CN101299029A - Preparation for detecting organophosphorus pesticide residual test paper and its use method - Google Patents
Preparation for detecting organophosphorus pesticide residual test paper and its use method Download PDFInfo
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- CN101299029A CN101299029A CNA2008100284328A CN200810028432A CN101299029A CN 101299029 A CN101299029 A CN 101299029A CN A2008100284328 A CNA2008100284328 A CN A2008100284328A CN 200810028432 A CN200810028432 A CN 200810028432A CN 101299029 A CN101299029 A CN 101299029A
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Abstract
The present invention discloses a preparation method of a test paper for testing the residual organphosphorous pesticide and a using method thereof, wherein the preparation method comprises the following procedures: (1) preparing acetylcholine esterase with fresh blood of hen or female duck as raw material; (2) fixing the acetylcholine esterase on the filter paper; (3) slicing when the filter paper is frozen and dried. A substrate liquid, a color developing liquid and a sensitization liquid are provided. The principle is that the inhibition of the organphosphorous pesticide which is processed by the sensitization liquid to the acetylcholine esterase in the chicken or duck blood serum to make that the substrate of naphthyl acetate of the acetylcholine esterase can not be decomposed thereby that the displayed colors are not consistent when the fast blue B is added into to obtain the purpose of determining whether the sample contains the residual organphosphorous pesticide. The method of the invention has the advantages of low cost, simple and fast operation. The method is suitable for determining whether the residual organphosphorous pesticide exists in vegetable, fruit and food on-site.
Description
Technical field:
The invention belongs to the analytical chemistry field and be specifically related to a kind of preparation and using method thereof that detects organophosphorus pesticide residual test paper.
Background technology:
At present, residues of pesticides have become the significant problem that threatens food security.Strengthen the Detecting Pesticide dynamics, the monitoring of strengthening food residues of pesticides such as vegetable and fruit on the market seems very urgent.Pesticide residue analysis mainly adopts technology such as gas chromatography, gas chromatography-mass spectrography and high performance liquid chromatography traditionally, can more accurately measure residual quantity, but need large-scale instrument, the pre-treatment complex operation, just can grasp by the personnel of specialized training, and instrument is inconvenient to carry mobile, be difficult to adapt to the requirement of on-the-spot simple quick and big quantity sample retention analysis.So fast and convenient, residues of pesticides are imperative in the fast survey technology detection vegetable and fruit with low cost.In recent years, the patent of domestic this respect application is a lot, the method of utilizing antigen-antibody is arranged, as " being used to detect the immune antiboidy and the application thereof of organophosphorus pesticide " (200410041895.X), have by oxyluminescence and measure the method that spectrum carries out colorimetric, (200310119273.X) as " chemiluminescent organophosphorus pesticide residual analyzer and detection method thereof " (200510041791.3), " a kind of analytical approach of vegetables organophosphorus pesticide " (200410026892.9), " rapid assay methods of organophosphorus pesticide residual quantity in vegetables and the fruit ".The similar method of utilizing enzyme to suppress to this project is also arranged, as " fish esterase reagent of fast detecting organophosphorus pesticide " (02115152.0), " a kind of preparation of reagent of qualitative reaction organophosphorus and content assaying method " (99109293.7), " plant-esterase tablet of fast measuring organophosphorus pesticide " (99114268.3), " quick test box of residual agricultural organophosphorus chemical and manufacture method " (O0135716.6), " method of fast detection of trace organophosphorus and carbamate pesticide " (01144343.X).But used enzyme source or the substrate of plant esterase that these patented methods are used or phenyl-(azo-1) 2-acetic acid naphthalene ester and this project is different.
Summary of the invention:
The present invention is a raw material with the acetylcholinesterase in fresh hen or the duck blood, by the inhibiting effect of organophosphorus pesticide to acetylcholinesterase, the substrate naphthyl acetate of enzyme can not be decomposed, thereby the color that adds behind the solid blue B shoals.The depth by color reaches the purpose that whether contains organophosphorus pesticide in the judgement sample at last.
The present invention is following realization:
The invention provides a kind of preparation and using method thereof that detects organophosphorus pesticide residual test paper.
The preparation method of detection organophosphorus pesticide residual test paper of the present invention comprises the steps: that (1) is the feedstock production acetylcholinesterase with fresh hen or duck blood; (2) acetylcholinesterase is fixed on the filter paper; (3) will cut into slices after the filter paper freeze drying.
The method for preparing acetylcholinesterase in the above-mentioned steps (1) is: after solidifying under the new blood room temperature of hen or duck, place to spend the night in 0～4 ℃ of refrigerator serum is separated out, get its serum supernatant, gained serum supernatant is again at the 16000r.p.m rotating speed, centrifugal 5min under 0～4 ℃, its supernatant is the thick enzyme of acetylcholinesterase; The triangular flask that the thick enzyme of acetylcholinesterase will be housed is put into frozen water then, slowly stirs the adding saturated ammonium sulfate in crude enzyme liquid, and the saturation degree that makes ammonium sulfate in the enzyme liquid is in 55%～60% scope; Gained solution is in 15000r.p.m, and 0～4 ℃ is descended centrifugal 8min, supernatant discarded; Precipitation is used dissolved in distilled water again, and the bag filter of the molecular weight 10,000 of packing into, in 4 ℃ of refrigerator dialysis 40 hours, changes liquid therebetween 8 times, and the enzyme liquid cooling freeze-drying of the back gained of will dialysing then is dry, is the acetylcholinesterase of purifying.
In the above-mentioned steps (2) acetylcholinesterase of freeze-drying being made into concentration with distilled water is 0.10～0.40mg/mL, and filter paper is immersed in the enzyme liquid, takes out after 3 fens kinds, places lyophilizer to carry out freeze-drying.
With specific tapping and plugging machine the filter paper enzyme sheet of freeze-drying is broken into the garden shape small pieces of diameter 5cm size, the bottle sealing preservation that places the band bottle stopper in the above-mentioned steps (3).
Detection organophosphorus pesticide residual test paper using method of the present invention has the following steps (1) with 50% methyl alcohol configuration acecoline substrate solution, concentration is 2.0mg/mL, with the solid blue B colour developing liquid of distilled water configuration, concentration is 1.0mg/mL, is 0.5～1.0% concentration with distilled water with the dilution of 3% bromine water, is used for enhanced sensitivity liquid; When (2) detecting, 0.5～1.0% the bromine water that the extract 500 μ L that get quantitative vegetables or fruit earlier add multiple proportions leaves standstill 10～15min, and contrast is left standstill 10～15min with 0.5～1.0% bromine water of distilled water 500 μ L adding multiple proportions; Get the enzyme sheet of 2 above-mentioned fixedly acetylcholinesterases, add sample extracting solution and each 40～60 μ L of contrast that above-mentioned bromine water was handled respectively, leave standstill 10min; Add 50～80 μ L acecoline substrate solutions again, leave standstill 10min; (3) add the solid blue B colour developing liquid of 30～50 μ L, observation sample enzyme sheet and control enzyme sheet shade, if solid colour, showing does not have residues of pesticides, if sample enzyme sheet is more of light color than control enzyme sheet, showing has residues of pesticides.
This patent method is compared with product with existing patent, and the preparation method of test paper is simple, and used enzyme source is abundant, and cost is low, uses more conveniently, is a kind of simple practical fast organophosphorus pesticide mensuration product.Can be suitable for on-the-spot to the organophosphorus pesticide fast measuring.
Embodiment
Implementation column 1:
Dispose the DDVP of 2.0 μ g/mL with distilled water, get the eppdof centrifuge tube of 2 1.5mL, the DDVP of adding 500 μ L2.0 μ g/mL and the bromine water of 500 μ L0.5% leave standstill 15min in one arm, the bromine water that adds 500 μ L distilled water and 500 μ L0.5% in another arm leaves standstill 15min, get the enzyme sheet of 2 acetylcholinesterases, add DDVP pesticide liquid and each 40 μ L of contrast that above-mentioned bromine water was handled respectively, leave standstill 10min; Add 60 μ L acecoline substrate solutions again, leave standstill 10min; Add the solid blue B colour developing liquid of 50 μ L at last, it is intimate colourless to observe the enzyme sheet that adds DDVP, and the control enzyme sheet shows light red.
Implementation column 2:
Dispose the acephatemet of 1.5 μ g/mL with distilled water, get the eppdof centrifuge tube of 2 1.5mL, the acephatemet of adding 500 μ L1.5 μ g/mL and the bromine water of 500 μ L0.5% leave standstill 15min in one arm, the bromine water that adds 500 μ L distilled water and 500 μ L0.5% in another arm leaves standstill 15min, get the enzyme sheet of 2 acetylcholinesterases, add methamidophos pesticide liquid and each 60 μ L of contrast that above-mentioned bromine water was handled respectively, leave standstill 10min; Add 70 μ L acecoline substrate solutions again, leave standstill 10min; Add the solid blue B colour developing liquid of 50 μ L at last, observation sample enzyme sheet and control enzyme sheet shade, the enzyme sheet that adds acephatemet is intimate colourless, and the control enzyme sheet shows light red.
Implementation column 3:
Buy back 5 parts of leaf vegetables at random from vegetables market, after getting the wherein continuous flushing of 100g leaf usefulness distilled water 100mL, the bromine water of numbering 5 1.5mL eppdof centrifuge tubes then and respectively adding different leaf vegetables washing fluids of 500 μ L and 500 μ L0.5% leaves standstill 15min, the bromine water that adds 500 μ L distilled water and 500 μ L0.5% in the 6th arm leaves standstill 15min, get the enzyme sheet of 6 acetylcholinesterases, add leaf vegetables sample wash liquid and each 60 μ L of contrast that above-mentioned bromine water was handled respectively, leave standstill 10min; Add 60 μ L acecoline substrate solutions again, leave standstill 10min; Add the solid blue B colour developing liquid of 50 μ L at last, observation sample enzyme sheet and control enzyme sheet shade, the consistent nearly light red of the color of 4 samples with the control enzyme sheet, the color comparison of one of them sample is according to shallow, show that this sample contains residues of pesticides, after repeated test and prove conclusively this sample with liquid chromatography to contain orthene residual again.
Claims (5)
1. a preparation method who detects organophosphorus pesticide residual test paper is characterized in that preparing this test paper and comprises the steps: that (1) is the feedstock production acetylcholinesterase with fresh hen or duck blood; (2) acetylcholinesterase is fixed on the filter paper; (3) will cut into slices after the filter paper freeze drying.
2. according to the preparation method of the described test paper of claim 1, it is characterized in that the method for preparing acetylcholinesterase in the above-mentioned steps (1) is: after solidifying under the new blood room temperature of hen or duck, place to spend the night in 0～4 ℃ of refrigerator serum is separated out, get its serum supernatant, gained serum supernatant is again at the 16000r.p.m rotating speed, centrifugal 5min under 0～4 ℃, its supernatant is the thick enzyme of acetylcholinesterase; The triangular flask that the thick enzyme of acetylcholinesterase will be housed is put into frozen water then, slowly stirs the adding saturated ammonium sulfate in crude enzyme liquid, and the saturation degree that makes ammonium sulfate in the enzyme liquid is in 55%～60% scope; Gained solution is in 15000r.p.m, and 0～4 ℃ is descended centrifugal 8min, supernatant discarded; Precipitation is used dissolved in distilled water again, and the bag filter of the molecular weight 10,000 of packing into, in 4 ℃ of refrigerator dialysis 40 hours, changes liquid therebetween 8 times, and the enzyme liquid cooling freeze-drying of the back gained of will dialysing then is dry, is the acetylcholinesterase of purifying.
3. according to the preparation method of the described test paper of claim 1, it is characterized in that in the above-mentioned steps (2) that it is 0.10～0.40mg/mL that acetylcholinesterase with freeze-drying is made into concentration with distilled water, filter paper is immersed in the enzyme liquid, take out after 3 fens kinds, place lyophilizer to carry out freeze-drying.
4. according to the preparation method of the described test paper of claim 1, it is characterized in that in the above-mentioned steps (3) the filter paper enzyme sheet of freeze-drying being broken into the garden shape small pieces of diameter 5cm size, the bottle sealing preservation that places the band bottle stopper with specific tapping and plugging machine.
5. the using method of a detection organophosphorus pesticide residual test paper as claimed in claim 1, it is characterized in that this using method has the following steps: (1) is with 50% methyl alcohol configuration acecoline substrate solution, concentration is 2.0mg/mL, with the solid blue B colour developing liquid of distilled water configuration, concentration is 1.0mg/mL, is 0.5～1.0% concentration with distilled water with the dilution of 3% bromine water, is used for enhanced sensitivity liquid; When (2) detecting, 0.5～1.0% the bromine water that the extract 500 μ L that get quantitative vegetables or fruit earlier add multiple proportions leaves standstill 10～15min, and contrast is left standstill 10～15min with 0.5～1.0% bromine water of distilled water 500 μ L adding multiple proportions; Get the enzyme sheet of 2 above-mentioned fixedly acetylcholinesterases, add sample extracting solution and each 40～60 μ L of contrast that above-mentioned bromine water was handled respectively, leave standstill 10min; Add 50～80 μ L acecoline substrate solutions again, leave standstill 10min; (3) add the solid blue B colour developing liquid of 30～50 μ L, observation sample enzyme sheet and control enzyme sheet shade, if solid colour, showing does not have residues of pesticides, if sample enzyme sheet is more of light color than control enzyme sheet, showing has residues of pesticides.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2008100284328A CN101299029A (en) | 2008-05-30 | 2008-05-30 | Preparation for detecting organophosphorus pesticide residual test paper and its use method |
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| CNA2008100284328A CN101299029A (en) | 2008-05-30 | 2008-05-30 | Preparation for detecting organophosphorus pesticide residual test paper and its use method |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102486459A (en) * | 2010-12-06 | 2012-06-06 | 中国科学院动物研究所 | Kit for rapidly detecting resistance mosquitoes through carboxylesterase development method |
| CN105044101A (en) * | 2015-07-31 | 2015-11-11 | 复旦大学 | Quick detection card for pesticide residues based on naked eye visual colorimetric determination |
| CN105319211A (en) * | 2015-11-13 | 2016-02-10 | 无锡艾科瑞思产品设计与研究有限公司 | Kit for quickly detecting organophosphorus residues in agricultural products and application method of kit |
| CN106093032A (en) * | 2016-08-31 | 2016-11-09 | 孟月志 | A kind of Fast Determination of Pesticide Residue eliminates false-positive detection method |
| CN108169220A (en) * | 2017-11-24 | 2018-06-15 | 武汉市农业科学院 | A kind of thick salt method and its production technology and application method based on horse serum cholinesterase |
| CN109781718A (en) * | 2019-01-17 | 2019-05-21 | 广东省食品工业研究所有限公司 | A kind of preparation method of the compound enzyme solution for fast detecting pesticide residue |
| CN110208256A (en) * | 2019-06-14 | 2019-09-06 | 智锐达仪器科技南通有限公司 | A kind of Detecting Pesticide pulvis and preparation method, cuvette, detection method |
| CN112198145A (en) * | 2020-09-27 | 2021-01-08 | 常州金坛江南制粉有限公司 | Method for detecting pesticide residue in raw material glutinous rice |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| CN117538318A (en) * | 2024-01-10 | 2024-02-09 | 山东利尔康医疗科技股份有限公司 | Residual peracetic acid test card and preparation method thereof |
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2008
- 2008-05-30 CN CNA2008100284328A patent/CN101299029A/en active Pending
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102486459A (en) * | 2010-12-06 | 2012-06-06 | 中国科学院动物研究所 | Kit for rapidly detecting resistance mosquitoes through carboxylesterase development method |
| CN105044101A (en) * | 2015-07-31 | 2015-11-11 | 复旦大学 | Quick detection card for pesticide residues based on naked eye visual colorimetric determination |
| CN105319211A (en) * | 2015-11-13 | 2016-02-10 | 无锡艾科瑞思产品设计与研究有限公司 | Kit for quickly detecting organophosphorus residues in agricultural products and application method of kit |
| CN106093032A (en) * | 2016-08-31 | 2016-11-09 | 孟月志 | A kind of Fast Determination of Pesticide Residue eliminates false-positive detection method |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| CN108169220A (en) * | 2017-11-24 | 2018-06-15 | 武汉市农业科学院 | A kind of thick salt method and its production technology and application method based on horse serum cholinesterase |
| CN108169220B (en) * | 2017-11-24 | 2021-02-12 | 武汉市农业科学院 | Horse serum cholinesterase-based pesticide residue rapid detection card and production process and use method thereof |
| CN109781718A (en) * | 2019-01-17 | 2019-05-21 | 广东省食品工业研究所有限公司 | A kind of preparation method of the compound enzyme solution for fast detecting pesticide residue |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11609042B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11740019B2 (en) | 2019-03-14 | 2023-08-29 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11747082B2 (en) | 2019-03-14 | 2023-09-05 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11815311B2 (en) | 2019-03-14 | 2023-11-14 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| CN110208256A (en) * | 2019-06-14 | 2019-09-06 | 智锐达仪器科技南通有限公司 | A kind of Detecting Pesticide pulvis and preparation method, cuvette, detection method |
| CN112198145A (en) * | 2020-09-27 | 2021-01-08 | 常州金坛江南制粉有限公司 | Method for detecting pesticide residue in raw material glutinous rice |
| CN117538318A (en) * | 2024-01-10 | 2024-02-09 | 山东利尔康医疗科技股份有限公司 | Residual peracetic acid test card and preparation method thereof |
| CN117538318B (en) * | 2024-01-10 | 2024-04-05 | 山东利尔康医疗科技股份有限公司 | Residual peracetic acid test card and preparation method thereof |
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Open date: 20081105 |