CN102053155A - Homogeneous chemiluminescence immunoassay method for measuring for organophosphorus pesticide Dursban - Google Patents
Homogeneous chemiluminescence immunoassay method for measuring for organophosphorus pesticide Dursban Download PDFInfo
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- CN102053155A CN102053155A CN2010102213659A CN201010221365A CN102053155A CN 102053155 A CN102053155 A CN 102053155A CN 2010102213659 A CN2010102213659 A CN 2010102213659A CN 201010221365 A CN201010221365 A CN 201010221365A CN 102053155 A CN102053155 A CN 102053155A
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Abstract
The invention discloses a homogeneous chemiluminescence immunoassay method for measuring an organophosphorus pesticide Dursban and belongs to the technical fields of pesticide residue analysis and chemiluminescence immunoassay. The method comprises the following steps: 1) activating the organophosphorus pesticide Dursban and introducing an active group carboxyl group so as to acquire the activated organophosphorus pesticide Dursban; 2) marking the chemiluminescence agent luminol onto bovine serum albumin (BSA) so as to acquire the luminol-BSA, wherein the coupling ratio reaches (1:20) to (1:40); 3) marking the activated organophosphorus pesticide Dursban onto the luminol-BSA, so as to acquire a Dursban chemiluminescence marker; 4) drawing a luminescence kinetics curve of the Dursban chemiluminescence marker, wherein a time axis is used as the horizontal ordinate and the relative luminous intensity is used as the vertical coordinate; and 5) establishing a homogeneous chemiluminescence immunoassay direct competition method to confirm the linear relationship between the luminous intensity and the antigen concentration of a sample to be identified. The method provided by the invention is suitable for the speedy qualitative and quantitative analysis detection for the Dursban on site.
Description
Technical field
The invention belongs to pesticide residue analysis and chemiluminescence immunoassay technology field, be specifically related to a kind of homogeneous chemistry luminescence immunoassay method that detects the organophosphorus pesticide chlopyrifos.
Background technology
Chlopyrifos (chlorpyrifos), O, O-diethyl-O-(3,5, the 6-trichloro-2-pyridyl) phosphorothionate.A kind of efficient pesticides that nineteen sixty-five is promoted the use of by Dow Chemical Company, worldwide be applied to the control of insect of grain, vegetables, fruit and industrial crops, be to substitute one of high malicious organic phosphorous insecticide principal item at present, also be the important kind of safe agricultural product residues of pesticides monitoring, the residue problem that causes on crops such as grain is existing to be reported.And, find in the environmental sample that the situation of chlopyrifos residue also is on the rise, people's health has been constituted potential threat.Chlopyrifos is 1mg/kg at the limit standard of China's residual vegetable amount, and Japan will be decided to be 0.01mg/kg from the chlopyrifos residue amount the bigger spinach of China's import volume when formulating the residual limit standard of chlopyrifos, and the standard in Europe also reaches 0.05mg/kg.These have all produced adverse influence to China's export food, agricultural product.Along with the growing interest of people, be badly in need of more science, detection means fast and efficiently to the toxicity and the environmental risk of chlopyrifos residue.Therefore develop a kind of simply, fast, the trace analysis method that is suitable for the residues of pesticides on-site supervision has important practical significance.
At present both at home and abroad the conventional sense method to residues of pesticides mainly is the physico-chemical analysis method, and utilize instrument such as gas chromatograph, liquid chromatograph, gas chromatograph-mass spectrometer, the liquid chromatograph/mass spectrometer etc. promptly said usually carry out the analyzing detecting method of persticide residue.The advantage of these methods be can be qualitative, quantitative the detection persticide residue, also be the state specified standards detection method.But these methods need be carried out complicated preprocessing process such as extract and separate to sample, need lot of manpower and material resources, and detection time, length and instrument cost an arm and a leg, need the professional and technical personnel to safeguard, detection cost height is unsuitable for on-the-spot the detection, can not satisfy miscellaneous agricultural product and detect demand.Therefore, press for development and application high-level efficiency express-analysis new technology.
Chemiluminescence immunoassay technology (CLIA) is a kind of analytical technology of highly sensitive, high specificity, is widely used in bioengineering, and during clinical medicine detects, but it is fewer both at home and abroad to be applied to the research of agricultural and veterinary chemicals.
Summary of the invention
The objective of the invention is to overcome the shortcoming and defect that prior art exists, a kind of homogeneous chemistry luminescence immunoassay method that detects the organophosphorus pesticide chlopyrifos is provided.
The object of the present invention is achieved like this:
The present invention has synthesized the chemiluminescent labels (Lu-BSA-AR) of chlopyrifos, and sample detected, the chemiluminescence intensity of chlopyrifos label and determined antigen concentration are directly proportional, and can draw the linear relationship between chemiluminescence intensity and the chlopyrifos concentration to be measured.
Big molecule can a plurality of micromolecule labels of mark, can amplify response signal like this.At first the molecular structure of chlopyrifos has been made small change, in the chlopyrifos structure, introduce the reactive group carboxyl (COOH), be carrier with bovine serum albumin(BSA) (BSA) then, chemiluminescence agent luminol is passed through diazo salt method mark to bovine serum albumin(BSA) (BSA), synthetic luminol-bovine serum albumin(BSA) compound, chlopyrifos after will activating again is coupled on luminol-bovine serum albumin(BSA) compound, thereby synthetic chlopyrifos chemiluminescent labels, with the chlopyrifos behind the mark and unlabelled chlopyrifos and quantitative antibody competition association reaction, can carry out quantitative measurement to unknown antigen according to the typical curve of setting up.
Particularly, the present invention includes the following step:
1. with organophosphorus pesticide chlopyrifos activation, introduce reactive group carboxyl (COOH), the chlopyrifos (AR) after obtaining activating;
2. with chemiluminescence agent luminol (Lu) mark to bovine serum albumin(BSA) (BSA), obtain luminol-bovine serum albumin(BSA) (Lu-BSA), coupling ratio requires to reach 1: 20～1: 40;
3. chlopyrifos (AR) mark after will activating obtains chlopyrifos chemiluminescent labels (Lu-BSA-AR) to luminol-bovine serum albumin(BSA) (Lu-BSA);
4. draw chlopyrifos chemiluminescent labels luminescence kinetics curve, be horizontal ordinate with time, be the ordinate mapping with the relative luminous intensity;
5. set up homogeneous phase immunity direct competition method, determine the linear relationship between luminous intensity and the testing sample antigen concentration.
The present invention has following advantage and good effect:
1, simple to operate, quick, cost is low;
2, having proposed first with the bovine serum albumin(BSA) is carrier, and big molecule can a plurality of micromolecule labels of mark, can amplify response signal like this, have increased the sensitivity of system;
3, be applicable to on-the-spot fast qualitative of chlopyrifos chemiluminescence immunoassay and quantitative analyzing and testing.
Description of drawings
Fig. 1 is the activation route map of chlopyrifos;
Fig. 2 is the chlopyrifos infrared scan spectrogram after the activation;
Fig. 3 is luminol-bovine serum albumin(BSA) (Lu-BSA) synthetic route chart;
Fig. 4 is chlopyrifos chemiluminescent labels (Lu-BSA-AR) synthetic route chart;
Fig. 5 is chlopyrifos chemiluminescent labels (Lu-BSA-AR) ultraviolet scanning spectrum figure;
Fig. 6 is chlopyrifos chemiluminescent labels (Lu-BSA-AR) fluorescence emission spectrogram;
Fig. 7 is chlopyrifos chemiluminescent labels (Lu-BSA-AR) luminescence kinetics curve map;
Fig. 8 is chlopyrifos chemiluminescent labels (Lu-BSA-AR) luminescence feature figure;
Fig. 9 is the working curve diagram of test sample chlopyrifos.
Embodiment
Describe in detail below in conjunction with drawings and Examples:
One, relevant step
1,1. about step
Rate of charge: chlopyrifos: 3-mercaptopropionic acid: NaOH=1: 1: 2;
Temperature of reaction: 65 ℃;
The magnetic agitation reaction time: 2h.
2,2. about step:
Temperature of reaction: 0～4 ℃.
3,3. about step
Selecting best coupling method is carbodlimide method.
4,5. about step
Fixing chlopyrifos chemiluminescent labels dilution ratio 1/50, anti-chlopyrifos antibody dilution multiple is 1500 times, temperature of reaction adds the H of 100 μ l 0.5mol/L at 37 ℃ of immune 1h down in the reacted mixed solution
2O
2
Two, experimental example
1, the activation of experimental example 1 chlopyrifos
As Fig. 1, be initiation material with former medicine of chlopyrifos and 3-mercaptopropionic acid, pass through nucleophilic substitution, synthesized O, O-diethyl-O-[3,5-two chloro-6-(2-carboxyethyl) sulfo--2-pyridine radicals] phosphorothionate (AR), thus introduce the reactive group carboxyl (COOH).
The 3-mercaptopropionic acid be dissolved in add three mouthfuls of flat bottom flasks behind the absolute ethyl alcohol, add NaOH then, add again be dissolved in absolute ethyl alcohol the former medicine of chlopyrifos, 60 ℃ of following back flow reaction 2h, filter reaction mixture, concentrating under reduced pressure evaporate to dryness.Use NaHCO then
3Solution is transferred to residue in the separating funnel, and normal hexane extraction 2 times discards organic phase.Use then about hydrochloric acid water transfer phase pH to 3, dichloromethane extraction 3 times, organic phase is crossed anhydrous MgSO
4, concentrating under reduced pressure is closely dried, gets faint yellow oily thing, and column chromatography is crossed silicagel column, collects the moving phase of eluent (normal hexane/acetate second fine wine, 1: 2), the concentrating under reduced pressure evaporate to dryness.Add a small amount of anhydrous alcohol solution product, obtain pale yellow crystals behind the recrystallization.
Assay: it is 121～123 ℃ that this crystal records m.p. with the fusing point instrument, and this reaction yield is 49.8% as calculated, by the successful as can be known introducing of its infrared scan spectrum carboxyl (COOH) group is as Fig. 2.
2, experimental example 2 luminols-bovine serum albumin(BSA) (Lu-BSA) is synthetic
As Fig. 3, with the 50mg luminol, add among the HCl of 5.0mL3.0mol/L, stir on magnetic stirring apparatus at 0～4 ℃, until dissolving.In 0～4 ℃ of acid luminol solution, add the NaNO of 50mg
2Solid, lucifuge is reacted 15min, and reaction makes mixed liquor that Congored test paper is blue when finishing, potassium iodide starch test paper is shown blue, is 8.5 borate buffer solution adjusting mixed liquor then with 0.50ml/L pH, and making its pH is about 8.5.The 0.10moI/L PBS damping fluid that adds a certain amount of BSA of containing then, lucifuge reaction 24h under 0～4 ℃ of condition.In the lucifuge place, reaction mixture to be dialysed by static gradient dialysis with borax buffer solution, the cut molecular weight of dialysis membrane is (8000～12000).Get dislysate every 12h, by fluorescence spectrum scanning, the Weak-luminescence measuring instrument detects the content of the luminol (Lu) of dislysate, dialyses and can not detect luminol to dislysate, can obtain the complex solution of luminol mark bovine serum albumin(BSA) (Lu-BSA).
Assay: the bovine serum albumin(BSA) of luminol mark has good luminescence kinetics curve, and from ultraviolet spectrogram mark success as can be known, calculating its coupling ratio is 1: 25.
3, experimental example 3 chlopyrifos chemiluminescent labels (Lu-BSA-AR) are synthetic
As Fig. 4, the chlopyrifos (AR) that takes by weighing after the activation is dissolved in N, in the dinethylformamide (DMF), add N-hydroxy-succinamide, on magnetic stirring apparatus, stir 1h, add 1-ethyl-(3-dimethyl aminopropyl) carbodiimide hydrochloride then, continue stirring reaction 2h.Get a certain amount of synthetic Lu-BSA solution and add above-mentioned mixed liquor, in the normal temperature lower magnetic force stirring reaction 5h of lucifuge place.After reaction finishes, reaction mixture, dialyse with phosphate buffer, get dislysate every 12h, detect the content of the chlopyrifos (AR) after the activation in the dislysate with ultraviolet spectrophotometry, dialysis only can not detect AR in the dislysate, can obtain luminol-BSA-chlopyrifos compound (Lu-BSA-AR) solution.The chlopyrifos chemiluminescent labels is made into certain density solution with the phosphoric acid phthalate buffer, carries out the scanning of ultraviolet spectrum and fluorescence spectrum.
4, experimental example 4 chlopyrifos chemiluminescent labels (Lu-BSA-AR) UV scanning
As Fig. 5, respectively to chlopyrifos (AR), bovine serum albumin(BSA) (BSA) after chlopyrifos chemiluminescent labels (Lu-BSA-AR), the activation, luminol (Lu) carries out sweep measuring to the employing ultraviolet spectrophotometer under 200-400nm.The result shows, the chlopyrifos chemiluminescent labels maximum absorption band occurs at 264nm, 282nm, 354nm place respectively, occurring maximum absorption band at 260nm, 280nm with the 348nm place respectively with improved chlopyrifos, BSA, luminol compares, obvious variation has taken place, and shows and has successfully synthesized chlopyrifos chemiluminescent labels (Lu-BSA-AR).
5, experimental example 5 chlopyrifos chemiluminescent labels (Lu-BSA-AR) fluorescent scanning spectrum
As Fig. 6, under selected experiment condition, respectively luminol (Lu), luminol mark bovine serum albumin(BSA) (Lu-BSA), chlopyrifos chemiluminescent labels (Lu-BSA-AR) being carried out the fluorescence spectrum emission scan measures, the result shows that the peak value of luminol (Lu), luminol mark bovine serum albumin(BSA) (Lu-BSA), chlopyrifos chemiluminescent labels (Lu-BSA-AR) fluorescence emission spectrum appears at 430nm, 435nm, 436nm place respectively.Obvious variation has taken place in Lu-BSA, Lu-BSA-AR compare fluorescence emission spectrum with Lu peak value, and Lu-BSA compares with Lu-BSA-AR, the peak change of fluorescence emission spectrum is little, and its luminous intensity is not affected, and shows that synthetic chlopyrifos chemiluminescent labels (Lu-BSA-AR) has good fluorescent characteristics.
6, experimental example 6 chlopyrifos chemiluminescent labels (Lu-BSA-AR) luminescence features
As Fig. 8, get a certain amount of Lu, Lu-BSA, Lu-BSA-AR and blank solution respectively under ultra-violet analysis, observe its feature luminescence phenomenon, the result shows to have good luminescence feature.
7, the drafting of embodiment 7 chlopyrifos chemiluminescent labels (Lu-BSA-AR) luminescence kinetics curves
As Fig. 7, get a certain amount of Lu, Lu-BSA, Lu-BSA-AR solution respectively, the carbonate buffer solution constant volume of using pH 9.6 is got 100 μ L solution then respectively and is joined in the measuring cup to 10ml, and original position is injected the H of 0.2mol/L
2O
2Solution detects on BPCL Weak-luminescence measuring instrument, the record chemiluminescence intensity, make Lu, Lu-BSA, Lu-BSA-AR luminescence kinetics curve.The result shows that Lu-BSA-AR compares luminous intensity with Lu-BSA with Lu and descends to some extent, and that Lu-BSA-AR and Lu-BSA get luminous intensity is close, though the luminous intensity of Lu-BSA-AR decreases than Lu, Lu-BSA-AR has still shown good chemiluminescence dynamics.
8, the preparation of experimental example 8 working curves
As Fig. 9, fixedly the amount of chlopyrifos chemiluminescent labels and anti-chlopyrifos antibody adds chlopyrifos haptens to be measured.Immune competitive reaction takes place in chlopyrifos chemiluminescent labels and chlopyrifos and a certain amount of anti-chlopyrifos antibody to be measured, at 37 ℃ of following immune response 1h, the mixed solution after the immune response is carried out chemical luminescent detecting.When fixing chlopyrifos chemiluminescent labels dilution ratio is 1/50, when anti-chlopyrifos antibody dilution multiple is 1500 times, add a certain amount of chlopyrifos solution to be measured, at 37 ℃ of following immune response 1h.The H that adds 100 μ l 0.5mol/L in the reacted mixed solution
2O
2, carry out the chemiluminescence hand hay cutter and decide.Added value according to chemiluminescence signal is carried out quantitative measurement to chlopyrifos.Concentration of chlopyrifos and chemiluminescence signal have good linear relationship in 18.9ng/mL～108ng/mL scope.Method detects and is limited to 1.27ng/mL.Chlopyrifos to 30ng/mLmL carries out 7 replicate determinations, and relative standard deviation is 5.1%.
Claims (5)
1. a homogeneous chemistry luminescence immunoassay method that detects the organophosphorus pesticide chlopyrifos is characterized in that comprising the following steps:
1. with the activation of organophosphorus pesticide chlopyrifos, introduce the reactive group carboxyl, the chlopyrifos after obtaining activating;
2. with chemiluminescence agent luminol mark to bovine serum albumin(BSA), obtain luminol-bovine serum albumin(BSA), coupling ratio requires to reach 1: 15～1: 40;
3. the chlopyrifos mark after will activating obtains the chlopyrifos chemiluminescent labels to luminol-bovine serum albumin(BSA);
4. drawing chlopyrifos chemiluminescent labels luminescence kinetics curve, is horizontal ordinate with time, and relative luminous intensity is the ordinate mapping;
5. set up homogeneous chemistry electrochemiluminescent immunoassay direct competition method, determine the linear relationship between luminous intensity and the testing sample antigen concentration.
2. by the described analytical approach of claim 1, its feature is characterized in that step is 1.:
Rate of charge: chlopyrifos: 3-mercaptopropionic acid: NaOH=1: 1: 2;
Temperature of reaction: 65 ℃;
Reaction time: magnetic agitation reaction 2h.
3. by the described analytical approach of claim 1, its feature is characterized in that step is 2.:
Temperature of reaction: 0～4 ℃.
4. by the described analytical approach of claim 1, its feature is characterized in that step is 3.:
Selecting best coupling method is carbodlimide method.
5. by the described analytical approach of claim 1, its feature is characterized in that step is 5.:
Fixing chlopyrifos chemiluminescent labels dilution ratio 1/50, anti-chlopyrifos antibody dilution multiple is 1500 times, temperature of reaction adds the H of 100 μ l 0.5mol/L at 37 ℃ of immune 1h down in the reacted mixed solution
2O
2
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Cited By (6)
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| CN105572391A (en) * | 2014-10-14 | 2016-05-11 | 镇江亿特生物科技发展有限公司 | Chemiluminescence enzyme-linked immunoassay (ELISA) method for detecting chlopyrifos in wheat |
| CN107163081A (en) * | 2017-05-26 | 2017-09-15 | 无锡中德伯尔生物技术有限公司 | A kind of chlopyrifos antigen and preparation method thereof |
| CN108196049A (en) * | 2017-12-28 | 2018-06-22 | 中国农业科学院哈尔滨兽医研究所 | The chemiluminescence antigen immue quantitative detection reagent box and its detection method of a kind of avian leukosis virus |
| CN108700584A (en) * | 2017-01-20 | 2018-10-23 | 深圳市新产业生物医学工程股份有限公司 | Labeled complex and preparation method thereof, kit, application and detecting system |
| CN112904003A (en) * | 2021-04-09 | 2021-06-04 | 华中农业大学 | Substrate concentration control type time-resolved homogeneous phase chemiluminescence biological detection method |
| CN113125421A (en) * | 2021-04-09 | 2021-07-16 | 华中农业大学 | Optical fiber biosensor and application thereof in homogeneous phase chemiluminescence biological detection |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105572391A (en) * | 2014-10-14 | 2016-05-11 | 镇江亿特生物科技发展有限公司 | Chemiluminescence enzyme-linked immunoassay (ELISA) method for detecting chlopyrifos in wheat |
| CN108700584A (en) * | 2017-01-20 | 2018-10-23 | 深圳市新产业生物医学工程股份有限公司 | Labeled complex and preparation method thereof, kit, application and detecting system |
| CN108700584B (en) * | 2017-01-20 | 2021-11-30 | 深圳市新产业生物医学工程股份有限公司 | Marker complex, preparation method thereof, kit, application and detection system |
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| CN108196049A (en) * | 2017-12-28 | 2018-06-22 | 中国农业科学院哈尔滨兽医研究所 | The chemiluminescence antigen immue quantitative detection reagent box and its detection method of a kind of avian leukosis virus |
| CN112904003A (en) * | 2021-04-09 | 2021-06-04 | 华中农业大学 | Substrate concentration control type time-resolved homogeneous phase chemiluminescence biological detection method |
| CN113125421A (en) * | 2021-04-09 | 2021-07-16 | 华中农业大学 | Optical fiber biosensor and application thereof in homogeneous phase chemiluminescence biological detection |
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Application publication date: 20110511 |