Since the physical state of the GOD / Fc-CS composite gel is an elastic semi-solid state without fluidity, it is not easy to drip directly. By turning on the second motor 176, the output shaft of the second motor 176 drives the bidirectional ball screw 178 to rotate, and the bidirectional ball screw 178 drives the moving plate 175 to move in the opposite direction. The moving plate 175 drives the squeezing roller 173 to move in the opposite direction. The squeezing roller 173 squeezes the spherical tube 171 when moving toward the middle and then releases it. During the air pressure change process, the GOD / Fc-CS composite gel in the mixing barrel 11 will drip onto the electrode through the discharge pipe 17 and the spherical tube 171.

[0071] By opening the second solenoid valve 242, when the pressure sensor 31 reaches the set value, the second solenoid valve 242 will be closed to stop discharging; the water level meter 16 is used to monitor the liquid position in the mixing barrel 11 when in use, and controls the output of the liquid in the first lower hopper 231 through the liquid level difference. When the specified liquid is reached, the first solenoid valve 232 is controlled to be closed;

[0072] When a saturated solution of ferrocene ethanol is poured into the filter cartridge 233, the spring 237 will expand and contract under the action of gravity, driving the lifting plate 236 to move up and down; the liquid in the first lower hopper 231 increases, and under the action of the float 239, the float 239 drives the lifting plate 236 to move upward through the limit rod 238, thereby prompting the lifting plate 236 to include a lifting movement. During the movement, impurities on the inner wall of the filter cartridge 233 are scraped off to prevent impurities from clogging the filter cartridge 233. After a period of time, a large amount of impurities are collected in the filter cartridge 233, and the filter cartridge 233 needs to be removed by rotating the buckle 235. The buckle 235 releases the fixation of the fixing rod 234, and the filter cartridge 233 is cleaned.

[0073] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing a glucose oxidase electrode, characterized in that: The following steps are involved: S1: filtering the saturated ferrocene ethanol solution, taking out appropriate amounts of the filtrate and the acetic acid solution in proportion, and mixing them evenly in a mixing container to obtain a mixed solution; S2: adding chitosan powder into the mixed solution in an amount of 5 to 7 g / L, stirring, and obtaining a ferrocene chitosan dispersion gel; S3: Glucose oxidase is added to ferrocene chitosan dispersion gel at a dosage of 0.5-2 g / L, and dripped on the electrode surface, dried at 3-7°C for 12-15 h, glutaraldehyde solution is dripped on the electrode surface, rinsed with PBS buffer after 15-20 min, and dried to obtain a glucose oxidase electrode.

2. The method for preparing a glucose oxidase electrode according to claim 1, characterized in that: The concentration of the saturated ferrocene ethanol solution in S1 is 16-22 g / L; the volume ratio of the filtrate to the acetic acid solution is 1:18-25, and the mass percentage of the acetic acid solution is 0.5%-1%.

3. The method for preparing a glucose oxidase electrode according to claim 2, characterized in that: The deacetylation degree of the chitosan in S2 is 85% to 95%, the pH of the chitosan solution is 5.0 to 6.0, and the mass percentage of the chitosan is 0.5% to 2%.

4. The method for preparing a glucose oxidase electrode according to claim 3, characterized in that: The concentration of glucose oxidase in the S3 is 180-280 U / mg, the pH value of the PBS buffer is 7.3-7.5, and the concentration is 0.05-0.1 mol / L; the glutaraldehyde solution is composed of 45% glutaraldehyde by mass and PBS buffer in a volume ratio of 2:98; the electrode uses a Pt electrode as a base electrode.

5. The method for preparing a glucose oxidase electrode according to claim 4, characterized in that: The mixing container comprises a mixing barrel (11), the bottom of the mixing barrel (11) is fixedly connected to a fixing seat (1); the bottom of the mixing barrel (11) is connected and fixedly connected to a discharge pipe (17), the middle of the discharge pipe (17) is connected and fixedly connected to a spherical tube (171), and the top of the discharge pipe (17) is installed with a discharge valve (172); two fixing plates (174) are fixedly connected to the fixing seat (1), and a bidirectional ball screw (178) is rotatably connected between the two fixing plates (174) via a bearing, and the bidirectional ball screw (178) is rotatably connected to the two fixing plates (174). ) are threadedly connected to two movable plates (175), one side wall of the movable plate (175) is rotatably connected to an extrusion roller (173) via a rotating shaft, and the two extrusion rollers (173) are symmetrically arranged on both sides of the spherical tube (171); a second motor (176) is fixedly connected to one of the fixed plates (174), and the output shaft of the second motor (176) is fixedly connected to one end of the reciprocating screw rod; two guide rods (177) are fixedly connected between the two fixed plates (174), and the movable plates (175) slide on the guide rods (177).

6. The method for preparing a glucose oxidase electrode according to claim 5, characterized in that: A bottom plate (3) is provided below the fixed seat (1), and a pressure sensor (31) is provided between the bottom plate (3) and the fixed seat (1); a conveyor (2) is fixedly connected to the top of the bottom plate (3), and a drive motor is provided on the conveyor (2); two photoelectric sensors (179) are symmetrically fixedly connected to the bottom of the discharge pipe (17) through a connecting plate; and the photoelectric sensors (179) are electrically connected to the drive motor.

7. The method for preparing a glucose oxidase electrode according to claim 6, characterized in that: A first bracket (23) and a second bracket (24) are fixedly connected to the frame of the conveyor (2), and the first bracket (23) and the second bracket (24) are respectively arranged on both sides of the mixing barrel (11); a second lower hopper (241) is fixedly connected to the second bracket (24), and the bottom end of the second lower hopper (241) extends through the interior of the mixing barrel (11); a second solenoid valve (242) is installed on the second lower hopper (241), and the second solenoid valve (242) is electrically connected to the pressure sensor (31).

8. The method for preparing a glucose oxidase electrode according to claim 7, characterized in that: A first lower hopper (231) is fixedly connected to the first bracket (23), and the bottom end of the first lower hopper (231) extends through the interior of the mixing barrel (11); a first solenoid valve (232) is installed on the first lower hopper (231), a filter cartridge (233) is inserted at the top of the first lower hopper (231), four fixing rods (234) are fixedly connected to the top of the first lower hopper (231), and the filter cartridge (233) is inserted on the four fixing rods (234); the top of the first lower hopper (231) is rotatably connected to four buckles (235) via a rotating shaft, The buckles (235) are respectively engaged with the fixing rods (234), and a torsion spring is installed on the buckles (235); the bottom of the filter cartridge (233) is slidably connected to a limit rod (238), the bottom of the limit rod (238) is fixedly connected with a floating ball (239), the top of the limit rod (238) is fixedly connected with a lifting plate (236), the lifting plate (236) is in contact with the inner wall of the filter cartridge (233), a spring (237) is provided between the lifting plate (236) and the bottom of the filter cartridge (233), and the spring (237) is sleeved on the outer side of the limit rod (238).

9. The method for preparing a glucose oxidase electrode according to claim 8, characterized in that: A first motor (12) is fixedly connected to the top of the mixing barrel (11); a rotating rod (13) is fixedly connected to the output shaft of the first motor (12); a plurality of stirring paddles (14) are fixedly connected to the rotating rod (13); two scraping rods (15) are fixedly connected to the rotating rod (13); one side of the scraping rod (15) is in contact with the inner wall of the mixing barrel (11); a water level gauge (16) is installed on one of the scraping rods (15) and the stirring paddle (14); and the water level gauge (16) is electrically connected to the first solenoid valve (232).

10. The method for preparing a glucose oxidase electrode according to claim 9, characterized in that: A collecting box (22) is provided on the top of the bottom plate (3), a scraper (21) is fixedly connected to the bottom end of the conveyor (2), the scraper (21) is in close contact with the bottom of the conveyor (2), and the collecting box (22) is provided below the scraper (21).

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