CN104007104B - Quickly detect kit and the using method thereof of organophosphorus pesticide - Google Patents

Abstract

The invention relates to a kit for rapidly detecting organophosphorus pesticide residues and a method for using the kit; the kit includes: C enzyme bottle, T enzyme bottle, C substrate reaction cup, T substrate reaction cup, the C enzyme bottle , T enzyme bottles are all sealed with enzyme bottle sealing caps, and are equipped with enzyme freeze-dried three-enzyme mixture; the C substrate reaction cup, T substrate reaction cup are equipped with acetylcholine, luminol and dimethyl Freeze-dried absorbent pads for benzidine. The present invention also relates to a method for rapidly detecting organophosphorus pesticide residues using the aforementioned kit. The invention uses acetylcholinesterase, choline oxidase and peroxidase as a catalytic system to hydrolyze and oxidize the acetylcholine chloride product with luminol to produce a chemiluminescent effect. Establish a method for sensitive testing of organophosphorus pesticides in agricultural products. The invention has the advantages of fast detection speed, simple operation process, good stability and repeatability, and does not require expensive instruments, and is very suitable for on-site monitoring of organophosphorus pesticides in farmers' markets.

Description

Kit for rapid detection of organophosphorus pesticide residues and its application method

technical field

The invention relates to a method for quickly detecting organophosphorus pesticide residues in food, in particular to a kit for rapidly detecting organophosphorus pesticide residues and a use method thereof.

Background technique

Pesticide residues in agricultural products, food and pharmaceuticals are still one of the important potential factors threatening human health. In China's agricultural production process, the annual use of pesticides still ranks first in the world, and the use of organophosphorus pesticides accounts for more than 70% of the total amount of pesticides used. According to reports in recent years, the unqualified rate of organophosphorus pesticides in vegetables and fruits sold in farmers’ markets in large provincial cities in China accounted for more than 14% in the first quarter, especially the pesticide residues in vegetables grown in greenhouses are higher . In the pesticide sampling monitoring, it was found that the detection rate of methamidophos, trichlorfon, omethoate and dichlorvos was higher. The current methods for detecting organophosphorus pesticides mainly include: ①spectrometry, mainly including spectrophotometry, chemiluminescence, infrared and fluorescence spectroscopy; ②chromatographic analysis, mainly including thin layer chromatography, gas chromatography, liquid chromatography method, supercritical fluid chromatography and chromatography/mass spectrometry; ③ immunological analysis methods, mainly including enzyme-linked immunosorbent assay, nano-gold immunoassay, immunofluorescence analysis, etc.; ④ sensor method, mainly including Pressed crystal biosensor method, optical fiber enzyme sensor, organophosphate hydrolase biosensor, cholinesterase sensor and bionic sensor based on molecular imprinting technology. ⑤Rapid detection method, mainly including rapid test card method and rapid test instrument method. Spectrometry and chromatography are national standard methods for confirmatory detection of organophosphorus pesticides in agricultural products. They have the characteristics of strong specificity and high sensitivity. It is quite complicated and not suitable for on-site detection of organophosphorus pesticide residues in agricultural products in large quantities. Immunological detection methods have high sensitivity and strong specificity, but they can only detect single or several organophosphorus pesticides, and the development of corresponding antibodies takes a long time and costs high. Another disadvantage of the immunological detection method is its poor repeatability and stability. The sensor method is a new type of detection technology developed in recent years, among which there are many reports on amperometric and potentiometric sensors using cholinesterase. Sensing technology is characterized by sensitivity and simple operation, but the research results are still in the laboratory stage, and there are still defects of poor repeatability and stability. The existing quick test card technology is to use cholinesterase to hydrolyze 2,6-dichloroindophenol acetate, an analogue of acetylcholine, to generate blue 2,6-dichloroindophenol acetic acid, so that according to the color of the quick test card Changes to determine the residues of organophosphorus pesticides. Its advantages are fast detection speed and simple operation. However, this method is still a simple qualitative method with low sensitivity. The detection limits of some types of organophosphorus and carbamate pesticides are higher than the national limit standards for vegetables and fruits. In addition, the organic pigments in the samples will seriously interfere with the determination. False negative results. However, the existing speed measuring instrument is still a portable spectrophotometer, which needs to cooperate with relatively complicated detection reagents, and is not easy to implement on site.

After searching the prior art documents, it is found that the patent application number is 2005100741791.3, and the name is "Chemiluminescent Organophosphorus Pesticide Residue Analyzer and Its Detection Method". The key technology is to use ultraviolet photocatalysis to convert organophosphorus pesticides on vegetables or fruits into inorganic phosphates in the presence of potassium persulfate and titanium dioxide nanoparticles, and then form phosphorus molybdenum vanadium with molybdic acid and vanadate Heteropolyacid, which produces chemiluminescence after oxidizing luminol, and is measured with a chemiluminescence instrument. However, its disadvantage is that the reaction system is susceptible to interference from external phosphates, resulting in false negative results. In addition, the defect of this detection method is that the repeatability and stability are still not ideal, and it cannot meet the needs of qualitative and quantitative detection.

Contents of the invention

The object of the present invention is to provide a kit for rapidly detecting organophosphorus pesticide residues and a method of use thereof in view of the defects in the prior art.

The present invention is achieved through the following technical solutions:

In the first aspect, the present invention relates to a kit for rapidly detecting organophosphorus pesticide residues, said kit comprising: C enzyme bottle, T enzyme bottle, C substrate reaction cup, T substrate reaction cup, said C enzyme bottle , T enzyme bottles are all sealed with enzyme bottle sealing caps, and the three enzyme mixtures of enzyme freeze-drying are housed; the C substrate reaction cup and the T substrate reaction cup are all set to contain acetylcholine chloride, luminol and dimethylformamide Freeze-dried absorbent pads based on benzidine.

In a second aspect, the present invention also relates to the method for rapidly detecting organophosphorus pesticide residues with the aforementioned kit, comprising the following steps:

Step 1: Preparation of enzyme cryoprotectant: add sucrose, bovine serum albumin, glycine, and glycerin to calcium chloride solution, stir until completely dissolved, and freeze for later use;

Step 2: Preparation of reaction enzyme solution: Dilute acetylcholinesterase, choline oxidase, and peroxidase with the enzyme cryoprotectant respectively, mix them, freeze-dry them, and put them into C enzyme bottle and T enzyme bottle respectively ;

The cholinesterase is commercial acetylcholinesterase, and its source can be acetylcholinesterase produced from insects, animal liver, fish brain, plant or genetic engineering technology, and the acetylcholinesterase from insects is preferred in this application. The choline oxidase is also a commercial choline oxidase, which is derived from the choline oxidase produced by Alcaligenes, Arthrobacter sphaeroides or genetic engineering technology, and the preferred Alcaligenes source of the present application is the choline oxidase . The peroxidase is commercially available horseradish peroxidase.

Step 3: Preparation of the substrate reaction cup: put the reaction substrate gasket into the mixed solution of luminol, acetylcholine chloride, and dimethylbenzidine, soak and drain, freeze-dry, and then put it into polyvinyl chloride Plastic cuvettes, marked as C substrate cuvettes, T substrate cuvettes;

Step 4: Sample testing: add pure water dropwise to C and T enzyme bottles respectively, shake well, add dropwise the sample solution to be tested into the T enzyme bottle with a plastic dropper, and drop Pure water or phosphate buffer solution, shake well, let it stand still, draw the reaction products from the C and T enzyme bottles with a dropper, drop them into the C and T substrate reaction cups respectively, and place them in the reaction cups within 2 to 8 minutes. Measure the luminescence intensity of C and T substrate cuvettes on a portable chemiluminescence instrument, and determine the concentration of organic phosphorus according to the relative inhibition rate of luminescence intensity.

Preferably, in the first step, the enzyme cryoprotectant includes the following components: 100 milliliters of 5-15 mmol/L calcium chloride solution, 1-3 grams of sucrose, 1-3 grams of glycine, and 5-15 milliliters of glycerol , 1 to 3 grams of bovine serum albumin.

Preferably, in the second step, the acetylcholinesterase is diluted 3000-8000 times, the choline oxidase is diluted 200-800 times, and the peroxidase is diluted 200-800 times.

Preferably, in the third step, the inner diameter of the bottom surface of the C substrate reaction cup and the T substrate reaction cup is 8-20mm, and the height is 10-25mm.

Preferably, in the third step, the thickness of the reaction substrate gasket is 3-6 mm.

Preferably, in the third step, the concentration of luminol is 1.0×10 ^-4 to 3.0×10 ^-4 mol/L, and the concentration of acetylcholine chloride is 3.0×10 ^-3 to 15.0×10 ^-3 mol/L L. The concentration of dimethylbenzidine is 2.0×10 ^-3 to 10.0×10 ^-3 mol/L.

Preferably, in the fourth step, the dropwise addition of pure water to C and T enzyme bottles is 50 to 200 microliters, the sample solution to be tested is 50 to 200 microliters, and the purified water in the C enzyme bottle is 50-200 microliters, the phosphate buffer solution is 0.1mol/L, and the pH is 7.0.

Preferably, in step 4, the reaction product in the C and T substrate cuvettes is 100-300 microliters.

The present invention has following beneficial effect:

(1) High detection sensitivity: the present invention selects a three-enzyme system containing acetylcholinesterase, choline oxidase and peroxidase, and establishes a chemiluminescence technology based on a sensitizer and luminol, and its sensitivity is higher than that of Tens of times and thousands of times, and the detection linear range is significantly widened. The minimum detection limit of the present invention for detecting dichlorvos and trichlorfon reaches 0.078ppm; the minimum detection limit for detecting fenamiphos reaches 0.035ppm; the detection limit for carbosulfan reaches 0.039ppm;

(2) The kit of the present invention is easy to use, simple to operate, does not need complex reagent preparation process in the test, and non-professionals can easily complete the detection according to the kit instructions;

(3) Rapid detection: the present invention can complete the sample pretreatment process and detection process within half an hour, and it only takes 3 to 5 minutes for the pretreatment of vegetables and fruit samples, and 10 to 25 minutes for the test process with the kit.

(4) Wide range of detectable organic phosphorus: the present invention can detect aldicarb, omethoate, dimethoate, acephate, carbaryl, trichlorfon, carbofuran, carbosulfan, dimethoate, Zinon, Dichlorvos, Dicrotophos, Thiamifos, Ethyl Parathion, Fenamiphos, Malathion, Methamidophos, Methyl Parathion, Monocrotophos, Dioxacarb, Imidophos Dozens of organophosphorus pesticides including phoxim, chlorpyrifos, isocarbophos, methylisothion, azinphos-methyl, etc. At the same time, the invention can also detect the residues of some heavy metals including cadmium, copper, lead, mercury and zinc on vegetables and fruits.

Description of drawings

Other characteristics, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

Fig. 1 is the schematic diagram of the kit of rapid detection organophosphorus pesticide residue of the present invention;

Among them, 1 is the three-enzyme reagent bottle group containing control and test, including C enzyme bottle and T enzyme bottle; 2 is the sealing cap of the enzyme bottle, 3 is the three-enzyme reagent bottle body; 4 is the three-enzyme mixture of enzyme freeze-drying ; 5 is the cup body of the substrate reaction cup; 6 is the freeze-dried absorbent pad containing acetylcholine chloride, luminol and dimethylbenzidine; 7 is the double substrate reaction cup group for control and test, including C substrate Reaction cup, T substrate reaction cup.

detailed description

The present invention will be described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

Example 1

This embodiment relates to a method for rapidly detecting organophosphorus pesticide residues using a kit, comprising the following steps:

This example detects the residues of organophosphorus pesticides in rapeseed sold in the market.

1. Sample pre-treatment, put 10 parts of rapeseed leaves on the market with a weight of less than 10 grams in a plastic or glass container with a screw cap, or use a commonly used can bottle. Add about 10 ml of pure water, shake for 3 minutes, and let it stand for 2 minutes, then take the supernatant and use it directly for testing;

2, the preparation of enzyme cryoprotectant: the preparation method of present embodiment is as follows: prepare 100 milliliters of 5mmol/L calcium chloride solutions earlier, then add 1 gram of sucrose, 1 gram of glycine, 5 milliliters of glycerol, bovine serum albumin 1 gram, Stir while adding the above ingredients until all the powdered reagents are completely dissolved, and then store in a freezer at -15°C until use;

3. Preparation of reaction enzyme liquid: Dilute the above three enzyme liquids with the above-mentioned enzyme cryoprotectant, that is, dilute the acetylcholinesterase containing 425 international enzyme activity units per mg by 3000 times, and dilute the acetylcholinesterase containing 10 international enzyme activity units per mg The acetylcholinesterase was diluted 200 times, and the horseradish peroxidase containing 113 international enzyme activity units per mg was diluted 200 times. Then take 100 microliters of the above-mentioned enzyme solutions and mix them together, pack them in brown ampoules, freeze-dry them on a freeze dryer, cover them and seal them, place them in aluminum foil bags, and store them at room temperature;

4. Preparation of the substrate reaction cup: In this embodiment, a polyvinyl chloride plastic reaction cup is used, the inner diameter of the bottom surface is 8 mm, the height is 10 mm, and the thickness of the reaction substrate gasket is 3 mm. In a mixed solution containing 1.0×10 ^-4 mol/L luminol, 3.0×10 ^-3 mol/L acetylcholine chloride and 2.0×10 ^-3 mol/L dimethylbenzidine, soak for 3 minutes and drain After drying and freeze-drying, put them into reaction cups, mark them as C cups and T cups respectively, seal them in packaging bags, and store them at room temperature for later use;

5. Sample test: add 50 microliters of purified water to the C and T enzyme bottles respectively, shake gently, then drop 50 microliters of the sample solution to be tested into the T enzyme bottle with a quantitative plastic dropper, and add 50 microliters of the sample solution to the C enzyme bottle. Use a quantitative plastic dropper to drop 50 microliters of pure water into the bottle, shake it gently, let it stand for 15 minutes, then use a pipette to draw 100 microliters of the reaction product from the enzyme bottles of C and T above, and add them dropwise to C and T respectively. In the T substrate cuvette, measure the luminescence intensity of the C and T substrate cuvettes on a portable chemiluminescence instrument within 2 minutes, and determine the concentration of organophosphorus according to the relative inhibition rate of the luminescence intensity.

The results showed that among the 10 tested rapeseed samples, the chemiluminescence inhibition rates of 2 rapeseed samples were 16% and 33%, respectively, which were judged as positive. The chemiluminescence inhibition rate of the remaining 8 rapeseed samples were all less than 15%. The control test positive sample was omethoate at 5ppm.

Example 2

This embodiment relates to a method for rapidly detecting organophosphorus pesticide residues using a kit, comprising the following steps:

This example detects the residues of organophosphorus pesticides in commercially available spinach.

1, sample pretreatment, step is the same as step 1 of embodiment 1;

2, the preparation of enzyme cryoprotectant: the preparation method of present embodiment is as follows: first prepare 100 milliliters of 10mmol/L calcium chloride solution, then add 2 grams of sucrose, 2 grams of glycine, 10 milliliters of glycerol, bovine serum albumin 2 grams. Stir while adding the above ingredients until all the powdered reagents are completely dissolved, and then store in a freezer at -25°C until use;

3. Preparation of reaction enzyme liquid: Dilute the above three enzyme liquids with the above-mentioned enzyme cryoprotectant, that is, dilute the acetylcholinesterase containing 425 international enzyme activity units per mg by 5000 times, and dilute the acetylcholinesterase containing 10 international enzyme activity units per mg The acetylcholinesterase was diluted 500 times, and the horseradish peroxidase containing 113 international enzyme activity units per mg was diluted 500 times. Then 200 microliters of the above enzyme solutions were mixed together, packed in brown ampoules, freeze-dried on a freeze dryer, sealed after being covered, placed in an aluminum foil bag, and stored at room temperature.

4. Preparation of the substrate reaction cup: In this embodiment, a polystyrene plastic reaction cup is used. The inner diameter of the bottom surface is 12mm, and the height is 15mm. The reaction substrate gasket has a thickness of 4 mm. Put this gasket into a mixed solution containing 2.0×10 ^-4 mol/L luminol, 8.0×10 ^-3 mol/L acetylcholine chloride and 6.0×10 ^-3 mol/L dimethylbenzidine , soaked for 3 minutes, drained, and freeze-dried, put into reaction cups, marked as C and T cups, sealed in packaging bags, and stored at room temperature for later use;

5. Sample testing: Add 100 microliters of purified water dropwise to the C and T enzyme bottles, shake gently, and drop 100 microliters of the sample solution to be tested into the T enzyme bottle with a quantitative plastic dropper. Add 100 microliters of pure water dropwise to the C enzyme bottle with a quantitative plastic dropper, shake it gently, and let it stand for 20 minutes. Then use a dropper to draw 200 microliters of the reaction product from the above C and T enzyme bottles, and drop them into the C and T substrate reaction cups respectively. Then measure the luminescence intensity of C and T substrate cuvettes on a portable chemiluminescence instrument within 5 minutes, and determine the concentration of organic phosphorus according to the relative inhibition rate of luminescence intensity.

The results showed that among the 10 tested spinach samples, 1 rape sample had a chemiluminescence inhibition rate of 21%, which was judged to be positive. The chemiluminescence inhibition rates of the remaining 9 spinach samples were all less than 15%. The control test positive sample was monocrotophos at 10 ppm.

Example 3

This embodiment relates to a method for rapidly detecting organophosphorus pesticide residues using a kit, comprising the following steps:

This example detects the residues of organophosphorus pesticides in crystal pears sold in the market.

1. Sample pretreatment: first cut the crystal pear into one-eighth size, and place it in a clean can. All the other steps are with the step 1 of embodiment 1;

2, the preparation of enzyme cryoprotectant: the preparation method of present embodiment is as follows: first prepare 100 milliliters of 15mmol/L calcium chloride solution, then add 3 grams of sucrose, 3 grams of glycine, 15 milliliters of glycerol, bovine serum albumin 3 grams. Stir while adding the above ingredients until all the powdered reagents are completely dissolved, and then store in a freezer at -40°C for later use;

3. Preparation of reaction enzyme liquid: Dilute the above three enzyme liquids with the above-mentioned enzyme cryoprotectant, that is, dilute the acetylcholinesterase containing 425 international enzyme activity units per mg by 8000 times, and dilute the acetylcholinesterase containing 10 international enzyme activity units per mg The acetylcholinesterase was diluted 800 times, and the horseradish peroxidase containing 113 international enzyme activity units per mg was diluted 800 times. Then 300 microliters of the above enzyme solutions were mixed together, packed in brown ampoules, freeze-dried on a freeze dryer, sealed after being covered, placed in an aluminum foil bag, and stored at room temperature.

4. Preparation of the substrate reaction cup: In this embodiment, a polycarbonate plastic reaction cup is used. The inner diameter of the bottom surface is 20mm, and the height is 25mm. When preparing reaction substrate gaskets. During the described reaction substrate pad, the used thickness is 6mm. Then put this gasket into a mixed solution containing 3.0×10 ^-4 mol/L luminol, 15.0×10 ^-3 mol/L acetylcholine chloride and 10.0×10 ^-3 mol/L dimethylbenzidine , soaked for 3 minutes, drained, and freeze-dried, put into reaction cups, marked as C and T cups, sealed in packaging bags, and stored at room temperature for later use;

5. Sample testing: add 200 microliters of purified water dropwise to the C and T enzyme bottles, shake gently, and drop 200 microliters of the sample solution to be tested into the T enzyme bottle with a quantitative plastic dropper. Add 200 microliters of pure water dropwise to the C enzyme bottle with a quantitative plastic dropper, shake it gently, and let it stand for 25 minutes. Then use a dropper to draw 300 microliters of the reaction product from the above C and T enzyme bottles, and drop them into the C and T substrate reaction cups respectively. Then measure the luminescence intensities of C and T substrate cuvettes on a portable chemiluminescence instrument within 8 minutes, and determine the concentration of organophosphorus according to the relative inhibition rate of luminescence intensities.

The results showed that the chemiluminescence inhibition rate of the 10 crystal pear samples tested were all less than 15%, and the results were judged as negative. The control test positive sample was methamidophos at 15 ppm.

The invention uses acetylcholinesterase, choline oxidase and peroxidase as a catalytic system to hydrolyze and oxidize the acetylcholine chloride product with luminol to produce a chemiluminescence effect. Since organophosphorus pesticides can inhibit the activity of the three enzymes in this reaction system, especially the activity of acetylcholinesterase, a method for sensitively testing organophosphorus pesticides in agricultural products can be established, and a detection method for organophosphorus pesticides can be developed using this principle Chemiluminescent Detection Kit. The invention has the advantages of fast detection speed, simple operation process, good stability and repeatability, and does not require expensive instruments, and is very suitable for on-site monitoring of organophosphorus pesticides in farmers' markets.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims (9)

1. the kit of a quick detection organophosphorus pesticide, it is characterised in that described kit includes: C enzyme Bottle, T enzyme bottle, C substrate reactions cup, T substrate reactions cup, described C enzyme bottle, T enzyme bottle all seal Gai Mi with enzyme bottle Envelope, and the three enzyme intermixtures being lyophilized equipped with enzyme；Described C substrate reactions cup, T substrate reactions cup are respectively provided with containing chlorine Change acetylcholine, luminol and the lyophilized adsorptive pads of dimethylbenzidine；

In the three enzyme intermixtures that described enzyme is lyophilized, the enzyme cryoprotector of employing is sucrose, bovine serum albumin(BSA), sweet ammonia Acid, glycerine and the mixed solution of calcium chloride；Three enzymes are acetylcholinesterase, choline oxidase, peroxidase.

2. applying the method that the kit described in claim 1 quickly detects organophosphorus pesticide, its feature exists In, comprise the following steps:

1st step: the preparation of enzyme cryoprotector: sucrose, bovine serum albumin(BSA), glycine, glycerine are added calcium chloride In solution, stirring is to being completely dissolved, and freezen protective is stand-by；

2nd step: the preparation of reaction enzymes liquid: described enzyme cryoprotector is diluted respectively acetylcholinesterase, choline oxidation Enzyme, peroxidase, mixing, after freeze-drying, it is respectively charged in C enzyme bottle and T enzyme bottle；

3rd step: the preparation of substrate reactions cup: reaction substrate pad is put into luminol, acecoline, dimethyl In benzidine mixed solution, immersion drains, and after freeze-drying, puts into igelite reaction cup, be labeled as at the bottom of C Thing reaction cup, T substrate reactions cup；

4th step: the test of sample: drip pure water respectively in C, T enzyme bottle, after shaking up, with plastic dropper to institute State dropping measuring samples solution in T enzyme bottle, in C enzyme bottle, drip pure water or phosphate buffer, shake up, stand, From described C, T enzyme bottle, draw product with dropper, be added drop-wise to respectively in C, T substrate reactions cup, at 2～8 points On portable chemical light-emitting appearance, the luminous intensity of C, T substrate reactions cup, relatively pressing down according to luminous intensity is measured in clock Rate processed, determines the concentration of organophosphor.

3. the method that kit as claimed in claim 2 quickly detects organophosphorus pesticide, it is characterised in that the 1st In step, enzyme cryoprotector includes each component of following content: 5～15mmol/L calcium chloride solutions 100 milliliters, sugarcane Sugar 1～3 gram, glycine 1～3 grams, glycerine 5～15 milliliters, bovine serum albumin(BSA) 1～3 grams.

4. the method that kit as claimed in claim 2 quickly detects organophosphorus pesticide, it is characterised in that the 2nd In step, described acetylcholinesterase dilutes 3000～8000 times, and described choline oxidase dilutes 200～800 times, Described peroxidase dilutes 200～800 times.

5. the method that kit as claimed in claim 2 quickly detects organophosphorus pesticide, it is characterised in that the 3rd In step, described C substrate reactions cup, the bottom surface internal diameter of T substrate reactions cup are 8～20mm, and height is 10～25mm.

6. the method that kit as claimed in claim 2 quickly detects organophosphorus pesticide, it is characterised in that the In 3 steps, the thickness of described reaction substrate pad is 3～6mm.

7. the method that kit as claimed in claim 2 quickly detects organophosphorus pesticide, it is characterised in that the In 3 steps, the concentration of described luminol is 1.0 × 10^-4～3.0 × 10^-4Mol/L, the concentration of acecoline are 3.0×10^-3～15.0 × 10^-3Mol/L, the concentration of dimethylbenzidine are 2.0 × 10^-3～10.0 × 10^-3mol/L。

8. the method that kit as claimed in claim 2 quickly detects organophosphorus pesticide, it is characterised in that the 4th In step, the described pure water that drips in C, T enzyme bottle is 50～200 microlitres, and described measuring samples solution is 50～200 Microlitre, in described C enzyme bottle, pure water is 50～200 microlitres, and phosphate buffer is 0.1mol/L, and pH It is 7.0.

9. the method that kit as claimed in claim 2 quickly detects organophosphorus pesticide, it is characterised in that the 4th Product 100～300 microlitre in step, in described C, T substrate reactions cup.