Preparation method of rapid detection reagent for organophosphorus and carbamate pesticide residues

Abstract

The invention discloses a preparation method of a rapid organophosphorus and carbamate pesticide residue test agent.The preparation method comprises the following steps that materials including a cuvette, a vacuum freeze dryer and a detection reagent bottle are prepared, and color developing agent freeze-dried microspheres and acetylcholin esterase freeze-dried microspheres are placed at the bottom in the detection reagent bottle; according to the rapid pesticide residue detection reagent prepared by using the method provided by the invention, the operation steps of pesticide residue detection can be simplified, standardization and rapidness of pesticide residue detection are facilitated, detection of fruit and vegetable pesticide residues can be automatically executed by a detection instrument, a base layer detector is separated from a heavy pesticide residue detection task, and the detection efficiency is improved. And meanwhile, due to automatic operation of the instrument, the problem of nonstandard detection operation of a detector is avoided.

Description

Technical Field

[0001] The invention relates to the technical field of rapid food safety detection, and in particular to a method for preparing a rapid test reagent for organophosphorus and carbamate pesticide residues. Background Art

[0002] Organophosphorus and carbamate pesticides have an inhibitory effect on the normal function of animal neurotransmitters cholinesterase, and the inhibition rate is positively correlated with the concentration of the pesticide. Under normal circumstances, cholinesterase catalyzes the hydrolysis of neurotransmitter analogs, and its hydrolysis products react with the color developer to produce yellow substances. The inhibition rate can be calculated by measuring the change in absorbance at 412nm with a spectrophotometer or pesticide residue tester, thereby determining whether there are high-dose organophosphorus or carbamate pesticide residues in the sample.

[0003] Before each use of the existing detection reagent, the cholinesterase, color developer, and substrate powder used need to be prepared before use. The order of addition is 2.5ml sample solution, 100ul cholinesterase, 100ul color developer, react at 37℃ for 10 minutes, then add 100ul substrate, mix well, and use a spectrophotometer or pesticide residue detector for on-machine testing.

[0004] Therefore, we proposed a method for preparing a rapid test reagent for organophosphorus and carbamate pesticide residues to solve the above problems. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for preparing a rapid test agent for organophosphorus and carbamate pesticide residues.

[0006] A method for preparing a rapid test reagent for organophosphorus and carbamate pesticide residues comprises the following steps:

[0007] S1: Material preparation: cuvette, vacuum freeze dryer, detection reagent bottle;

[0008] S2: freeze-dried microsphere preparation;

[0009] a: Raw material preparation: acetylcholinesterase solution, color developer, substrate and protective agent, and the volume ratio of acetylcholinesterase solution, color developer and protective agent is 1:1:0.3;

[0010] b: Preparation of acetylcholinesterase freeze-dried microspheres: placing the acetylcholinesterase solution and the protective agent in a cuvette, and placing the cuvette in a vacuum freeze dryer for vacuum freeze drying to obtain acetylcholinesterase freeze-dried microspheres;

[0011] c: Preparation of chromogen freeze-dried microspheres: placing the chromogen solution and the protective agent in a cuvette, and placing the cuvette in a vacuum freeze dryer for vacuum freeze drying to obtain chromogen freeze-dried microspheres;

[0012] d: Preparation of substrate freeze-dried microspheres: placing the substrate and the protective agent in a cuvette, and placing the cuvette in a vacuum freeze dryer for vacuum freeze drying to obtain substrate freeze-dried microspheres;

[0013] S3: A mesh is placed on the top of the detection reagent bottle, and the color developer freeze-dried microspheres and acetylcholinesterase freeze-dried microspheres are placed on the bottom of the detection reagent bottle. The substrate freeze-dried microspheres are placed on the mesh and screwed with a bottle cap, and sealed for storage to prepare a pesticide residue rapid test reagent that can be tested once alone.

[0014] Preferably, in S2, the diameter of the cuvettes used for preparing the freeze-dried microspheres of acetylcholinesterase and the freeze-dried microspheres of the color developer are both 5 mm.

[0015] Preferably, in S2, the vacuum freeze-drying process includes a pre-freezing stage, which includes 13 temperature sections. The cold trap temperature of the freeze drying box is set to a first temperature, the vacuum is turned on and maintained for T1 time, then heated to the second temperature and maintained for T2 time, and finally naturally sublimated for T3 time.

[0016] Preferably, the first temperature is -40°C, the second temperature is -20°C, T1 is 2 hours, T2 is 2 hours, and T3 is 10 hours.

[0017] Preferably, in S2, the vacuum freeze-drying treatment also includes a sublimation drying stage, wherein the frozen acetylcholinesterase solution, color developer, and substrate are respectively placed in reagent bottles for heating treatment, and the acetylcholinesterase solution, color developer, and substrate are dehydrated and dried. When the drying is completed, about 90% of the total water is removed, and all ice crystals are removed.

[0018] Preferably, in S2, the vacuum freeze-drying treatment also includes analytical drying, and the acetylcholinesterase solution, the color developer, and the substrate are further dehydrated and dried. After the dehydration and drying treatment, the residual water content in the acetylcholinesterase solution, the color developer, and the substrate is ensured to be 0.5%-4%.

[0019] The beneficial effects of the present invention are:

[0020] In the present invention, a rapid pesticide residue test reagent is prepared by placing freeze-dried microspheres of a color developer and freeze-dried microspheres of acetylcholinesterase at the bottom of a detection reagent bottle, placing freeze-dried microspheres of a substrate on a filter net, and screwing the bottle cap together. The operation steps of pesticide residue detection can be simplified, and the standardization and rapidization of pesticide residue detection can be facilitated. Moreover, the detection of pesticide residues in fruits and vegetables can be automatically performed by a detection instrument, so that grassroots inspectors are freed from the heavy task of pesticide residue detection. At the same time, the automated operation of the instrument avoids the problem of non-standard detection operations by inspectors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the disassembled structure of the detection reagent bottle. DETAILED DESCRIPTION

[0022] The present invention will be further explained below in conjunction with specific embodiments.

[0023] Reference Figure 1 , a method for preparing a rapid test reagent for organophosphorus and carbamate pesticide residues, comprising the following steps:

[0024] S1: Material preparation: cuvette, vacuum freeze dryer, detection reagent bottle;

[0025] S2: freeze-dried microsphere preparation;

[0026] a: Raw material preparation: acetylcholinesterase solution, color developer, substrate and protective agent, and the volume ratio of acetylcholinesterase solution, color developer and protective agent is 1:1:0.3;

[0027] b: Preparation of acetylcholinesterase freeze-dried microspheres: placing the acetylcholinesterase solution and the protective agent in a cuvette, and placing the cuvette in a vacuum freeze dryer for vacuum freeze drying to obtain acetylcholinesterase freeze-dried microspheres;

[0028] c: Preparation of chromogen freeze-dried microspheres: placing the chromogen solution and the protective agent in a cuvette, and placing the cuvette in a vacuum freeze dryer for vacuum freeze drying to obtain chromogen freeze-dried microspheres;

[0029] d: Preparation of substrate freeze-dried microspheres: placing the substrate and the protective agent in a cuvette, and placing the cuvette in a vacuum freeze dryer for vacuum freeze drying to obtain substrate freeze-dried microspheres;

[0030] S3: A mesh is placed on the top of the detection reagent bottle, and the color developer freeze-dried microspheres and acetylcholinesterase freeze-dried microspheres are placed on the bottom of the detection reagent bottle. The substrate freeze-dried microspheres are placed on the mesh and screwed with a bottle cap, and sealed for storage to prepare a pesticide residue rapid test reagent that can be tested once alone.

[0031] Furthermore, in S2, the diameters of the cuvettes used for preparing the freeze-dried microspheres of acetylcholinesterase and the freeze-dried microspheres of the color developer are both 5 mm.

[0032] At the same time, in S2, the vacuum freeze-drying process includes a pre-freezing stage, which includes 13 temperature sections. The cold trap temperature of the freeze drying box is set to the first temperature, the vacuum is turned on and maintained for T1 time, then heated to the second temperature and maintained for T2 time, and finally naturally sublimated for T3 time.

[0033] Meanwhile, the first temperature is -40°C, the second temperature is -20°C, T1 is 2 hours, T2 is 2 hours, and T3 is 10 hours.

[0034] Furthermore, in S2, the vacuum freeze drying process also includes a sublimation drying stage, wherein the frozen acetylcholinesterase solution, the color developer, and the substrate are placed in a reagent bottle for heating treatment, and the acetylcholinesterase solution, the color developer, and the substrate are dehydrated and dried. When the drying is completed, about 90% of the total water is removed, and all ice crystals are removed. When the frozen acetylcholinesterase solution, the color developer, and the substrate are placed in a reagent bottle for heating treatment, the ice crystals will sublimate into water vapor and escape, thereby dehydrating and drying the product. The drying starts from the outer surface and gradually moves inward, and the gaps left after the ice crystals sublimate become escape channels for the sublimated water vapor.

[0035] Furthermore, in S2, the vacuum freeze-drying treatment also includes analytical drying, and the acetylcholinesterase solution, the color developer, and the substrate are further dehydrated and dried. After the dehydration and drying treatment, the residual water content in the acetylcholinesterase solution, the color developer, and the substrate is ensured to be 0.5%-4%.

[0036] Each material has a different freeze-drying curve. The freeze-drying curve table is as follows:

[0037]

[0038]

[0039] In the present invention, a rapid pesticide residue test reagent is prepared by placing freeze-dried microspheres of a color developer and freeze-dried microspheres of acetylcholinesterase on the bottom of a detection reagent bottle, placing freeze-dried microspheres of a substrate on a filter screen, and screwing the bottle cap. The operation process is to take 2.5 ml of a sample solution into the pesticide residue test reagent, react at 37° C. for 10 minutes, put a funnel containing substrate microspheres into the test reagent, cover the funnel with a lid, mix well, and then perform an on-machine test using a spectrophotometer or a pesticide residue detector. The entire operation process omits the steps of adding cholinesterase, a color developer and a substrate, simplifies the operation steps of pesticide residue detection, facilitates the standardization and rapidity of pesticide residue detection, and can automatically perform the detection of pesticide residues in fruits and vegetables by the detection instrument, so that grassroots inspectors are freed from the heavy task of pesticide residue detection. At the same time, the automated operation of the instrument avoids the problem of non-standard detection operation by the inspectors.

[0040] In the present invention, a rapid pesticide residue test reagent is prepared by placing freeze-dried microspheres of a color developer and freeze-dried microspheres of acetylcholinesterase at the bottom of a detection reagent bottle, placing freeze-dried microspheres of a substrate on a filter net, and screwing the bottle cap together. The operation steps of pesticide residue detection can be simplified, and the standardization and rapidization of pesticide residue detection can be facilitated. Moreover, the detection of pesticide residues in fruits and vegetables can be automatically performed by a detection instrument, so that grassroots inspectors are freed from the heavy task of pesticide residue detection. At the same time, the automated operation of the instrument avoids the problem of non-standard detection operations by inspectors.

[0041] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a rapid test reagent for organophosphorus and carbamate pesticide residues, characterized in that: The following steps are involved: S1: Material preparation: cuvette, vacuum freeze dryer, detection reagent bottle; S2: freeze-dried microsphere preparation; a: Raw material preparation: acetylcholinesterase solution, color developer, substrate and protective agent, and the volume ratio of acetylcholinesterase solution, color developer and protective agent is 1:1:0.3; b: Preparation of acetylcholinesterase freeze-dried microspheres: placing the acetylcholinesterase solution and the protective agent in a cuvette, and placing the cuvette in a vacuum freeze dryer for vacuum freeze drying to obtain acetylcholinesterase freeze-dried microspheres; c: Preparation of chromogen freeze-dried microspheres: placing the chromogen solution and the protective agent in a cuvette, and placing the cuvette in a vacuum freeze dryer for vacuum freeze drying to obtain chromogen freeze-dried microspheres; d: Preparation of substrate freeze-dried microspheres: placing the substrate and the protective agent in a cuvette, and placing the cuvette in a vacuum freeze dryer for vacuum freeze drying to obtain substrate freeze-dried microspheres; S3: A mesh is placed on the top of the detection reagent bottle, and the color developer freeze-dried microspheres and acetylcholinesterase freeze-dried microspheres are placed on the bottom of the detection reagent bottle. The substrate freeze-dried microspheres are placed on the mesh and screwed with a bottle cap, and sealed for storage to prepare a pesticide residue rapid test reagent that can be tested once alone.

2. The method for preparing a rapid test reagent for organophosphorus and carbamate pesticide residues according to claim 1, characterized in that: In S2, the diameter of the cuvettes used for preparing the freeze-dried microspheres of acetylcholinesterase and the freeze-dried microspheres of the color developer are both 5 mm.

3. The method for preparing a rapid test reagent for organophosphorus and carbamate pesticide residues according to claim 1, characterized in that: In S2, the vacuum freeze-drying process includes a pre-freezing stage, which includes 13 temperature sections. The cold trap temperature of the freeze drying box is set to a first temperature, vacuuming is turned on and maintained for T1 time, then heated to a second temperature and maintained for T2 time, and finally naturally sublimated for T3 time.

4. The method for preparing a rapid test reagent for organophosphorus and carbamate pesticide residues according to claim 3, characterized in that: The first temperature is -40°C, the second temperature is -20°C, T1 is 2 hours, T2 is 2 hours, and T3 is 10 hours.

5. The method for preparing a rapid test reagent for organophosphorus and carbamate pesticide residues according to claim 1, characterized in that: In the S2, the vacuum freeze-drying process also includes a sublimation drying stage, in which the frozen acetylcholinesterase solution, the color developer, and the substrate are placed in reagent bottles for heating treatment, and the acetylcholinesterase solution, the color developer, and the substrate are dehydrated and dried. When the drying is completed, about 90% of the total water is removed and all ice crystals are removed.

6. The method for preparing a rapid test reagent for organophosphorus and carbamate pesticide residues according to claim 5, characterized in that In S2, the vacuum freeze-drying treatment also includes analytical drying, and the acetylcholinesterase solution, the color developer, and the substrate are further dehydrated and dried. After the dehydration and drying treatment, the residual water content in the acetylcholinesterase solution, the color developer, and the substrate is ensured to be 0.5%-4%.

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