CN103792350A - Preparation method of fast detection reagent plate for detecting carbofuran - Google Patents
Preparation method of fast detection reagent plate for detecting carbofuran Download PDFInfo
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- CN103792350A CN103792350A CN201210435691.9A CN201210435691A CN103792350A CN 103792350 A CN103792350 A CN 103792350A CN 201210435691 A CN201210435691 A CN 201210435691A CN 103792350 A CN103792350 A CN 103792350A
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
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Abstract
The invention discloses a preparation method of a fast detection reagent plate for detecting carbofuran. The fast detection reagent plate is mainly used for detecting whether carbofuran residue exists in crops, trees and the like. The product mainly comprises an upper plastic clamp shell, a lower plastic clamp shell and a test strip, wherein the bottom layer of the test strip serves as a support layer; the middle layer serves as an adsorption layer and is fixed on the support layer; the outer layer serves as a protection layer and is fixed on the adsorption layer; a fiber layer, a gold mark carrier protein fiber layer for adsorbing and coupling carbofuran, an NC membrane layer and a water absorption material layer at a handle end are sequentially arranged on the adsorption layer from a test sample end; the NC membrane layer is provided with a detection blot (called as a T line) scratched with a carrier protein solution of carbofuran and a control blot (called as a C line) scratched with a goat anti-rabbit antibody solution; the final test result is developed by the C line and T line to obtain color judgment. The product is low in cost, and the method is fast, simple and convenient and high in accuracy, and is suitable for large-scale fast sample detection of enterprises.
Description
Technical field
The invention belongs to food safety detection field, be specifically related to detect on rice, cotton, corn, Chinese sorghum, beet, sugarcane, tobacco, soybean, peanut and other crops and forest, flowers, whether to contain the residual of Furadan.
Background technology
Furadan is carbamate insecticides and nematicide.Within 1963, formulated by the U.S., within 1967, promote.Sterling is white crystals, and 25 ℃ time, in water, solubleness is 700 ppm, and more stable under neutral and acid condition, unstable in alkaline medium, hydrolysis rate is accelerated with the rising of pH value and temperature.In paddy field, the half life period is 1～2 day, is 30～60 days in soil.Furadan is systemics, has action of contace poison concurrently, can be absorbed by plant roots, stem, leaf, and conduction in vivo.Impose on soil pest control more.Insecticidal spectrum is wide, effective to the various pests on rice, cotton, corn, Chinese sorghum, beet, sugarcane, tobacco, soybean, peanut and other crops and forest, flowers and nematode, but to rice leaf roller weak effect.Lasting period is long, generally can reach 30～40 days.Formulation is mainly granule.Control is 35～60 grams of effective constituents for insect mu on the ground; Prevent and treat subterranean pest-insect and 200～250 g for nematode mu.The multiplex soil in rice field or water surface dispenser method; When control dry crop insect and subterranean pest-insect, nematode, be sown in soil with seed; Crop growth period can row replacement or during ditch spread buries.Granule is sprayed after can not soaking.Furadan oral toxicity is large, and skin contact toxicity is little, and people's intoxication is to it suppresses cholinesterase, and atropine sulfate can be used as toxinicide.Safety interval when use is generally 21 days.Fish and other aquatic animals are had to severe toxicity.
Furadan mainly suppresses cholinesterase activity in body, makes acetylcholine in tissue, accumulate and cause poisoning.Mechanism of action is similar with organic phosphorus pesticide poisoning.Poisoning manifestations has salivation, sheds tears, myosis and spasm.But compared with organophosphorus pesticide, suppress the lasting time of the effect of cholinesterase shorter.Stop after contact, it is very fast that cholinester recovers.Its toxicity per os belongs to hypertoxic class; Belong to medium malicious class through skin.Furadan belongs to high-toxic pesticide, also very high to environmental organism toxicity.In various environmental organisms, the harmfulness maximum of Furadan to birds, as long as bird is looked for food, a Furadan may be fatal.Be subject to the poisoning lethal bird of furans or other insect, after being looked for food by bird of prey, Small Mammals or reptile, can cause secondary poisoning and lethal.Once found in the U.S. that more than 30 played the bird of prey (hawk, falcon, cinereous vulture) round Furadan secondary poisoning accident.Another environmental behaviour feature of Furadan is to grow large (water solubility is 700 mg/L) of (degradation half life is 1～2 month), travelling performance in soil its residual life in soil, the sand soil area large in quantity of precipitation, underground water table is shallow easily causes the pollution to underground water, the U.S. has made to be used as some districts to Furadan and has easily caused the pollution to underground water for this reason, and the U.S. makes to be used as some provincialism restrictions to Furadan for this reason.Furadan is met naked light, high heat is flammable.Decomposes is emitted poisonous nitrogen oxide flue gas.Its burning (decomposition) product has carbon monoxide, carbon dioxide, nitrogen oxide.
Summary of the invention
The present invention is one and conducts a research for the Furadan in grain crops and forest is residual, object is in order to overcome the deficiencies in the prior art, a kind of high specificity is provided, highly sensitive, and simple to operation, being applicable to on-the-spot rapid screening Furadan positive sample, need be only the quick test paper quantitative detecting method of detectable Furadan through simple process to sample.
The immune colloid gold of made of the present invention detects agent plate and is mainly got stuck by upper and lower two blocks of plastics, test strips composition.Test strips comprises sample pad, gold mark pad, NC film, thieving paper.On NC film, draw and have detection line (T line), nature controlling line (C line).On reagent strip, closely pasting successively sample pad, gold mark pad, NC film and thieving paper.Wherein sample pad, gold mark pad, NC film have that 2 mm's is overlapping between adjacent with thieving paper.Its effect has two aspects: the firstth, in order to make there is the sufficient reaction time between sample solution and sample pad, the buffer system in sample pad can in and the potential of hydrogen of sample solution, guarantee the smooth combination of antibody on effective constituent in sample solution and gold mark pad; In order to allow sample solution smoothly by the hole of NC film, to be extended to thieving paper always on the other hand; On gold mark pad, be coated with the bond of Furadan antibody and collaurum, on detection line and nature controlling line, drawn respectively Furadan carrier protein couplet thing and goat-anti rabbit antiantibody.
The prepared quick detection reagent plate application chromatography type antibody mediated immunity of the present invention competition principle, by the antibody response colour developing on antigen and gold mark pad, detects Furadan in grain residual fast.If contain Furadan in sample solution, the antibody first combination of Furadan first and on gold mark pad, until be diffused into detection line.Because the antibody activity site on gold mark pad is because being occupied and cannot be combined by Furadan specific antigen on detection line by the Furadan carrier protein couplet thing in sample solution.In the time that the Furadan content in sample exceedes agent plate detection limit, the detection line colour developing in agent plate is than the shallow not even colour developing of nature controlling line colour developing, and now result is positive.Otherwise when Furadan content in sample is below agent plate detection limit or when noresidue, the detection line colour developing in agent plate is close with control line or partially dark, is judged to be feminine gender.
Immune colloid gold quick detection reagent plate prepared by the present invention, each several part constituent and the function of agent plate are as follows:
1, plastics get stuck: play fixing test strips and referential function district, show well, indicate detection line, nature controlling line;
2, test strips is the toughness material not absorbing water that one side scribbles adhesive sticker, as PVC plate, plays fixing test paper and other accessories supported;
3, sample pad is made up of glass fibre, works to absorb sample solution and buffering sample solution;
4, gold mark pad is made up of glass, is marked with the bond of anti-Furadan antibody and collaurum on it, is the place that effective constituent in sample solution and golden labeling antibody react;
5, NC membrane portions Main Function is by reaction result with macroscopic characterization out;
6, thieving paper is thicker filter paper, and its effect is that the mobile unnecessary solution coming up is absorbed.
Agent plate of the present invention has following beneficial effect:
(1) specificity is good.The anti-Furadan antibody of agent plate of the present invention is 100% to the cross reacting rate of Furadan, there is no cross reaction with the pesticide of other kinds;
(2) easy and simple to handle quick.Most of raw material that agent plate of the present invention is reacted required by immunochromatography has been incorporated in reagent strip, and after a sample, antigen-antibody reaction is carried out fast on immobilon-p, has greatly shortened the sample time, after a sample, in 5-8 min, gets final product reading result.The operation of agent plate of the present invention does not need any professional training, and ordinary person all can operate, and only needs by with the naked eye interpretation after an explanation sample;
(3) do not rely on experimental facilities.Agent plate of the present invention is run after plate directly dripping sample, and the shade that judge detection line and nature controlling line on NC film by naked eyes is to sentence read result recently, and whole testing process, without using any experimental facilities, is suitable for field and execute-in-place, is easy to popularization;
(4) cost is low, profitable.Agent plate production technology of the present invention is simple, can realize single pattern detection, and production cost is low, greatly reduces testing cost.
Accompanying drawing explanation
The structural representation that accompanying drawing 1 is test strip of the present invention: in figure, 1 is that sample liquid absorption portion 2 is that colloid gold label part 3 is that detection reaction part 4 is that detection line 5 is that reference line 6 is the part that absorbs water.
Accompanying drawing 2 detects the testing result schematic diagram of the quick detection reagent plate of Furadan, and A is negative to be shown, B is positive to be shown, the invalid demonstration of C.
embodiment
1, the preparation of colloidal gold solution
The mean size of required colloid gold particle is 30 nm, and its preparation method is: preparation 1 L, measure ultrapure water 900 mL in conical flask, and be heated to boiling.Add again 20 mL HAuCl
4solution, continues heating 15 min after being heated to add again the colors such as 20 mL sodium citrate solutions to change into redness after fierce boiling again, is cooled to after room temperature and is settled to 1 L with ultrapure water, puts into 4 ℃ of Refrigerator stores.
2, the preparation of gold mark pad
Get colloidal gold solution 100 mL that prepared, with 0. l moL/L solution of potassium carbonate tune pH to 8. 0.Add while stirring the anti-Furadan monoclonal antibody of 2 mg, stir 20 min, centrifugal 15 min of 20000 r/min, abandon supernatant, add the PBS buffer solution for cleaning 2 times of l0 mL pH7. 4.Precipitation is dissolved containing the PBS damping fluid (pH 7. 4) of 2 % BSA with l0 mL, and after filtering with 0.22 μ m sterilizing filter, 4 ℃ save backup.
3. the preparation of carbofuran half-antigen, the preparation of Furadan-BSA conjugate, the preparation of Furadan antibody.
(1) preparation of carbofuran half-antigen
The one red haptens of muttering of barking, is characterized in that molecular structure is:
NC is from 1 to 20 random natural number
A preparation method for carbofuran half-antigen, is characterized in that its step is:
1. 1 part of benzofuranol is dissolved in 1 ~ 4 part of toluene, passes into 1 ~ 2 part, phosgene, and hydro-oxidation sodium to solution is alkalescence, and temperature of reaction be-10 ~ 10 ℃, reacts and obtains a potpourri;
2. said mixture is carried out to liquid-liquid and distribute, then obtain the oily mixture of benzofuranol and product through reduced pressure concentration, this oily mixture is separated out a colourless crystallization after cooling, is 2,3-dihydro-2,2-dimethyl-7-benzofuranyl chloro-carbonate;
3. by 1 part 2,3-dihydro-2,2-dimethyl-7-benzofuranyl chloro-carbonate reacts and can obtain 4-[[(2 in Jian dioxane solution with 1 ~ 2 part of aminobutyric acid or aminocaproic acid, 3-dihydro-2,2-dimethyl-7-benzofuranyl oxygen) carboxyl] amino] butyric acid or 6-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyl oxygen) carboxyl] amino] caproic acid.
(2) coupling of Furadan and carrier protein
Adopt carbodiimide (EDC-HCl) method that Furadan and carrier protein couplet are prepared to immunizing antigen and envelope antigen.Take 20 mgBSA, add 1 mL distilled water.Dissolve 20 mg EDC-HCl and 20 mg Furadans with 1 mL distilled water in addition, join in above-mentioned solution.After solution is mixed, be placed in lucifuge at 4 ℃ and react 12 h (can put upside down and mix during this time).With PBS damping fluid 4 d that dialyse, change liquid 1 time at interval of 8 h during this time afterwards.Again by solution centrifugal, collect supernatant, frozen for subsequent use at-20 ℃.
(3) preparation of Furadan antibody
1. with physiological saline by antigen diluent to desired concn, then with the emulsification complete with it of isopyknic not formula Freund's complete adjuvant, with the male new zealand white rabbit of subcutaneous multi-point injection method immunity 2 ~ 3 kg;
2. after 4 weeks with physiological saline by antigen diluent to desired concn, then with the emulsification complete with it of isopyknic not formula Freund's incomplete adjuvant, carry out booster immunization immunity by subcutaneous multi-point injection method;
3. after 2 weeks physiological saline by antigen diluent to desired concn, then with the emulsification complete with it of isopyknic not formula Freund's incomplete adjuvant, carry out booster immunization immunity again by subcutaneous multi-point injection method, and after immunity the 8th day survey antibody titer;
4. after this carried out booster immunization every 2 weeks, till tiring of antibody reaches requirement, rabbit arteria carotis is adopted whole blood, and separation of serum obtains antibody after purifying.
4. the assembling of Furadan immune colloid gold quick detection reagent plate
With drawing film machine, the Furadan carrier protein couplet thing of debita spissitudo and goat-anti rabbit antiantibody are drawn on film, respectively as detection line and nature controlling line, 37 ℃ of oven drying 8 h.Detecting reagent set becomes a PVC test strips, is stained with in order sample pad, gold mark pad, NC film and thieving paper thereon.The test paper plate posting is cut into the wide bar of 4 mm with cutting machine, pack plastics into and make detection agent plate in getting stuck, then put into the aluminium foil bag sealed storage with drying agent.
5. Furadan immune colloid gold quick detection reagent plate detects implementation and operation method
1, sample preparation
(1) determinand forest class is squeezed the juice processing, for subsequent use;
(2) feed, cereal, Fructus Hordei Germinatus sample are pulverized so that 75% sample can pass through 20 mesh sieves, size particles and the instant coffee of sample are suitable.Take the sample that 3.0 g crush, add (the extract preparation: 50 mL methyl alcohol+50 mL water of 9 mL sample extracting solutions, add 4 g sodium chloride and mix and obtain sample extracting solution, can configure as required), fully be uniformly mixed (10 min), gentle aspiration supernatant after standing 20 min, carries out sample detection after mixing in 1:1 ratio with sample dilution.
2, detecting step
Slowly vertically drip 2 samples (approximately 50 μ L) with Dispette and, in well, after application of sample, start timing.After application of sample, be sure not mobile check-out console, 5-8 min reads result.
3, result judgement
Negative (-): T line (detection line, near well one end) colour developing, shows in sample that Furadan concentration is too low or containing Furadan; Positive (+) T line, without colour developing, shows Furadan excessive concentration in sample; Null result: not occurring C line (control line), may be that misoperation or check-out console lost efficacy.Applying new check-out console retests.
Claims (5)
1. this is one and detects the preparation method who whether has the residual quick detection reagent plate of Furadan on rice, cotton, corn, Chinese sorghum, beet, sugarcane, tobacco, soybean, peanut and other crops and forest, flowers, it is characterized in that posting on NC film the collaurum gold mark pad that has been coated with Furadan antibody; Be followed successively by detection line and nature controlling line from sample pad to thieving paper direction, drawn respectively Furadan carrier protein couplet thing and goat-anti rabbit antiantibody above.
2. the method for preparing reagent plate as described in claims Article 1, is characterized in that, by collaurum and Furadan antibody mixed mark by a certain percentage, making it form stable gold grain, is made into gold mark pad.
3. the method for preparing reagent plate as described in claims Article 1, in the present utility model, detect with antigen is the conjugates of Furadan and carrier mass formation, wherein carrier mass comprises protein, protein fragments, synthetic polypeptide, semi-synthetic polypeptide, polysaccharide, as seralbumin, globulin, lipoprotein, polyamino acid, glucosan, exemplary carrier mass comprises bovine serum albumin(BSA), oralbumin, keyhole limpet hemocyanin, thyroglobulin, L-poly-D-lysine etc.; In addition, carrier mass can be also other synthetic or natural polymers with reactive group, as diphtheria toxin, tetanus toxin, yeast, nylon, dextrorotation glucosan etc., equally also can be used for preparing detection antigen; The second kind animal protein refers to that non-antibody source belongs to the albumen of animal, and for example, antibody is mouse, and the second kind animal protein can be the chicken such as oralbumin, albumin rabbit serum, rabbit or other non-mouse animal proteins.
4. the method for preparing reagent plate as described in claims Article 1, it is characterized in that Furadan in sample number relevant with the detection line colour developing depth on check-out console, if detection line colour developing is more shallow or do not develop the color, predicate the positive, on the contrary negative.
5. the method for preparing reagent plate as described in claims Article 1, is characterized in that technological process comprises and prepares Furadan antibody, prepares colloidal gold solution, prepares colloid gold label Furadan antibody and assembling Furadan immune colloid gold quick detection reagent plate.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107462715A (en) * | 2017-07-31 | 2017-12-12 | 深圳市药品检验研究院（深圳市医疗器械检测中心） | A kind of carbofuran and Mobucin duplex inspection Immunofluorescence test paper strip and kit and its application |
| CN113238043A (en) * | 2020-12-14 | 2021-08-10 | 黑龙江大学 | Preparation method and application of furadan test paper based on SERS immunochromatographic technique |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107462715A (en) * | 2017-07-31 | 2017-12-12 | 深圳市药品检验研究院（深圳市医疗器械检测中心） | A kind of carbofuran and Mobucin duplex inspection Immunofluorescence test paper strip and kit and its application |
| CN107462715B (en) * | 2017-07-31 | 2018-10-02 | 深圳市药品检验研究院（深圳市医疗器械检测中心） | A kind of carbofuran and Mobucin duplex inspection Immunofluorescence test paper strip and kit and its application |
| CN113238043A (en) * | 2020-12-14 | 2021-08-10 | 黑龙江大学 | Preparation method and application of furadan test paper based on SERS immunochromatographic technique |
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Application publication date: 20140514 |