CN103048455A - Herbicide 2, 4-D and pesticide CHI bigeminy detection card and preparation method thereof - Google Patents
Herbicide 2, 4-D and pesticide CHI bigeminy detection card and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a herbicide 2, 4-D and pesticide CHI bigeminy detection card and a preparation method thereof, relating to the technical field of environmental hormone detection. A test paper strip is arranged in an outer shell of the detection card, and the detection card consists of a PVC (Poly Vinyl Chloride) rubber plate, a sample pad, a colloidal gold combining pad, a wrapping membrane and a water absorbing pad, wherein the colloidal gold membrane is a glass fiber cellulose membrane containing 2, 4-D monoclonal antibody and CHI monoclonal antibody colloidal gold markers; the wrapping membrane is a nitric acid cellulose membrane; two detection lines T and a quality control line C are arranged on the wrapping membrane; the detection lines T are wrapped by a 2, 4-D protein conjugate and a CHI protein conjugate; and the quality control line C is wrapped by a rabbit anti-mouse IgG antibody. By utilizing the method, the herbicide 2, 4-D and pesticide CHI in a water sample can be detected at the same time, and the method is simple, convenient, rapid, and the result is accurate.
Description
Technical field
The present invention relates to environmental hormone detection technique field, particularly relate to a kind of herbicide 2,4-D-pesticide CHI bigeminy test card and preparation method thereof.
Background technology
In the last few years, scientists finds that in succession some are present in outside the living organism, have and people and the similar material of biological endocrine hormone effect, thus these materials can simulate the endocrine hormone function and cause the biosome endocrine system disorder, just it is called environmental hormone or Environmental Hormone.Environmental hormone has become the third-largest environmental problem in the whole world after ozonosphere, terrestrial climate warm, becomes the heat subject in the Research of Environmental Sciences field.
Environmental hormone refers to that exogen disturbs the endocrine chemical substance of biosome.Owing to mankind's activity is discharged into chemical substance in the environment, the similar estrogenic effect of performance in animal and human's body.It can disturb the normal physiological metabolism of biosome, endocrine and Reproductive Performance by being combined with hormone receptor, causes all negative biological effects.Environmental hormone enters in the human or animal body by surrounding medium and food chain, disturbs its internal system and reproductive function system, even the harm that causes malignant tumour and biological extinction is arranged.
Modern industrial and agricultural production has also brought white elephant to environment when constantly satisfying human living needs, and has threatened human healthy reproduction, even the procreation of species.China is pesticide producing and use big country in the world, and data shows that China's pesticide original medicine output in 2011 is 264.87 ten thousand tons, and wherein the raw insecticide drug prods is 70.9 ten thousand tons, accounts for 44.35% of total production; Herbicide output is 117.5 ten thousand tons, accounts for 26.77% of total production; The bactericide original drug prods is 150,000 tons, accounts for 5.67% of total production.This type of agricultural chemicals is except section port, and major part is sold at home and used.China has 50 ~ 600,000 tons of organo-chlorine pesticides directly to be disposed in the environment every year approximately according to the related data estimation, such as herbicide 2,4-drips (2,4-Dichlorophenoxyacetic Acid, 2,4-D), pesticide chlopyrifos (Chlorpyrifos, CHI) and epiphyte pharmaceutical pentachlorophenol (Pentachlorophenol, PCP).And most organo-chlorine pesticides have stronger endocrine.In case this type of agricultural chemicals entered environment will enter in the biosome by multiple route of exposure, be enriched in the adipose tissue of biosome and then the healthy reproduction of harm humans and animals.
At present, the analytical approach of Environmental Hormone Pollution thing mainly be divided into take GC-MS, HPLC and LC-MS technology as representative instrument analytical method and based on the immune analysis method of antigen-antibody reaction, the common running program of these conventional residue analysis methods is complicated, efficient is lower, analysis cost is higher, be unfavorable for promoting the use of, be difficult to adapt to the requirement of great amount of samples and Site Detection.Harmfulness, representativeness and present study hotspot based on the Environmental Hormone Pollution thing the present invention relates to a kind of herbicide 2,4-D and pesticide CHI bigeminy test card and preparation method thereof.That test card of the present invention has is simple and efficient, easy operating, the advantage such as with low cost, and can detect simultaneously these two kinds of Environmental Hormone Pollution materials in the water sample, has produced good Social benefit and economic benefit.
Summary of the invention
The object of the present invention is to provide and a kind ofly can detect simultaneously herbicide 2 in the water sample, the test card of 4-D and pesticide CHI is fit to carry out Site Detection and selectivity is good, stability is strong, simple and convenient, with low cost advantage.
(1) colloidal gold solution preparation: get 1 of 1 L Erlenmeyer flask, add ultrapure water 495 mL, then add 1% gold chloride (HAuCl
43H
2O) 5 mL are mixed with 500 mL, 0.01% aqueous solution of chloraurate, are continuing to add 1% trisodium citrate (Na in the situation about stirring after heating is boiled
3C
6H
5O
72H
2O) solution 5-7 mL continues agitating heating, when the color of the solution becomes transparent aubergine fully, keeps stopped heating behind 5 min, and moisturizing is cooled to room temperature to original volume, and 2-8 ℃ saves backup;
(2) pre-service of antibody: will mark 2,4-D or CHI monoclonal antibody are at 1000 r/min, under 4 ℃ of conditions, centrifugal 20 min get supernatant, are diluted to 1 mg/mL with 0.01 mol/L PBS; Use perhaps that 0.01 mol/L PBS will mark 2,4-D or CHI monoclonal antibody are diluted to 1 mg/mL, cross 0.22 μ m filter membrane;
(3) preparation of colloid gold label thing: get colloidal gold solution 40 mL in the step (1), with 0.25 mol/L K
2CO
3Regulate colloidal gold solution pH to 8.5, magnetic stirrer 250 r/min stir, and dropwise add 4 mL and contain 0.3 mg 2, and the protein solution of 4-D or CHI monoclonal antibody reacts 10 min; Dropwise add 4 mL, 10% BSA, continue stirring reaction 10 min; Centrifugal 20 min of gold labeling antibody solution normal temperature low speed (1500 r/min) discard the precipitation that is formed by the gold grain that condenses; 4 ℃ of red supernatant solution, centrifugal 20 min of 12000 r/min abandon supernatant, and collecting precipitation will precipitate with golden labeling antibody dilution and be settled to 1 mL, be prepared into the colloid gold label thing of 2,4-D monoclonal antibody or CHI monoclonal antibody;
(4) preparation of collaurum film: with step (3) 2, the colloid gold label thing of 4-D monoclonal antibody and CHI monoclonal antibody evenly is sprayed on the carrier glass fibre element film with drawing the concentration of film instrument with 5 μ L/cm successively, room temperature is dried or 37 ℃ of oven dry 3 h naturally, make the collaurum film that contains 2,4-D monoclonal antibody and CHI monoclonal antibody;
(5) coated film preparation: the rabbit anti-mouse igg antibody dilution is become 1 mg/mL, 2, the 4-D antigen diluent becomes 0.3 mg/mL to become 0.5 mg/mL with the CHI antigen diluent, be sprayed on the nitrocellulose filter with the concentration of 1 μ L/cm successively with drawing the film instrument, be prepared into coated film, behind 37 ℃ of coated 2 h, room temperature is dried or 37 ℃ of oven dry naturally;
(6) sample pad pre-treatment: the sample pad treating fluid is uniformly coated on the glass fibre element film, is lower than in air humidity under 60% the condition, room temperature is dried naturally;
(7) assembling of test card: the PVC backing is pasted sample pad, collaurum film, coated film and adsorptive pads from top to bottom successively, is assembled into test strips, and it is rectangular to cut into one fixed width, test strips is installed in the test card shell of rectangular flat shelly again.
Golden labeling antibody dilution in the described step (3) is by 5 g Bovine serum albumins (BSA), 10 g sucrose, and it is formulated to add the dissolving of 100 mL, 0.01 mol/L PBS solution, crosses 0.22 μ m filter membrane, now with the current; Gold labeling antibody lavation buffer solution is by 10 g Bovine serum albumins (BSA) and 1 g PEG-20000, is settled to 100 mL gained with 0.01 mol/L PBS pH7.4;
Sample pad treating fluid in the described step (6) is by 1 g Bovine serum albumin (BSA) and 0.8 g sodium chloride (NaCl), is settled to 100 mL with the 0.01 mol/L PBS that contains 0.5%TRITON-100;
The present invention can be effective to measure simultaneously herbicide 2 in the water sample, two kinds of Environmental Hormone Pollution materials of 4-D and pesticide CHI, and method is simple, and is convenient, fast, and the result is accurate.
Description of drawings
Fig. 1 is herbicide 2 of the present invention, the structural drawing of 4-D-pesticide CHI bigeminy test card: 1. wells 2. 2 among the figure, and 4-D detection line 3. CHI detection lines 4. nature controlling lines 5. detect hole 6. test strips;
Fig. 2 is herbicide 2 of the present invention, the sectional structure chart of the test strips of 4-D-pesticide CHI bigeminy test card, 7. test card shells, 8. sample pad, 9. collaurum pads, 10. PVC backings, 11. nitrocellulose filters among the figure.
Embodiment
Fig. 1 is herbicide 2 of the present invention, and structural drawing, Fig. 2 of 4-D-pesticide CHI bigeminy test card are herbicide 2 of the present invention, the sectional structure chart of the test strips of 4-D-pesticide CHI bigeminy test card, and 10 is the PVC backing among the figure; 8 is sample pad; 9 is the collaurum pad, has been coated with 2,4-D monoclonal antibody and CHI monoclonal antibody colloid gold label thing on this collaurum pad; 11 is coated film, and namely nitrocellulose filter has been coated with 2,4-D protein conjugate, CHI protein conjugate and rabbit anti-mouse igg on this nitrocellulose filter.
(sample pipetting volume end) adheres to sample pad 8, collaurum pad 9 on an end of PVC backing 10, and sample pad 8 is overlapped on the collaurum pad 9.
In the middle of PVC backing 10, adhere to nitrocellulose filter 11.Be provided with 2,4-D protein conjugate detection line 2, CHI protein conjugate detection line 3 and rabbit anti-mouse igg nature controlling line 4 at nitrocellulose filter 11.
The other end at PVC backing 10 adheres to adsorptive pads 12.One end of nitrocellulose filter 11 slightly intersects with collaurum pad 9, and the other end slightly intersects with adsorptive pads 12.This test strips 6 can be incorporated with in the test card shell 7 that mould of plastics makes, and is provided with well 1 and detects hole 5 covering of test card shell 7, and sample pad 8 is over against well 1, and nitrocellulose filter 11 is over against detecting hole 5.
(1) colloidal gold solution preparation: get 1 of 1 L Erlenmeyer flask, add ultrapure water 495 mL, then add 1% gold chloride (HAuCl
43H
2O) 5 mL are mixed with 500 mL, 0.01% aqueous solution of chloraurate, are continuing to add 1% trisodium citrate (Na in the situation about stirring after heating is boiled
3C
6H
5O
72H
2O) solution 5-7 mL continues agitating heating, when the color of the solution becomes transparent aubergine fully, keeps stopped heating behind 5 min, and moisturizing is cooled to room temperature to original volume, and 2-8 ℃ saves backup;
(2) pre-service of antibody: will mark 2,4-D or CHI monoclonal antibody are at 1000 r/min, under 4 ℃ of conditions, centrifugal 20 min get supernatant, are diluted to 1 mg/mL with 0.01 mol/L PBS; Use perhaps that 0.01 mol/L PBS will mark 2,4-D or CHI monoclonal antibody are diluted to 1 mg/mL, cross 0.22 μ m filter membrane;
(3) preparation of colloid gold label thing: get colloidal gold solution 40 mL in the step (1), with 0.25 mol/L K
2CO
3Regulate colloidal gold solution pH to 8.5, magnetic stirrer 250 r/min stir, and dropwise add 4 mL and contain 0.3 mg 2, and the protein solution of 4-D or CHI monoclonal antibody reacts 10 min; Dropwise add 4 mL, 10% BSA, continue stirring reaction 10 min; Centrifugal 20 min of gold labeling antibody solution normal temperature low speed (1500 r/min) discard the precipitation that is formed by the gold grain that condenses; 4 ℃ of red supernatant solution, centrifugal 20 min of 12000 r/min abandon supernatant, and collecting precipitation will precipitate with golden labeling antibody dilution and be settled to 1 mL, be prepared into the colloid gold label thing of 2,4-D monoclonal antibody or CHI monoclonal antibody;
(4) preparation of collaurum film: with step (3) 2, the colloid gold label thing of 4-D monoclonal antibody and CHI monoclonal antibody evenly is sprayed on the carrier glass fibre element film with drawing the concentration of film instrument with 5 μ L/cm successively, room temperature is dried or 37 ℃ of oven dry 3 h naturally, make the collaurum film that contains 2,4-D monoclonal antibody and CHI monoclonal antibody;
(5) coated film preparation: the rabbit anti-mouse igg antibody dilution is become 1 mg/mL, 2, the 4-D antigen diluent becomes 0.3 mg/mL to become 0.5 mg/mL with the CHI antigen diluent, be sprayed on the nitrocellulose filter with the concentration of 1 μ L/cm successively with drawing the film instrument, be prepared into coated film, behind 37 ℃ of coated 2 h, room temperature is dried or 37 ℃ of oven dry naturally;
(6) sample pad pre-treatment: the sample pad treating fluid is uniformly coated on the glass fibre element film, is lower than in air humidity under 60% the condition, room temperature is dried naturally;
(7) assembling of test card: the PVC backing is pasted sample pad, collaurum film, coated film and adsorptive pads from top to bottom successively, is assembled into test strips, and it is rectangular to cut into one fixed width, test strips is installed in the test card shell of rectangular flat shelly again.
Embodiment 3
The test of minimum detectability amount.
Prepare respectively the herbicide 2 of standard, 4-D and pesticide CHI standard solution concentration are: 0,5 ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL; Drip 2 samples to the test card well, observe the colour developing result of detection line T and nature controlling line C behind 5 min.Through repeatedly check, the recall rate of 2,4-D and CHI reaches more than 99%, and the minimum detectability amount is less than 15 ng/mL.
。
Claims (2)
1. herbicide 2, and 4-D and pesticide CHI bigeminy colloidal-gold detecting-card is characterized in that preparing according to following step:
Colloidal gold solution preparation: get 1 of 1 L Erlenmeyer flask, add ultrapure water 495 mL, then add 1% gold chloride HAuCl
43H
2O 5 mL are mixed with 500 mL, 0.01% aqueous solution of chloraurate, are continuing to add 1% trisodium citrate Na in the situation about stirring after heating is boiled
3C
6H
5O
72H
2O solution 5-7 mL continues agitating heating, when the color of the solution becomes transparent aubergine fully, keeps stopped heating behind 5 min, and moisturizing is cooled to room temperature to original volume, and 2-8 ℃ saves backup;
The pre-service of antibody: will mark 2,4-D or CHI monoclonal antibody are at 1000 r/min, under 4 ℃ of conditions, centrifugal 20 min get supernatant, are diluted to 1 mg/mL with 0.01 mol/L PBS; Use perhaps that 0.01 mol/L PBS will mark 2,4-D or CHI monoclonal antibody are diluted to 1 mg/mL, cross 0.22 μ m filter membrane;
The preparation of colloid gold label thing: get colloidal gold solution 40 mL in the step (1), with 0.25 mol/L K
2CO
3Regulate colloidal gold solution pH to 8.5, magnetic stirrer 250 r/min stir, and dropwise add 4 mL and contain 0.3 mg 2, and the protein solution of 4-D or CHI monoclonal antibody reacts 10 min; Dropwise add 4 mL, 10% BSA, continue stirring reaction 10 min; Centrifugal 20 min of gold labeling antibody solution normal temperature 1500 r/min discard the precipitation that is formed by the gold grain that condenses; 4 ℃ of red supernatant solution, centrifugal 20 min of 12000 r/min abandon supernatant, and collecting precipitation will precipitate with golden labeling antibody dilution and be settled to 1 mL, be prepared into the colloid gold label thing of 2,4-D monoclonal antibody or CHI monoclonal antibody;
The preparation of collaurum film: with step (3) 2, the colloid gold label thing of 4-D monoclonal antibody and CHI monoclonal antibody evenly is sprayed on the carrier glass fibre element film with drawing the concentration of film instrument with 5 μ L/cm successively, room temperature is dried or 37 ℃ of oven dry 3 h naturally, make the collaurum film that contains 2,4-D monoclonal antibody and CHI monoclonal antibody;
Coated film preparation: the rabbit anti-mouse igg antibody dilution is become 1 mg/mL, 2, the 4-D antigen diluent becomes 0.3 mg/mL to become 0.5 mg/mL with the CHI antigen diluent, be sprayed on the nitrocellulose filter with the concentration of 1 μ L/cm successively with drawing the film instrument, be prepared into coated film, behind 37 ℃ of coated 2 h, room temperature is dried or 37 ℃ of oven dry naturally;
The sample pad pre-treatment: the sample pad treating fluid is uniformly coated on the glass fibre element film, is lower than in air humidity under 60% the condition, room temperature is dried naturally;
The assembling of test card: the PVC offset plate is pasted sample pad, collaurum film, coated film and adsorptive pads from top to bottom successively, is assembled into test strips, and it is rectangular to cut into one fixed width, test strips is installed in the test card shell of rectangular flat shelly again.
2. herbicide 2 according to claim 1,4-D and pesticide CHI bigeminy colloidal-gold detecting-card, it is characterized in that golden labeling antibody dilution in the described step (3) is by 5 g Bovine serum albumins, 10 g sucrose, it is formulated to add the dissolving of 100 mL, 0.01 mol/L PBS solution, cross 0.22 μ m filter membrane, now with the current; Gold labeling antibody lavation buffer solution is by 10 g Bovine serum albumins and 1 g PEG-20000, is settled to 100 mL gained with 0.01 mol/L PBS pH7.4;
Sample pad treating fluid in the described step (6) is by 1 g Bovine serum albumin and 0.8 g sodium chloride, is settled to 100 mL with the 0.01 mol/L PBS that contains 0.5%TRITON-100.
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CN108226505A (en) * | 2016-12-15 | 2018-06-29 | 南京亿特生物科技有限公司 | Immune colloid gold detection card of sodium sulfocyanate and preparation method thereof |
CN113030467A (en) * | 2021-03-13 | 2021-06-25 | 河南省农业科学院 | Self-amplification precisely-controlled indirect competition immunochromatography test strip and application thereof |
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