A sulfite biosensor and its preparation method and measurement method

Abstract

The present invention relates to the field of biosensor technology, and in particular to a sulfite biosensor and a preparation method and a measuring method thereof, wherein the sulfite biosensor comprises a dissolved oxygen electrode body and a coating coated on the surface of the electrode membrane of the dissolved oxygen electrode body, and the coating comprises the following raw materials by mass fraction: 25-35 parts of polyphenol oxidase, 35-45 parts of modified gelatin, 20-25 parts of sugar alcohol, 1-5 parts of modified graphite and 1-5 parts of epoxy resin. On the dissolved oxygen electrode membrane, the stability of the coating is effectively improved due to the addition of modified gelatin and epoxy resin, and the addition of modified graphite gives the electrode material excellent conductivity, so that electrons can be directly exchanged between the target substance, the electrode material and the electrode. Therefore, the sulfite biosensor prepared by the present invention can be continuously and stably operated for 3 hours at an operating temperature of 40°C, has good stability, no activity loss, and the sensitivity of the sensor is improved by about 30% compared with traditional biosensors.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to a sulfite biosensor and a preparation method and a measurement method thereof. Background Art

[0002] Wet flue gas desulfurization (WFGD) is a process for removing sulfur dioxide from flue gas by contacting the flue gas with an alkaline slurry dispersed in a spray tower. After WFGD of the flue gas, the slurry collected or accumulated in the tank absorbs sulfur dioxide and reacts with water to produce sulfite (SO3 2- ), the generated sulfite reacts with the oxygen supplied to the reaction slurry to produce sulfate (SO4 2- ). This method of ensuring that all sulfite reacts to form sulfate is called forced oxidation. The disadvantage of forced oxidation is that it is very difficult to measure or assess the sulfite concentration in the reaction slurry.

[0003] The basic methods for sulfite detection include the Monier Williams method (acid distillation followed by titration) and the colorimetric method based on hydrochloric acid pararosaniline dye. Other commonly used methods include iodine titration, gas chromatography, liquid chromatography, ion chromatography, etc. Masoom and Townshend proposed an enzymatic method for the determination of sulfite by amperometry in 1986, and derived other detection systems (voltammetry, photometry, etc.), which enzymatically oxidize sulfite by sulfite oxidase (SOD) and monitor the formed hydrogen peroxide with a hydrogen peroxide biosensor to calculate the sulfite content. Masoom et al. developed a flow injection analysis system to determine sulfite based on the current measurement of enzymatically generated hydrogen peroxide.

[0004] However, amperometric biosensors based on hydrogen peroxide detection have the following disadvantages: 1. The electrochemical oxidation of hydrogen peroxide depends on pH and oxygen concentration, and these two parameters must be strictly controlled; 2. In aqueous samples, oxygen concentration is a limiting factor that affects the linear range of the sensor; 3. The redox potential required for the electrochemical oxidation of hydrogen peroxide is relatively high (+0.7 V), which can oxidize other interfering compounds.

[0005] To address this problem, researchers have improved the current-type biosensor by using immobilized enzyme media and derivatized electrode surfaces to enable it to operate at low redox potentials. Hlavay et al. developed a chemiluminescent fiber optic biosensor that relies on immobilizing sulfite oxidase and peroxidase on different membranes and detecting chemiluminescent signals. The hydrogen peroxide produced by the sulfite oxidase-catalyzed reaction reacts with the substrate 3-amino-benzenedicarboxylic acid hydrazide in the presence of alkaline media and horseradish peroxidase.

[0006] However, the existing methods for measuring the concentration of sulfite in substances and sulfite sensors have many major problems, such as many limiting factors, difficult control, narrow sensor linear range, and high cost. In addition, if the sensor is operated at an operating temperature above 40°C for more than half an hour, the biosensor will lose activity and have poor stability. On the other hand, the sensor has low sensitivity, resulting in a slow sensor response speed, which affects the use effect of the sensor.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The purpose of the present invention is to provide a sulfite biosensor and its preparation method and measurement method, which is a sulfite concentration measurement method based on dissolved oxygen, which is different from the method of calculating the sulfite content by monitoring hydrogen peroxide. In addition, the biosensor can operate stably for 3 hours at an operating temperature of 40°C, has good stability, no activity loss, and the sensitivity of the sensor is improved by about 30% compared with traditional biosensors.

[0009] In a first aspect, the present invention provides a sulfite biosensor, comprising a dissolved oxygen electrode body and a coating coated on the surface of an electrode membrane of the dissolved oxygen electrode body, wherein the coating comprises the following raw materials in mass fractions: 25-35 parts of polyphenol oxidase, 35-45 parts of modified gelatin, 20-25 parts of sugar alcohol, 1-5 parts of modified graphite and 1-5 parts of epoxy resin.

[0010] The principle of the dissolved oxygen-based sulfite biosensor for measuring sulfite concentration is that polyphenol oxidase (PPO) enzyme catalyzes the O-hydroxylation of monophenol to O-diphenol, which can further catalyze the oxidation of O-diphenol to produce O-quinone. Sulfite has an inhibitory effect on polyphenol oxidase, and in the presence of sulfite, the oxidation of catechol catalyzed by polyphenol oxidase copper can be delayed. Therefore, the standard curve obtained by the oxygen consumption level of the polyphenol oxidase reaction in the biological material and the reduction of the oxygen consumption level by sulfite can achieve the measurement of sulfite in the sample to be tested.

[0011] Coating a coating containing polyphenol oxidase, modified gelatin, sugar alcohol, and modified graphite epoxy resin on the dissolved oxygen electrode membrane can give the biosensor excellent temperature resistance and sensitivity. This is because the addition of modified gelatin and epoxy resin can effectively improve the stability of the coating and prevent the coating from falling off during the detection process; and the addition of sugar alcohol and modified graphite gives the electrode material excellent conductivity, allowing electrons to be directly exchanged between the target substance, the electrode material and the electrode, thereby giving the biosensor higher sensitivity and anti-interference ability. Therefore, the sulfite biosensor prepared by the present invention can operate continuously and stably for 3 hours at an operating temperature of 40°C, has good stability, no activity loss, and the sensitivity of the biosensor is improved by about 30% compared with traditional biosensors.

[0012] As a preferred embodiment of the present technical solution, the modified gelatin used in the present invention is gelatin modified with quaternary ammonium salt. Modifying the gelatin with quaternary ammonium salt can give the coating certain antibacterial properties, stability and water resistance. During the modification process, the present invention does not strictly limit the mass ratio of gelatin to quaternary ammonium salt, and preferably is 6: (0.8-1.2).

[0013] In the present invention, the specific preparation method of modified gelatin includes: dissolving a gelatin solution in an organic solvent, then adding a quaternary ammonium salt to modify the gelatin, and after the reaction is complete, separating the organic solvent to obtain modified gelatin; wherein the organic solvent includes any one of acetone, ethanol and isopropanol, and the concentration of the gelatin solution is 110-160g / L;

[0014] When the gelatin solution is dissolved in the organic solvent, in order to improve the dissolution effect of gelatin, it can be stirred and dissolved at 25-45°C for 1-3 hours; similarly, during the modification, in order to obtain a higher yield, the reaction can be stirred at 30-40°C for 3-7 hours; finally, after the reaction is completed, the organic solvent is distilled and separated at 55-75°C to obtain modified gelatin with higher purity.

[0015] As a preferred embodiment of the present technical solution, in order to further improve the conductivity of the coating, the modified graphite used in the present invention is spherical graphite modified with ethanol. The improvement of the spherical graphite by ethanol can effectively improve the continuous dispersion of the spherical graphite, thereby ensuring the excellent conductivity of the electrode material, so that electrons can be directly exchanged between the target substance, the electrode material and the electrode, thereby improving the sensitivity of the biosensor. During the modification process, the present invention does not strictly limit the mass ratio of spherical graphite to ethanol, and preferably is 3: (5-9).

[0016] In the present invention, the specific preparation method of modified graphite comprises: firstly grinding spherical graphite with a ball mill, pouring the ground spherical graphite into an ultra-low temperature refrigeration device and cooling it to -40°C to -60°C; at the same time, heating ethanol to keep the temperature of ethanol at 66-70°C, and sprinkling low-temperature graphite powder into ethanol under a vacuum environment. Under the action of thermal expansion and contraction, the graphite powder is broken again, and after repeatedly rinsing with clean water, the modified graphite can be obtained.

[0017] To facilitate reaction detection, the polyphenol oxidase used in the present invention is derived from plant tissue homogenate or homogenized extraction.

[0018] In a second aspect, the present invention further provides a method for preparing the above-mentioned sulfite biosensor, which should also fall within the scope of protection of the present invention and comprises the following steps:

[0019] The polyphenol oxidase, modified gelatin, sugar alcohol, modified graphite and epoxy resin are stirred, dissolved and mixed at 35-42° C., coated on the surface of the electrode membrane of the dissolved oxygen electrode body, and dried to obtain a sulfite biosensor.

[0020] The present invention does not strictly limit the specific drying conditions. Specifically, the temperature can be controlled to be 1-5°C and the time can be 30-90 minutes. Low-temperature drying is performed to avoid uneven heating affecting the stability of the coating.

[0021] In a third aspect, the present invention also discloses a method for measuring sulfite using the above sulfite biosensor, comprising the following steps:

[0022] S1. Add phenols to the standard sample and record the dissolved oxygen content;

[0023] S2. Add sulfite standard solution to the standard sample, and record the change in dissolved oxygen content, and draw a standard curve based on the change in dissolved oxygen content corresponding to sulfite standard solutions of different concentrations;

[0024] S3. Determine the sulfite concentration in the sample by the change in dissolved oxygen content.

[0025] As a preferred embodiment of the present technical solution, the phenols include any one of monophenols and bisphenols. Among them, monophenols include phenol, guaiacol, 2-methoxyphenol, 2-methoxy-4-methylphenol and 2,6-dimethoxyphenol, and bisphenols include catechol, resorcinol, and the like.

[0026] The sulfite biosensor of the present invention has at least the following technical effects:

[0027] The present invention prepares a novel sulfite biosensor based on dissolved oxygen by coating a coating material made of polyphenol oxidase, modified gelatin, sugar alcohol, modified graphite and epoxy resin on a dissolved oxygen electrode membrane. The test principle of the biosensor is that sulfite has an inhibitory effect on the oxidation of phenol. Therefore, the standard curve obtained by the oxygen consumption level of the polyphenol oxidase reaction in the biomaterial and the reduction of the oxygen consumption level by sulfite can realize the measurement of sulfite in the sample to be tested. On the dissolved oxygen electrode membrane, the addition of modified gelatin and epoxy resin effectively improves the stability of the coating, and the addition of modified graphite gives the electrode material excellent conductivity, so that electrons can be directly exchanged between the target substance, the electrode material and the electrode, thereby giving the biosensor higher sensitivity and anti-interference ability;

[0028] 2. The sulfite biosensor prepared by the present invention obtains a linear range of analytical response between 0.01-10mM sulfite, and the biosensor can operate stably and continuously for 3 hours at an operating temperature of 40°C, has good stability, and has no activity loss. The sensitivity of the sensor is improved by about 30% compared with traditional biosensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 The present invention is a flow chart of the method for measuring sulfite based on the dissolved oxygen biosensor. DETAILED DESCRIPTION

[0031] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0032] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this description, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0033] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1

[0035] The coating material comprises: 33 parts of polyphenol oxidase, 37 parts of modified gelatin, 23 parts of sugar alcohol, 5 parts of modified graphite and 2 parts of epoxy resin.

[0036] Preparation method of modified gelatin:

[0037] A gelatin solution with a concentration of 150 g / L is dissolved in acetone and poured into a reactor. The mixture is stirred and reacted at 35° C. for 2 hours. A quaternary ammonium salt is then added and stirred and reacted at 37° C. for 5 hours. After the reaction is completed, acetone is distilled and separated at 65° C. to obtain modified gelatin, wherein the mass ratio of the gelatin solution, the quaternary ammonium salt and the acetone solvent is controlled at 6:1:3.

[0038] Preparation method of modified graphite:

[0039] Obtain natural spherical graphite, use a ball mill to grind the natural spherical graphite, pour the ground graphite powder into an ultra-low temperature refrigeration device, cool to -50°C, heat ethanol and keep the temperature of ethanol at 69°C, sprinkle the low-temperature graphite powder into ethanol in a vacuum environment, break the graphite powder again under the action of thermal expansion and contraction, rinse repeatedly with clean water, and dry to obtain modified graphite, wherein the ratio of natural spherical graphite to ethanol is controlled at 3:7.

[0040] Preparation method of sensor:

[0041] Polyphenol oxidase, modified gelatin, sugar alcohol, modified graphite and epoxy resin are stirred and mixed at a temperature of 38°C, and coated on the surface of the dissolved oxygen electrode membrane after being completely dissolved. After coating, the membrane is dried at a temperature of 4°C for 70 minutes to obtain a sulfite biosensor.

[0042] The test method includes the following steps:

[0043] S1: Get a sulfite biosensor, add a certain amount of phenols, the dissolved oxygen decreases, and record the dissolved oxygen content;

[0044] S2: Add 0.01-10mM different concentrations of sulfite standard solutions respectively, the dissolved oxygen rises, calculate the change in dissolved oxygen, and draw a standard curve based on the change in dissolved oxygen content corresponding to different concentrations of sulfite standard solutions;

[0045] S3: Add the sample to be tested and determine the sulfite concentration in the sample by the change in dissolved oxygen content.

[0046] Example 2

[0047] The coating material comprises: 31 parts of polyphenol oxidase, 39 parts of modified gelatin, 24 parts of sugar alcohol, 4 parts of modified graphite and 2 parts of epoxy resin.

[0048] Preparation method of modified gelatin:

[0049] A gelatin solution with a concentration of 120 g / L is dissolved in acetone and poured into a reactor. The mixture is stirred and reacted at 35° C. for 1 hour. A quaternary ammonium salt is then added and stirred and reacted at 32° C. for 6 hours. After the reaction is completed, acetone is distilled and separated at 65° C. to obtain modified gelatin, wherein the mass ratio of the gelatin solution, the quaternary ammonium salt and the acetone solvent is controlled at 6:1:3.

[0050] Preparation method of modified graphite:

[0051] Obtain natural spherical graphite, use a ball mill to grind the natural spherical graphite, grind it into graphite powder, pour it into an ultra-low temperature refrigeration device, cool it to -40°C, heat ethanol and keep the temperature of ethanol at 66°C, sprinkle the low-temperature graphite powder into ethanol in a vacuum environment, break the graphite powder again under the action of thermal expansion and contraction, rinse it repeatedly with clean water, and dry it to obtain modified graphite, wherein the ratio of natural spherical graphite to ethanol is controlled at 3:6.

[0052] Preparation method of sensor:

[0053] Polyphenol oxidase, modified gelatin, sugar alcohol, modified graphite and epoxy resin are stirred and mixed at a temperature of 40°C, and coated on the surface of the dissolved oxygen electrode membrane after being completely dissolved. After coating, the membrane is dried at a temperature of 5°C for 60 minutes to obtain a sulfite biosensor.

[0054] The test method is the same as in Example 1.

[0055] Example 3

[0056] The coating material comprises: 29 parts of polyphenol oxidase, 42 parts of modified gelatin, 23 parts of sugar alcohol, 3 parts of modified graphite and 3 parts of epoxy resin.

[0057] Preparation method of modified gelatin:

[0058] A gelatin solution with a concentration of 160 g / L is dissolved in acetone and poured into a reactor. The mixture is stirred and reacted at 40° C. for 2 hours. A quaternary ammonium salt is then added and stirred and reacted at 40° C. for 4 hours. After the reaction is completed, acetone is distilled and separated at 65° C. to obtain modified gelatin, wherein the mass ratio of the gelatin solution, the quaternary ammonium salt and the acetone solvent is controlled at 6:1.2:3.

[0059] Preparation method of modified graphite:

[0060] Obtain natural spherical graphite, use a ball mill to grind the natural spherical graphite, pour the ground graphite powder into an ultra-low temperature refrigeration device, cool to -50°C, heat ethanol and keep the temperature of ethanol at 70°C, sprinkle the low-temperature graphite powder into ethanol in a vacuum environment, break the graphite powder again under the action of thermal expansion and contraction, rinse repeatedly with clean water, and dry to obtain modified graphite, wherein the ratio of natural spherical graphite to ethanol is controlled at 3:8.

[0061] Preparation method of sensor:

[0062] Polyphenol oxidase, modified gelatin, sugar alcohol, modified graphite and epoxy resin are stirred and mixed at a temperature of 35°C, and coated on the surface of the dissolved oxygen electrode membrane after being completely dissolved. After coating, the membrane is dried at a temperature of 2°C for 90 minutes to obtain a sulfite biosensor.

[0063] The test method is the same as in Example 1.

[0064] Example 4

[0065] The coating material comprises: 27 parts of polyphenol oxidase, 43 parts of modified gelatin, 22 parts of sugar alcohol, 4 parts of modified graphite and 4 parts of epoxy resin.

[0066] Preparation method of modified gelatin:

[0067] A gelatin solution with a concentration of 160 g / L is dissolved in acetone and poured into a reactor. The mixture is stirred and reacted at 45° C. for 2 hours. A quaternary ammonium salt is then added and stirred and reacted at 35° C. for 5 hours. After the reaction is completed, acetone is distilled and separated at 65° C. to obtain modified gelatin, wherein the mass ratio of the gelatin solution, the quaternary ammonium salt and the acetone solvent is controlled at 6:1.2:3.

[0068] Preparation method of modified graphite:

[0069] Obtain natural spherical graphite, use a ball mill to grind the natural spherical graphite, pour the ground graphite powder into an ultra-low temperature refrigeration device, cool to -60°C, heat ethanol and keep the temperature of ethanol at 65°C, sprinkle the low-temperature graphite powder into ethanol in a vacuum environment, break the graphite powder again under the action of thermal expansion and contraction, rinse repeatedly with clean water, and dry to obtain modified graphite, wherein the ratio of natural spherical graphite to ethanol is controlled at 3:7.

[0070] Preparation method of sensor:

[0071] Polyphenol oxidase, modified gelatin, sugar alcohol, modified graphite and epoxy resin are stirred and mixed at a temperature of 40°C, and coated on the surface of the dissolved oxygen electrode membrane after being completely dissolved. After coating, the membrane is dried at a temperature of 4°C for 70 minutes to obtain a sulfite biosensor.

[0072] The test method is the same as in Example 1.

[0073] Example 5

[0074] The coating material comprises: 25 parts of polyphenol oxidase, 45 parts of modified gelatin, 23 parts of sugar alcohol, 2 parts of modified graphite and 5 parts of epoxy resin.

[0075] Preparation method of modified gelatin:

[0076] A gelatin solution with a concentration of 140 g / L is dissolved in acetone and poured into a reactor. The mixture is stirred and reacted at 35° C. for 2 hours. A quaternary ammonium salt is then added and stirred and reacted at 37° C. for 5 hours. After the reaction is completed, acetone is distilled and separated at 65° C. to obtain modified gelatin, wherein the mass ratio of the gelatin solution, the quaternary ammonium salt and the acetone solvent is controlled at 6:1:3.

[0077] Preparation method of modified graphite:

[0078] Obtain natural spherical graphite, use a ball mill to grind the natural spherical graphite, grind it into graphite powder, pour it into an ultra-low temperature refrigeration device, cool it to -50°C, heat ethanol, keep the temperature of ethanol at 70°C, sprinkle the low-temperature graphite powder into ethanol in a vacuum environment, break the graphite powder again under the action of thermal expansion and contraction, rinse it repeatedly with clean water, and dry it to obtain modified graphite, wherein the ratio of natural spherical graphite to ethanol is controlled at 3:5.

[0079] Preparation method of sensor:

[0080] Polyphenol oxidase, modified gelatin, sugar alcohol, modified graphite and epoxy resin are stirred and mixed at a temperature of 38°C, and coated on the surface of the dissolved oxygen electrode membrane after being completely dissolved. After coating, the membrane is dried at a temperature of 4°C for 70 minutes to obtain a sulfite biosensor.

[0081] The test method is the same as in Example 1.

[0082] Comparative Example 1

[0083] A hydrogen peroxide biosensor was used to monitor hydrogen peroxide concentration to calculate sulfite content.

[0084] In order to study the performance indicators such as detection limit and sensitivity of the above biosensors, the performance of different biosensors was tested by cyclic voltammetry and chronoamperometry on the LK2005 electrochemical workstation, with the biosensor as the working electrode, platinum wire and saturated calomel electrode as the counter electrode and reference electrode respectively. , Table 1 Performance comparison of different sensors

[0085]

[0086] In summary, the sensitivity of the biosensor of the present invention is improved by about 30% compared with the traditional biosensor, and it can operate stably and continuously for 3 hours at an operating temperature of 40° C., with good stability and no activity loss.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for measuring sulfite using a sulfite biosensor, characterized in that: The sulfite biosensor comprises a dissolved oxygen electrode body and a coating coated on the surface of an electrode membrane of the dissolved oxygen electrode body. The coating comprises the following raw materials in mass fractions: 25-35 parts of polyphenol oxidase, 35-45 parts of modified gelatin, 20-25 parts of sugar alcohol, 1-5 parts of modified graphite and 1-5 parts of epoxy resin; The method for measuring sulfite by the sulfite biosensor comprises the following steps: S1. Add phenols to the standard sample and record the dissolved oxygen content; S2. Add sulfite standard solution to the standard sample, and record the change in dissolved oxygen content, and draw a standard curve based on the change in dissolved oxygen content corresponding to sulfite standard solutions of different concentrations; S3, determining the sulfite concentration in the sample to be tested by the change value of dissolved oxygen content; The standard curve obtained by the oxygen consumption level of the polyphenol oxidase reaction in the biological material and the reduction of the oxygen consumption level by sulfite can realize the measurement of sulfite in the sample to be tested; The modified gelatin is gelatin modified with a quaternary ammonium salt, and the mass ratio of gelatin to the quaternary ammonium salt is 6:(0.8-1.2); The modified graphite is spherical graphite modified by ethanol, and the mass ratio of spherical graphite to ethanol is 3:(5-9).

2. The method for measuring sulfite by a sulfite biosensor according to claim 1, characterized in that: The phenols include any one of monophenol and bisphenol.

3. The method for measuring sulfite by a sulfite biosensor according to claim 1, characterized in that: The polyphenol oxidase is extracted from plant tissues.

4. The method for measuring sulfite by a sulfite biosensor according to claim 1, characterized in that: The preparation method of the modified gelatin comprises: The gelatin solution is dissolved in an organic solvent, and then a quaternary ammonium salt is added to modify the gelatin. After the reaction is complete, the organic solvent is separated to obtain modified gelatin; The organic solvent includes any one of acetone, ethanol and isopropanol, and the concentration of the gelatin solution is 110-160 g / L; When the gelatin solution is dissolved in the organic solvent, stirring is performed at 25-45° C. for 1-3 hours; During the modification, stirring at 30-40°C for 3-7h; During the separation, the organic solvent is separated by distillation at 55-75°C.

5. The method for measuring sulfite using a sulfite biosensor according to claim 1, characterized in that: The preparation method of the modified graphite comprises: In a vacuum environment, spherical graphite powder at -40℃~-60℃ is sprinkled into ethanol at 66-70℃, and after repeated washing, modified graphite is obtained.

6. The method for measuring sulfite using a sulfite biosensor according to claim 1, characterized in that: The preparation method of the sulfite biosensor comprises the following steps: The polyphenol oxidase, modified gelatin, sugar alcohol, modified graphite and epoxy resin are stirred, dissolved and mixed at 35-42° C., coated on the surface of the electrode membrane of the dissolved oxygen electrode body, and dried to obtain a sulfite biosensor.

7. The method for measuring sulfite using a sulfite biosensor according to claim 6, characterized in that: During the drying, the temperature is controlled at 1-5°C and the time is 30-90 minutes.

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