A method for detecting pesticide residues in food using enzyme biosensor

Abstract

The present application proposes a method for detecting pesticide residues in food using an enzyme biosensor, which relates to the field of pesticide detection technology. The method comprises: cleaning and drying an electrode substrate and placing it in an incubator; performing multiple silver mirror reactions on the electrode substrate to form a silver reference electrode and a corresponding silver conductive tape; placing carbon slurry on the silver reference electrode and covering the electrode's protective layer with polyvinyl chloride insulating tape; cleaning the carrier surface, drying it, dripping a fixative, and then cutting it to obtain an enzyme sheet; fixing the enzyme sheet on a thin-layer base electrode to obtain an enzyme biosensor; dripping a pesticide solution onto the carbon slurry surface of the thin-layer base electrode, dispersing it onto the enzyme sheet, and removing the enzyme sheet after the reaction is completed; dripping a buffer solution onto the electrode, applying a voltage to obtain a response signal, and analyzing the composition of the pesticide solution. The enzyme biosensor of the present application detects pesticide residue oxidation signals with stable and regular results and can be widely used in the detection of pesticide residues in food.

Description

Technical Field

[0001] The present application relates to the technical field of pesticide detection, and in particular to a method for detecting pesticide residues in food using an enzyme biosensor. Background Art

[0002] With increasing consumer demand, agricultural production growth has become a challenge for farmers. To ensure economic returns, highly toxic pesticides and pesticide residues are widely used in daily crop cultivation, undoubtedly posing a serious threat to human life. According to statistics, global annual production of chemical pesticides has reached 2 million tons, including over 1,000 synthetic pesticides. Pesticide residues in vegetables, fruits, and grain crops are primarily absorbed and accumulated immediately after use. Pesticides applied to crops adhere to them, while others are dispersed around them. Some of these pesticides are dispersed into the atmosphere, where they are metabolized and the levels in the body and environment decrease to a certain extent. In agricultural production, the misuse or overuse of pesticides has a cumulative impact on crops. It is reported that 10% of cancer cases are caused by long-term exposure to pesticide residues. Therefore, routine testing for pesticide residues in agricultural products is gaining increasing attention.

[0003] At present, there are many studies on pesticide detection. Liu Mei reviewed aptamers that specifically bind to various pesticides and aptamer sensors for pesticide detection. Lee Minwoo used alkylene dimers to improve SERS (surface enhanced Raman scattering) sensor filter paper, which can be used for high-sensitivity pesticide detection. Wei Wei developed a flexible, durable and reusable silver-functionalized SERS matrix for pesticide residue detection. Xu Xiaoyu developed a glove-based wearable sensor for non-invasive monitoring of organophosphorus pesticides. Chen Jie developed a flexible gel matrix based on nanocellulose-modified silver nanoparticles and explored its application in SERS rapid in situ detection of pesticides in fruits and vegetables. Farooq Saqib reviewed the pretreatment extraction of pesticides by different types of molecular imprinting polymers and described their specific extraction efficiency for single and multiple pesticides.

[0004] Pesticides are primarily used to protect plants, forest fruits, and their products from bacteria, pests, and weeds, thereby regulating plant growth and development. To more effectively monitor pesticide use and prevent direct damage caused by pesticide residues, it is necessary to promote and adopt rapid pesticide residue detection technologies suitable for on-site testing. Summary of the Invention

[0005] The purpose of this application is to provide a method for detecting pesticide residues in food using an enzyme biosensor, which has the advantages of being simple and having stable detection effects.

[0006] The present application solves the technical problem by adopting the following technical solutions.

[0007] The present invention provides a method for detecting pesticide residues in food using an enzyme biosensor, which comprises the following steps:

[0008] Preparation of a thin-layer base electrode: After cleaning and drying the electrode base, place it in an incubator, perform multiple silver mirror reactions on the electrode base to form a silver reference electrode and a corresponding silver conductive tape, and finally place carbon paste on the silver reference electrode, and cover the protective layer of the electrode with polyvinyl chloride insulating tape to obtain the thin-layer base electrode;

[0009] Preparation of enzyme tablets: clean the carrier surface, dry it, drip a fixative, dry it naturally, and cut it into the shape of the fixed agent to obtain enzyme tablets;

[0010] Detection of pesticide components: An enzyme sheet is fixed on a thin-layer base electrode to obtain an enzyme biosensor; pesticide solution is dripped onto the carbon slurry surface of the thin-layer base electrode and dispersed onto the enzyme sheet. The enzyme biosensor is reacted at 60°C and the enzyme sheet is removed after the reaction is completed; buffer solution is dripped onto the electrode, voltage is applied to obtain a response signal, and the composition of the pesticide solution is analyzed.

[0011] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0012] This application uses the silver mirror reaction method to prepare a silver reference electrode and an immobilized enzyme sheet, and establishes an enzyme biosensor, and verifies its performance; the results show that the enzyme biosensor of the present application obtains stable and regular results for the detection of pesticide residue oxidation signals, and can be widely used in the detection of pesticide residues in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0014] Figure 1 This is a schematic diagram of the production process of the thin-layer base electrode according to an embodiment of the present application;

[0015] Figure 2 This is a schematic diagram of the production process of the enzyme tablets according to the embodiment of the present application;

[0016] Figure 3 This is a diagram showing the effect of different amounts of silver mirror reaction on electrode potential in Experimental Example 1 of this application;

[0017] Figure 4 This is the effect of temperature on electrode potential in Experimental Example 1 of this application;

[0018] Figure 5 This is the electrode potential change curve of the silver reference electrode at different pH values ​​in Experimental Example 1 of this application;

[0019] Figure 6 This is the performance test process of the enzyme biosensor in Experimental Example 2 of this application;

[0020] Figure 7 is the response signal state of sensor 1 in Experimental Example 2 of this application;

[0021] Figure 8 is the response signal state of sensor 2 in Experimental Example 2 of this application;

[0022] Figure 9 This is the effect of temperature on sensor 1 in Experimental Example 2 of this application;

[0023] Figure 10 This is the effect of temperature on sensor 2 in Experimental Example 2 of this application;

[0024] Figure 11 This is the effect of enzyme concentration on sensor 1 in Experimental Example 2 of this application;

[0025] Figure 12 This is the effect of enzyme concentration on sensor 2 in Experimental Example 2 of this application;

[0026] Figure 13 This is a graph showing the consistency of the response signal of the enzyme biosensor in Experimental Example 2 of this application;

[0027] Figure 14 This is the effect of placement time on sensor 1 in Experimental Example 2 of this application;

[0028] Figure 15 This is the effect of placement time on sensor 2 in Experimental Example 2 of this application;

[0029] Figure 16 This is a graph showing the monitoring results of the oxidation signal in Experimental Example 2 of this application;

[0030] Figure 17 This is the monitoring result of the restored signal in Experimental Example 2 of this application. DETAILED DESCRIPTION

[0031] To make the purpose, technical solutions and advantages of the examples of the present application clearer, the technical solutions in the examples of the present application will be described clearly and completely below. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0032] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present application will be described in detail below with reference to specific embodiments.

[0033] A method for detecting pesticide residues in food using an enzyme biosensor comprises the following steps:

[0034] Preparation of a thin-layer base electrode: After cleaning and drying the electrode base, place it in an incubator, perform multiple silver mirror reactions on the electrode base to form a silver reference electrode and a corresponding silver conductive tape, and finally place carbon paste on the silver reference electrode, and cover the protective layer of the electrode with polyvinyl chloride insulating tape to obtain the thin-layer base electrode;

[0035] Preparation of enzyme tablets: cleaning the carrier surface, drying it, and then dripping a fixative containing bovine serum albumin and organophosphorus hydrolase. After natural drying, the tablets are cut according to the shape of the fixed agent to obtain immobilized enzyme tablets.

[0036] Detection of pesticide components: An immobilized enzyme sheet is fixed on a thin-layer base electrode to obtain an enzyme biosensor; a pesticide solution is dripped onto the carbon slurry surface of the thin-layer base electrode and dispersed onto the enzyme sheet. The enzyme biosensor is reacted at 60°C and the enzyme sheet is removed after the reaction is completed; a buffer solution is dripped onto the electrode, a voltage is applied to obtain a response signal, and the composition of the pesticide solution is analyzed.

[0037] In some embodiments of the present application, the electrode substrate is cleaned by sequentially using alcohol and dilute nitric acid.

[0038] In some embodiments of the present application, the silver mirror reaction is performed 9-11 times, and the electrode substrate is cleaned with distilled water and dried after each silver mirror reaction.

[0039] In some embodiments of the present application, the specific steps of each silver mirror reaction are: adding 5% sodium hydroxide solution to 2% silver nitrate solution, adding 2% ammonia water after the reaction, adding 10% glucose solution after dissolution is complete, and mixing to obtain a mixture; the mixture is evenly applied on the electrode substrate, and then reacted at a temperature of 42-48°C for 5-10 minutes, and the volume ratio of silver nitrate solution, sodium hydroxide solution, ammonia water, and glucose solution is 1.5:0.1:1.15:0.65.

[0040] In some embodiments of the present application, the carbon slurry is prepared from dimethyl silicone oil / liquid paraffin and high-purity graphite powder in a ratio of 3:7.

[0041] In some embodiments of the present application, the carrier of the enzyme sheet is a nitrocellulose membrane or a cellulose acetate membrane, and the natural drying temperature is below 25°C.

[0042] In some embodiments of the present application, the enzyme-containing fixative is prepared by mixing 10% bovine serum albumin solution and pure enzyme solution in a volume ratio of 1:1.

[0043] In some embodiments of the present application, the reaction time of the enzyme biosensor is 3-5 minutes.

[0044] In some embodiments of the present application, the buffer solution is a 50 mM phosphate buffer solution with a pH of 8.0, the applied voltage is 0.9-1 V, and a response signal is obtained after applying the voltage for 60 seconds.

[0045] The features and performance of the present application are further described in detail below with reference to the embodiments.

[0046] The materials and equipment used in each step of this application are as follows:

[0047] Preparation of thin-layer base electrodes: base plate (25.4 × 76.2 mm, Dikma, USA); high-purity graphite powder (≥1000 mesh, Merck, Germany); silver nitrate (Tianjin Third Chemical Reagent Factory); glucose (Tianjin Comeo); dimethyl silicone oil (Tianjin Ruijinte Chemical Co., Ltd.); p-nitrophenol (Shanghai Yuanju Biotechnology Co., Ltd.); ammonia (Sinopharm Chemical Reagent Co., Ltd.); sodium hydroxide (Luoyang Chemical Reagent Factory); polyvinyl chloride insulating tape (Shanghai Wokai).

[0048] WYJ-3A transistor-regulated DC power supply (Jiangsu Hanbang Technology Co., Ltd.); AL-204 electronic balance (Shanghai Ailang Instrument Co., Ltd.); PHS-25C pH meter (Agilent, USA); Miller-Q ultrapure water machine (Millipore, USA); 101-1AB temperature chamber (Jinhuanbao Instrument Factory).

[0049] Preparation of enzyme tablets: nitrocellulose membrane, cellulose acetate membrane (Zhejiang Taizhou Sijia Biochemical Plastic Factory); bovine serum albumin (Huamei Bioengineering Company); organophosphorus hydrolase (Huamei Bioengineering Company).

[0050] PHS-25C pH meter (Ningbo Haihai Saifu Experimental Instrument Factory); WYJ-3A transistor-regulated DC power supply (Shanghai Chenhua Instrument Co., Ltd.); 101-1AB temperature chamber (Jin Huanbao Instrument Factory).

[0051] Example 1

[0052] A method for detecting pesticide residues in food using an enzyme biosensor comprises the following steps:

[0053] Preparation of thin-layer base electrodes, such as Figure 1As shown: The electrode base is cleaned with alcohol and dilute nitric acid in sequence, then placed in an incubator after drying. The silver mirror reaction is performed on the electrode base 10 times. After each silver mirror reaction, the electrode is rinsed with distilled water, dried, and the next silver mirror reaction is performed to form a silver reference electrode and a corresponding silver conductive tape. A carbon paste is prepared by mixing dimethyl silicone oil and high-purity graphite powder in a mass ratio of 3:7. The carbon paste is placed on the silver reference electrode. The upper conductive part of the tape is pressed in and scraped off to make it shiny. The protective layer of the electrode is then covered with polyvinyl chloride insulating tape to obtain a thin-layer base electrode.

[0054] The specific process of the silver mirror reaction in this example is as follows: 1.5 mL of 2% silver nitrate solution is placed in a clean beaker, followed by the addition of 2 drops (0.1 mL) of 5% sodium hydroxide solution. After mixing, 23 drops (1.15 mL) of 2% ammonia solution are added and stirred until the brown precipitate is completely dissolved. 13 drops (0.65 mL) of 10% glucose solution are then added and mixed to obtain a mixture. The mixture is evenly applied to the electrode substrate with a rubber tip and then reacted at 45°C for 7 minutes.

[0055] The process steps for preparing enzyme tablets are as follows: Figure 2 As shown: a 10 cm × 10 cm nitrocellulose membrane was used as a carrier for the enzyme sheet, which was divided into 4 × 4 = 16 parts. The surface of the carrier was washed with distilled water, dried and stored below 4°C. When used, 10 μL of a fixative (prepared by a 1:1 volume ratio of 10% bovine serum albumin solution and organophosphorus hydrolase) was dropped into the membrane. After natural drying at 25°C, the membrane was cut according to the shape of the dried fixative to obtain 16 enzyme sheets.

[0056] Detecting pesticide components: An enzyme sheet was immobilized on a thin-film electrode to create an enzyme biosensor. 30 μL of an organophosphorus pesticide (0.1 g / L) was dripped onto the carbon paste surface of the thin-film electrode, dispersing it throughout the enzyme sheet. The enzyme biosensor was then placed in a 60°C oven for a 3-minute reaction. After the reaction, the enzyme biosensor was removed and the enzyme sheet was removed. 200 μL of a 50 mM, pH 8.0 phosphate buffer solution was quickly dripped onto the electrode. A voltage of 0.95 V was quickly applied to the thin-film electrode, and a response signal was obtained after 60 seconds. The response signal was read to determine the material and reagent composition of the immobilized enzyme sheet.

[0057] Example 2

[0058] A method for detecting pesticide residues in food using an enzyme biosensor comprises the following steps:

[0059] Preparation of a thin-layer base electrode: The electrode substrate is cleaned sequentially with alcohol and dilute nitric acid, then dried and placed in an incubator. Eleven silver mirror reactions are performed on the electrode substrate. After each silver mirror reaction, the electrode is rinsed with distilled water, dried, and subjected to the next silver mirror reaction to form a silver reference electrode and corresponding silver conductive tape. A carbon paste is prepared by mixing dimethyl silicone oil and high-purity graphite powder in a mass ratio of 3:7. The carbon paste is placed on the silver reference electrode, and the upper conductive portion of the tape is pressed in and scraped off to make it shiny. The protective layer of the electrode is then covered with polyvinyl chloride insulating tape to obtain a thin-layer base electrode.

[0060] The specific process of the silver mirror reaction in this example is as follows: 1.5 mL of 2% silver nitrate solution is placed in a clean beaker, followed by the addition of 2 drops (0.1 mL) of 5% sodium hydroxide solution. After mixing, 23 drops (1.15 mL) of 2% ammonia solution are added and stirred until the brown precipitate is completely dissolved. 13 drops (0.65 mL) of 10% glucose solution are then added and mixed to obtain a mixture. The mixture is evenly applied to the electrode substrate with a rubber tip and then reacted at 42°C for 8 minutes.

[0061] Preparation of enzyme tablets: A 10 cm × 10 cm nitrocellulose membrane was used as a carrier for the enzyme tablets, divided into 4 × 4 = 16 sections. The surface of the carrier was washed with distilled water, dried, and stored below 4°C. Upon use, 10 μL of a fixative (prepared with 10% bovine serum albumin solution and organophosphorus hydrolase in a volume ratio of 1:1) was added. After natural drying at 25°C, the tablets were cut according to the shape of the dried fixative to obtain 16 enzyme tablets.

[0062] Detecting pesticide components: An enzyme sheet was immobilized on a thin-film electrode to create an enzyme biosensor. 30 μL of an organophosphorus pesticide (0.1 g / L) was dripped onto the carbon paste surface of the thin-film electrode, dispersing it throughout the enzyme sheet. The enzyme biosensor was then placed in a 60°C oven for a 5-minute reaction. After the reaction, the enzyme biosensor was removed and the enzyme sheet was removed. 200 μL of a 50 mM, pH 8.0 phosphate buffer solution was quickly dripped onto the electrode. A voltage of 0.9 V was quickly applied to the thin-film electrode, and a response signal was obtained after 60 seconds. The response signal was read to determine the material and reagent composition of the immobilized enzyme sheet.

[0063] Example 3

[0064] A method for detecting pesticide residues in food using an enzyme biosensor comprises the following steps:

[0065] Preparation of a thin-layer base electrode: The electrode substrate is cleaned sequentially with alcohol and dilute nitric acid, then dried and placed in an incubator. Eleven silver mirror reactions are performed on the electrode substrate. After each silver mirror reaction, the electrode is rinsed with distilled water, dried, and subjected to the next silver mirror reaction to form a silver reference electrode and corresponding silver conductive tape. A carbon paste is prepared by mixing dimethyl silicone oil and high-purity graphite powder in a mass ratio of 3:7. The carbon paste is placed on the silver reference electrode, and the upper conductive portion of the tape is pressed in and scraped off to make it shiny. The protective layer of the electrode is then covered with polyvinyl chloride insulating tape to obtain a thin-layer base electrode.

[0066] The specific process of the silver mirror reaction in this example is as follows: 1.5 mL of 2% silver nitrate solution is placed in a clean beaker, followed by the addition of 2 drops (0.1 mL) of 5% sodium hydroxide solution. After mixing, 23 drops (1.15 mL) of 2% ammonia solution are added and stirred until the brown precipitate is completely dissolved. 13 drops (0.65 mL) of 10% glucose solution are then added and mixed to obtain a mixture. The mixture is evenly applied to the electrode substrate with a rubber tip and then reacted at 48°C for 5 minutes.

[0067] Preparation of enzyme tablets: A 10 cm × 10 cm nitrocellulose membrane was used as a carrier for the enzyme tablets, divided into 4 × 4 = 16 sections. The surface of the carrier was washed with distilled water, dried, and stored below 4°C. Upon use, 10 μL of a fixative (prepared with 10% bovine serum albumin solution and organophosphorus hydrolase in a volume ratio of 1:1) was added. After natural drying at 25°C, the tablets were cut according to the shape of the dried fixative to obtain 16 enzyme tablets.

[0068] Detecting pesticide components: An enzyme sheet was immobilized on a thin-film electrode to create an enzyme biosensor. 30 μL of an organophosphorus pesticide (0.1 g / L) was dripped onto the carbon paste surface of the thin-film electrode, dispersing it throughout the enzyme sheet. The enzyme biosensor was then placed in a 60°C oven for a 5-minute reaction. After the reaction, the enzyme biosensor was removed and the enzyme sheet was removed. 200 μL of a 50 mM, pH 8.0 phosphate buffer solution was quickly dripped onto the electrode. A voltage of 0.9 V was quickly applied to the thin-film electrode, and a response signal was obtained after 60 seconds. The response signal was read to determine the material and reagent composition of the immobilized enzyme sheet.

[0069] Experimental Example 1

[0070] Detection of thin-layer base electrodes:

[0071] 1. The potential values ​​obtained by different amounts of silver mirror reaction on the silver reference electrode are as follows Figure 3 As shown, Figure 3The potential curves obtained at the silver reference electrode for different amounts of silver mirror reaction are shown below. The results show that the amplitude of the electrode potential change gradually stabilizes with increasing number of reactions. As the silver mirror reaction time increases, the change in electrode potential decreases. After nine reactions, the electrode potential change stabilizes. The anode potential change is approximately 5mV, and the cathode potential change is approximately -16mV. Based on data analysis, the number of silver mirror reactions should be set to 10.

[0072] 2. Place the silver reference electrode in the p-nitrophenol solution. The electrode potential change curve of the silver reference electrode at different temperatures is as follows: Figure 4 shown.

[0073] from Figure 4 As can be seen from the graph, when the temperature is below 25°C, the electrode potential maintains a relatively large rate of decrease. When the temperature exceeds 25°C, the electrode potential changes slowly. When the temperature exceeds 30°C, the electrode potential changes gradually, but the rate of increase is relatively slow. Based on the analyzed data, the electrode temperature should be set above 25°C.

[0074] 3. Use PBS (polybutyl succinate) buffer solution to conduct pH test on silver reference electrode. The electrode potential change curve of silver reference electrode at different pH values ​​is as follows: Figure 5 shown

[0075] At a pH of 3.3, the electrode potential of the silver reference electrode changed by 16.3 mV. At a pH of 4.5, the electrode potential of the silver reference electrode changed by 11.7 mV. When the pH was below 5.7, the electrode potential of the silver reference electrode decreased rapidly with increasing pH.

[0076] At a pH of 5.7, the electrode potential of the silver reference electrode changed by 8.2 mV. At a pH of 6.5, the electrode potential of the silver reference electrode changed by 8.5 mV. At a pH of 7, the electrode potential of the silver reference electrode changed by 8.9 mV. At a pH of 7.6, the electrode potential of the silver reference electrode changed by 9.4 mV. At a pH of 8.2, the electrode potential of the silver reference electrode changed by 9.8 mV. When the pH value was between 5.7 and 8.2, the electrode potential change of the silver reference electrode increased with increasing pH value. However, the growth rate was relatively slow, and the change in electrode potential remained stable below 10 mV.

[0077] At a pH of 8.7, the electrode potential of the silver reference electrode changed by 11.9 mV. At a pH of 9.5, the electrode potential of the silver reference electrode changed by 13.7 mV. At a pH of 9.8, the electrode potential of the silver reference electrode changed by 14.8 mV. When the pH was greater than 8.2, the electrode potential of the silver reference electrode increased rapidly with increasing pH.

[0078] When the buffer solution is too acidic or too alkaline, the potential of the silver reference electrode changes significantly and becomes unstable. When the pH is between 5.7 and 8.2, the electrode potential change of the reference electrode is relatively stable and the change value is relatively low. This is the appropriate pH for the silver reference electrode. Therefore, the optimal pH of the phosphate buffer in the pesticide detection step is 5.7-8.2.

[0079] In summary, the experimental results show that the optimal conditions for electrode preparation are a silver mirror reaction number of 10, a temperature of at least 25°C, and a buffer pH of 5.7-8.2.

[0080] Experimental Example 2

[0081] Enzyme biosensor detection, materials and equipment: silver reference electrode, immobilized enzyme sheet (homemade); PHS-25C pH meter (Ningbo Haihai Saifu Experimental Instrument Factory); WYJ-3A transistor regulated DC power supply (Shanghai Chenhua Instrument Co., Ltd.); 101-1AB temperature chamber (Jin Huanbao Instrument Factory). The process is as follows Figure 6 As shown;

[0082] 1. Determination of optimal response signal acquisition time

[0083] Two sensors are used for the experiment. The response signals of the two sensors are as follows: Figure 7 and Figure 8 As shown, from Figure 7 and Figure 8 As can be seen, the sensor's response signal gradually decreases as the response time increases, and the change trend of the response signals of the two sensors is the same. During the first 30 seconds of the reaction, the sensor's response signal drops rapidly and fluctuates greatly. The sensor's response signal does not have a reference value during this period.

[0084] At 40 seconds, the signal of sensor 1 was 5.6 mV, and the signal of sensor 2 was 3.9 mV. At 60 seconds, the signal of sensor 1 was 4.2 mV, and the signal of sensor 2 was 2.5 mV. After the reaction lasted for 30 seconds, the response signals of the sensors slowly decreased.

[0085] After 60 seconds of reaction, the response signals of the sensors remain basically unchanged. The response signal of sensor 1 is stable at around 3.5mV, and the response signal of sensor 2 is stable at around 2.0mV. Therefore, the response signal of the sensor with a response time of 60 seconds can be used.

[0086] 2. Study the effect of enzyme degradation temperature on response signal

[0087] Two sensors were used for the experiment. The effect of temperature on the two sensors is as follows: Figure 9 and Figure 10As shown in the data, the sensor response signal increases first and then decreases as the temperature rises. At lower reaction temperatures, the sensor response signal is also lower. As the temperature gradually rises, the sensor signal also gradually increases, reaching a peak at 60°C, indicating that 60°C is the optimal temperature for the enzyme.

[0088] At 70°C, the response signal of sensor 1 was 3.4mV, and that of sensor 2 was 3.6mV. At 80°C, the response signal of sensor 1 was 2.9mV, and that of sensor 2 was 2.9mV. At 90°C, the response signal of sensor 1 was 2.5mV, and that of sensor 2 was 2.3mV. When the reaction temperature exceeded 60°C, the response signals of the sensors gradually decreased. The analysis results showed that 60°C is the ideal temperature for enzyme degradation reaction.

[0089] 3. Effect of different enzyme concentrations on sensor response signals

[0090] 140U enzyme tablets and 280U enzyme tablets were prepared using pure enzyme preparations of organophosphorus hydrolase. The sensor was placed in organophosphorus pesticides and the response signals of the sensors at two different enzyme concentrations were monitored. Figure 11 and Figure 12 shown.

[0091] Under the action of 280 U of enzyme, the response signal of sensor 1 was 4.9 mV and the response signal of sensor 2 was 4.3 mV, which were the maximum response signals of the sensors under these conditions.

[0092] At 140 U of enzyme, the response signal of sensor 1 was 3.8 mV, and that of sensor 2 was 3.4 mV. At 70 U of enzyme, the response signal of sensor 1 was 3.2 mV, and that of sensor 2 was 2.7 mV. As the enzyme dosage decreased, the sensor response signals also decreased, likely due to a decrease in the amount of enzyme adsorbed on the support. These comparative data indicate that pure enzyme preparations should be used as the enzyme solution when preparing immobilized enzyme tablets.

[0093] 4. Sensor consistency

[0094] The enzyme biosensor was prepared using the method of Example 1 and used for the detection of organophosphorus pesticides. The reaction signals of 10 enzyme biosensors are as follows: Figure 13 shown.

[0095] For organophosphorus pesticides, the response signals of the 10 enzyme sensors ranged from 3.8 to 4.7 mV, with an average of 4.24 mV. The response signals of the 10 enzyme sensors showed little variation. The variance of the 10 sample data was calculated to be 0.0784, and the standard deviation was 0.28. The low variance and standard deviation of this data set indicate a low degree of dispersion, indicating minimal variation in the response signals of the 10 enzyme sensors and high sensor consistency.

[0096] 5. Sensor stability

[0097] The enzyme biosensor was prepared according to the experimental conditions of Example 1. The enzyme biosensor was placed at a specific temperature for a specific time, and then placed in an organophosphorus pesticide to detect the sensor response signal. The test results are as follows: Figure 14 and Figure 15 shown.

[0098] When the dwell time was 0.5 days, the signal of sensor 1 was 4.5mV, and that of sensor 2 was 4.9mV. When the dwell time was 1 day, the signal of sensor 1 was 3.8mV, and that of sensor 2 was 4.1mV. After 2 days, the signal of sensor 1 was 2.9mV, and that of sensor 2 was 2.7mV. After 3 days, the signal of sensor 1 was 2.1mV, and that of sensor 2 was 1.8mV. After 4 days, the signal of sensor 1 was 1.2mV, and that of sensor 2 was 0.9mV. After 5 days, the signal of sensor 1 was 0.5mV, and that of sensor 2 was 0.4mV. As can be seen from the data, the longer the sensors are left in the water, the smaller their response signals become. The sensor response signals drop too quickly, and the detection capability rapidly deteriorates. Therefore, this sensor can only be used once.

[0099] 6. Detection characteristics of enzyme biosensors

[0100] The enzyme biosensor was prepared according to the above experimental conditions. The enzyme biosensor was placed in different concentrations of organophosphorus pesticides and the generated oxidation and reduction signals were recorded respectively. The reaction results of the enzyme biosensor to the oxidation and reduction signals are shown in Figure 2. Figure 16 and Figure 17 shown.

[0101] When the enzyme biosensor detected oxidation signals, the response signals for 0.1g / L of organophosphorus pesticide were 4.9mV, and 0.01g / L was 2.4mV. The response signals for 0.001g / L and 0.0001g / L were 1.4mV and 0.8mV, respectively. The enzyme biosensor's oxidation response signal gradually decreased with increasing organophosphorus pesticide concentration, and the changes in the response signal showed a certain regularity.

[0102] When the enzyme sensor detects a reduction signal, the reduction response signal for 0.1g / L organophosphorus pesticide is 10mV, and for 0.01g / L it is 7mV. The reduction response signals for 0.001g / L and 0.0001g / L are 4mV, respectively. When the enzyme sensor detects a reduction signal, it exhibits a high response signal. However, when detecting organophosphorus pesticides at concentrations of 0.001g / L and 0.0001g / L, the response signal remains unchanged, exhibiting irregular changes. This makes it suitable for early warning and monitoring of pesticides with high concentrations. Therefore, an oxidation signal should be detected during pesticide residue testing.

[0103] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

Claims

1. A method for detecting pesticide residues in food using an enzyme biosensor, characterized in that: The following steps are involved: Preparation of a thin-layer base electrode: After cleaning and drying the electrode base, place it in an incubator, perform multiple silver mirror reactions on the electrode base to form a silver reference electrode and a corresponding silver conductive tape, and finally place carbon paste on the silver reference electrode, and cover the protective layer of the electrode with polyvinyl chloride insulating tape to obtain the thin-layer base electrode; Preparation of enzyme tablets: Clean the surface of the carrier, dry it, drip the enzyme-containing fixative, dry it naturally, and cut it into the shape of the fixed agent to obtain the immobilized enzyme tablets; Detection of pesticide components: An immobilized enzyme sheet is fixed on a thin-layer base electrode to obtain an enzyme biosensor; pesticide solution is dripped onto the carbon slurry surface of the thin-layer base electrode and dispersed onto the enzyme sheet. The enzyme biosensor is reacted at 60°C and the enzyme sheet is removed after the reaction is completed; buffer solution is dripped onto the electrode, voltage is applied to obtain a response signal, and the composition of the pesticide solution is analyzed.

2. The method for detecting pesticide residues in food using an enzyme biosensor according to claim 1, characterized in that: The electrode substrate is cleaned by sequentially using alcohol and dilute nitric acid.

3. The method for detecting pesticide residues in food using an enzyme biosensor according to claim 1, wherein: The number of times of the silver mirror reaction is 9-11 times. After each silver mirror reaction, the electrode substrate is cleaned with distilled water and dried.

4. The method for detecting pesticide residues in food using an enzyme biosensor according to claim 3, characterized in that: The specific steps of each silver mirror reaction are: adding 5% sodium hydroxide solution to 2% silver nitrate solution, adding 2% ammonia water after the reaction, adding 10% glucose solution after dissolution is complete, and mixing to obtain a mixture; evenly applying the mixture on the electrode substrate, and then reacting at a temperature of 42-48°C for 5-10 minutes. The volume ratio of the silver nitrate solution, sodium hydroxide solution, ammonia water, and glucose solution is 1.5:0.1:1.15:0.

65.

5. The method for detecting pesticide residues in food using an enzyme biosensor according to claim 1, wherein: The carbon slurry is prepared from dimethyl silicone oil / liquid paraffin and high-purity graphite powder in a ratio of 3:

7.

6. The method for detecting pesticide residues in food using an enzyme biosensor according to claim 1, wherein: The carrier of the enzyme sheet is a nitrocellulose membrane or a cellulose acetate membrane, and the natural drying temperature is below 25°C.

7. The method for detecting pesticide residues in food using an enzyme biosensor according to claim 1, wherein: The enzyme-containing fixative is prepared by mixing 10% bovine serum albumin solution and pure enzyme solution in a volume ratio of 1:

1.

8. The method for detecting pesticide residues in food using an enzyme biosensor according to claim 1, wherein: The reaction time of the enzyme biosensor is 3-5 minutes.

9. The method for detecting pesticide residues in food using an enzyme biosensor according to claim 1, wherein: The buffer solution is a 50 mM phosphate buffer solution with a pH of 8.

0. The applied voltage is 0.9-1 V, and a response signal is obtained after applying the voltage for 60 seconds.

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