阅读说明：本技术 有机磷农残的快速检测试剂盒、其制备方法和检测方法 (Rapid detection kit for organophosphorus pesticide residues, preparation method and detection method thereof ) 是由 叶升锋 王文珺 桑华春 傅晓春 严可以 于 2020-04-28 设计创作，主要内容包括：本发明涉及一种有机磷农药残留的快速检测试剂盒、其制备方法和检测方法。本发明的方法基于N-溴代丁二酰亚胺增强的酶抑制法,具体采用马血清提取的丁酰胆碱酯酶作为催化酶、抗坏血酸作为还原剂、无水乙醇作为提取剂。使用的N-溴代丁二酰亚胺作为增强剂,可提高检测灵敏度1-3个数量级。本发明的酶抑制法,极大的提高了有机磷农药的检出率,具有灵敏、快速、准确、高通量等优点,用于粮谷大量样本的农残筛查分析,适用于粮站、企业等基层农药残留检测实验室。(The invention relates to a rapid detection kit for organophosphorus pesticide residues, a preparation method and a detection method thereof. The method is based on an N-bromosuccinimide enhanced enzyme inhibition method, and specifically adopts butyrylcholinesterase extracted from horse serum as catalytic enzyme, ascorbic acid as a reducing agent and absolute ethyl alcohol as an extracting agent. The N-bromosuccinimide is used as a reinforcing agent, and the detection sensitivity can be improved by 1 to 3 orders of magnitude. The enzyme inhibition method greatly improves the detection rate of the organophosphorus pesticide, has the advantages of sensitivity, rapidness, accuracy, high flux and the like, is used for screening and analyzing pesticide residues of a large number of grain samples, and is suitable for basal pesticide residue detection laboratories of grain stations, enterprises and the like.)

1. An enzyme inhibition method kit for detecting organophosphorus pesticide residues, which comprises the following reagents A, B, C and D:

reagent A: n-bromosuccinimide;

and (3) reagent B: ascorbic acid;

and (3) reagent C: butyrylcholinesterase enzyme of EC 3.1.1.8;

and (3) reagent D: and (4) extracting the solvent.

2. The kit of claim 1, wherein,

the working concentration of N-bromosuccinimide is 0.1 to 1.5 volume%; and/or

The working concentration of the ascorbic acid solution is from 0.5% to 5% by volume; and/or

Butyrylcholinesterase solution is extracted from horse serum; and/or

The extraction solvent is absolute ethyl alcohol.

3. A method for enhancing enzyme inhibition for detecting organophosphorus pesticide residues, characterized in that the detection is carried out by using the kit of any one of claims 1 to 2 through the following steps:

step 1: placing a sample to be tested in a test tube, adding a reagent D, shaking and centrifuging;

step 2: placing the supernatant centrifuged in the step 1 in a new test tube, drying by using nitrogen at 60 ℃, adding the reagent A, and standing;

and step 3: adding the reagent B, shaking and centrifuging;

and 4, step 4: adding a reagent C into the supernatant solution after centrifugation in the step 3, adding a color developing agent, uniformly mixing, standing, and adding a substrate;

and 5: and detecting the absorbance.

4. The method of claim 3, wherein the sample to be tested is cereal grain, and 1g to 5g of the sample to be tested is taken in the step 1.

5. The method of claim 3, wherein the shaking time of steps 1 and 3 is 1 to 5 minutes, and/or the centrifugation conditions of steps 1 and 3 are 4000rpm centrifugation for 3 to 8 minutes, and/or the standing time of steps 2 and 4 is 3 to 8 minutes.

6. The method of claim 3, wherein the substrate is iodobutyrylcholine and/or the chromogenic agent is 5, 5-dithiobis (2-nitrobenzoic acid).

7. The method of claim 3, wherein the absorbance detected is absorbance at a wavelength of 410 nm.

8. A method of making the kit of any one of claims 1 to 2, the method comprising separately packaging reagents A, B, C and D in separate containers.

Technical Field

The invention belongs to the fields of biological medicine and chemistry, and relates to a high-sensitivity enzyme inhibition enhancement method for detecting organophosphorus pesticide residues in cereals.

Background

The grains need to use pesticides to control pests in both planting and storage stages. The organophosphorus pesticide is an insecticide widely used in grain production at present, and the residue of the organophosphorus pesticide seriously harms human, livestock and environmental safety. The organophosphorus pesticides have the characteristics of multiple types, large toxicity difference, unstable chemical properties, large detection difficulty and the like. At present, the organophosphorus pesticide residue detection mainly comprises large-scale instrument analysis methods such as Gas Chromatography (GC), liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and the like and an enzyme inhibition method (spectrophotometry). The instrument analysis method has accurate and sensitive detection results, but has high detection cost, time consumption and high professional requirements on technical personnel, and is suitable for laboratory analysis; the enzyme inhibition method has the advantages of simple and convenient operation, rapidness, suitability for on-site detection and the like, so the enzyme inhibition method is recommended by a series of national standards and agricultural industry standards such as GB/T5009.199-2003 rapid detection of organophosphorus and carbamate pesticide residues in vegetables [1 ]. At present, the research and application of the enzyme inhibition method mainly focuses on the detection of pesticide residues in vegetables and fruits, and the application of the enzyme inhibition method in food is less. Conventional enzyme inhibition methods have been controversial for many years due to problems of accuracy and sensitivity. For organophosphorus pesticides, the detection limit of enzyme inhibition method often cannot reach the national safety limit of organophosphorus pesticides in cereals in China, and the main technical reason is insensitivity of thio-type phosphate pesticides to cholinesterase [2 ]. In fact, the organophosphorus pesticide comprises a part of thio-type organophosphate pesticides, and the organophosphorus pesticide is insensitive to acetylcholinesterase, often causes omission, and greatly influences the detection rate and the applicability of an enzyme inhibition method.

Cholinesterase is the basis of enzyme inhibition method application, and the sensitivity, specificity and stability of cholinesterase directly influence the accuracy of detection results, so that the selection of proper enzyme is always a hotspot of enzyme inhibition method research. Acetylcholinesterase (AChE) is often used as a catalytic enzyme, but the inhibition effect of the thio-type organic phosphate pesticide on the AChE catalytic hydrolysis function is poor, so that the condition of omission detection often occurs. Therefore, improvement of detection sensitivity is a key to the enzyme inhibition method.

For enzyme inhibition, chloride has been used as an enhancer to increase sensitivity, but is now no longer used due to its toxicity. At present, bromine water is adopted as an enhancer, but the enhancing effect is limited.

Through intensive research, the invention selects Butyrylcholinesterase (BChE) which can be well inhibited by the organophosphorus pesticide, adopts a specific extraction solvent and particularly takes N-bromosuccinimide as an enhancer, thereby obviously improving the detection sensitivity of the organophosphorus pesticide in an enzyme inhibition method, realizing the effective detection of the organophosphorus pesticide in grains and providing powerful technical support for the safety supervision of the grain quality in China.

Reference documents:

[1] and (4) rapidly detecting organophosphorus and carbamate pesticide residues in GB/T5009.199-2003 vegetables [ S ].

[2] The donkey serum cholinesterase inhibition method is used for rapidly detecting pesticide residue [ J ] in vegetables, and the food industry science and technology is 2013,34(13): 293-.

Disclosure of Invention

Technical problem to be solved

The invention aims to provide a rapid and high-sensitivity enhanced enzyme inhibition method kit and a detection method for detecting organophosphorus pesticide residues, which are used for high-throughput screening of organophosphorus pesticide residues in agricultural products, particularly cereals.

Technical scheme

In order to achieve the above object, the present invention provides an enzyme inhibition method kit for detecting organophosphorus pesticide residues, the kit comprising: the following reagents A, B, C and D were used,

reagent A: n-bromosuccinimide;

and (3) reagent B: ascorbic acid;

and (3) reagent C: butyrylcholinesterase enzyme of EC 3.1.1.8;

and (3) reagent D: and (4) extracting the solvent.

In an embodiment of the kit of the invention, the working concentration of N-bromosuccinimide is from 0.1% to 1.5% by volume; and/or the working concentration of the ascorbic acid solution is 0.5% to 5% by volume; and/or the butyrylcholinesterase solution is extracted from horse serum, and/or the extraction solvent is ethanol.

The invention also provides an enhanced enzyme inhibition method for detecting organophosphorus pesticide residues, which is characterized in that the kit of the above embodiment of the invention is used for detection through the following steps:

step 1: placing a sample to be tested in a test tube, adding a reagent D, shaking and centrifuging;

step 2: placing the centrifuged supernatant in the step 1 into a new test tube, drying by blowing nitrogen at 60 ℃, adding a reagent A, and standing;

and step 3: adding the reagent B, shaking and centrifuging;

and 4, step 4: adding a reagent C into the supernatant solution after centrifugation in the step 3, adding a color developing agent, uniformly mixing, standing, and adding a substrate;

and 5: and detecting the absorbance.

In an embodiment of the method of the present invention, the sample to be tested is cereal grain.

In an embodiment of the method of the present invention, in step 1, 1 to 5g of the sample to be tested is taken.

In an embodiment of the process of the invention, the shaking time of steps 1 and 3 is between 1 and 5 minutes.

In an embodiment of the method of the invention, the centrifugation conditions of steps 1 and 3 are 4000rpm centrifugation for 3 to 8 minutes.

In an embodiment of the method of the invention, the standing time of said steps 2 and 4 is 3-8 minutes.

In an embodiment of the method of the invention, the substrate is iodothiobutyrylcholine.

In an embodiment of the method of the invention, the developer is 5, 5-dithiobis (2-nitrobenzoic acid).

In an embodiment of the method of the invention, the absorbance detected is the absorbance at a wavelength of 410 nm.

The present invention also provides a method of preparing the kit according to the above embodiment of the present invention, which comprises encapsulating the reagents A, B, C and D in different solvents, respectively.

The invention has the advantages of

Through intensive research, the inventor selects a specific combination of N-bromosuccinimide, ascorbic acid and butyrylcholinesterase with EC 3.1.1.8 to prepare the kit of the invention by matching with an extraction solvent, and can detect organophosphorus pesticide residues in agricultural products, particularly grains, with extremely high sensitivity.

On the basis, the detection effect of the method on the organophosphorus pesticide residues can be further improved by selecting ethanol as a specific extraction solvent.

The traditional enzyme inhibition method is improved, butyrylcholinesterase with good inhibition effect on organophosphorus pesticides is provided, and N-bromosuccinimide is provided as a reinforcing agent, so that the detection sensitivity is greatly improved, and the bottleneck that the traditional enzyme inhibition method is low in detection rate of thio-type organic phosphate pesticides is overcome; and provides a suitable sample pretreatment method, and can be effectively applied to the detection of organophosphorus pesticides in grains. The method has important practical significance for solving the problem of on-site monitoring of organophosphorus pesticide residues of large-batch grain samples.

Drawings

FIG. 1 is a standard inhibition curve for a portion of organophosphorus pesticides.

Detailed Description

The present invention is described in detail by the following embodiments, but they are not intended to limit the scope of the present invention.

The invention provides an enzyme inhibition method kit for detecting organophosphorus pesticide residues, which comprises the following components: reagent A: n-bromosuccinimide; and (3) reagent B: ascorbic acid; and (3) reagent C: butyrylcholinesterase enzyme of EC 3.1.1.8; and (3) reagent D: and (4) extracting the solvent.

The reagent A is used as an enhancer, and N-Bromosuccinimide (N-Bromosucinimide) is adopted, and has a chemical formula: c₄H₄BrNO₂CAS registry number: 128-08-5,

structural formula (xvi):

the working concentration of the reagent A can be 0.1-1.5%. If the concentration is below the lower limit, the enhancement effect is not significant; if the concentration is higher than the upper limit, the reinforcing effect is lost.

Reagent B as a reducing agent, Ascorbic Acid (Ascorbic Acid) is adopted, and the chemical formula is as follows: c₆H₈O₆CAS registry number: 50-81-7, structural formula:

the working concentration of the reagent B can be 0.1-1.5%. If the concentration is below the lower limit, the enhancement effect is not significant; if the concentration is higher than the upper limit, the reinforcing effect is lost.

Reagent C is a hydrolysis catalytic enzyme, preferably Butyrylcholinesterase (BChE), preferably Butyrylcholinesterase extracted from horse serum, CAS accession No.: 9001-08-5, EC 3.1.1.8.

The working concentration of reagent C may be 1-3%. If the concentration is below the lower limit, the enzyme activity is insufficient; if the concentration is higher than the upper limit, the absorbance will be too high to affect the detection.

Reagent D, an extraction solvent, preferably ethanol (ethanol), of formula: c₂H₆O, CAS accession number: 64-17-5, structural formula:

the working concentration of reagent D was 100%. If the concentration is less than 100%, no enhancing effect or an enhancing effect is not significant.

The invention also provides an enhanced enzyme inhibition method for detecting organophosphorus pesticide residues, which is characterized in that the kit of the above embodiment of the invention is used for detection through the following steps:

step 1: placing a sample to be tested in a test tube, adding a reagent D, shaking and centrifuging;

step 2: placing the centrifuged supernatant in the step 1 into a new test tube, drying by blowing nitrogen at 60 ℃, adding a reagent A, and standing;

and step 3: adding the reagent B, shaking and centrifuging;

and 4, step 4: adding a reagent C into the supernatant solution after centrifugation in the step 3, adding a color developing agent, uniformly mixing, standing, and adding a substrate;

and 5: and detecting the absorbance.

In an embodiment of the method of the present invention, the sample to be tested is cereal grain, including but not limited to: wheat, corn, rice, brown rice and coarse cereals.

In an embodiment of the method of the present invention, in step 1, 1 to 5g, more preferably 2g, of the sample to be tested is taken.

In an embodiment of the process of the invention, the shaking time of steps 1 and 3 is 1 to 5 minutes, more preferably 2 minutes.

In an embodiment of the method of the invention, the centrifugation conditions of steps 1 and 3 are 4000rpm centrifugation for 3 to 8 minutes, more preferably for 5 minutes.

In an embodiment of the process of the invention, the standing time of steps 2 and 4 is 3 to 8 minutes, more preferably 5 minutes.

In an embodiment of the method of the invention, the substrate is iodothiobutyrylcholine. Preferably, the preparation method of the substrate solution comprises the following steps: 137mg of thiobutyrylcholine iodide is weighed, dissolved fully in 15mL of deionized water and stored in a refrigerator at 4 ℃ for later use.

In an embodiment of the method of the invention, the developer is 5, 5-dithiobis (2-nitrobenzoic acid). Preferably, the color developing agent preparation method comprises the following steps: 160mg of 5, 5-dithiobis (2-nitrobenzoic acid) and 15.6mg of sodium hydrogencarbonate were weighed out, respectively, and sufficiently dissolved in 20mL of PBS buffer solution and stored at 4 ℃.

In an embodiment of the method of the invention, the absorbance detected is the absorbance at a wavelength of 410 nm.