CN101451997A - Reagent strip for rapidly detecting colloidal gold for 2,4-D residual - Google Patents
Reagent strip for rapidly detecting colloidal gold for 2,4-D residual Download PDFInfo
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- CN101451997A CN101451997A CNA2008102469600A CN200810246960A CN101451997A CN 101451997 A CN101451997 A CN 101451997A CN A2008102469600 A CNA2008102469600 A CN A2008102469600A CN 200810246960 A CN200810246960 A CN 200810246960A CN 101451997 A CN101451997 A CN 101451997A
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Abstract
The invention relates to a fast detection colloidal gold test strip used for 2, 4-D residual, belonging to the technical field of immunity analysis. The invention overcomes the defects of complex operation, high detection cost, slow speed and secondary pollution and the like in the 2,4-D physical and chemical analysis, and fast and accurately detects 2, 4-D residual in garden spgarden stuff, cereal crops and water samples. During the detection process, 2,4-D in the sample is reacted with specificity of gold marking 2,4-D antibody (quantification), 2, 4-D content in the sample is determined (coloration is feminine, namely not exceeding controlling quantity; otherwise exceeding the controlling quantity) through specificity fixation reaction of peridium 2, 4-D envelope antigen (detection line, T line) on the test strip cellulose acetate; pre-custodite goat anti-rabbit antibody on the test strip cellulose acetate is used as a quality control line (C line, coloration is effective, otherwise ineffective). Shelf life of the test strip is at least six months.
Description
One, technical field
The present invention relates to a kind ofly be used for 2, the colloidal gold strip of 4-D residue detection mainly is applicable in the samples such as fruits and vegetables, water and grain 2, the fast measuring that 4-D is residual.Belong to the immuno analytical method field.
Two, background technology
2,4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic acid, 2,4-D) be a kind of phenoxy carboxylic acid herbicides and plant growth regulator, be widely used in wheat, barley, cereal, lawn and pasture etc. and remove the growth regulator of broad leaved weeds and vegetables, contain 2 in the agricultural chemicals of nearly kind more than 1500, the active component of 4-D.2, the acute toxicity of 4-D mainly shows as nerve toxicity; 2,4-D can also cause nausea, vomit and suffer from diarrhoea in the stimulating gastrointestinal road; Skin contact 2,4-D may cause fash or dermatitis.According to another report 2,4-D may have potential " three cause " effect.Because its being extensive use of in agricultural production has been caused the pollution of fruits and vegetables, grain, underground water and potable water.
Herbicide 2, the residual conventional method of analysis of 4-D mainly are gas chromatography (GC) and high performance liquid chromatography instrumental methods such as (HPLC).Use these physico-chemical analysis technology to trace 2 in the samples such as environment, biology, food, 4-D is residual to be analyzed, not only the instrumentation degree is had relatively high expectations, and need through loaded down with trivial details separation, extraction, the pre-treatment process such as purify, derive, analysis speed is slow, cost is high, and the pre-treatment process need uses a large amount of organic solvents, has caused secondary pollution.Along with sample to be checked, particularly require increasing sharply of field quick detection sample size, traditional pesticide residue analysis means are difficult to adapt to requirement, therefore, press for exploitation high-level efficiency residues of pesticides express-analysis technology.Based on 2 of ELISA adsorption analysis method, the residual quick colloidal gold test of 4-D has high specificity, highly sensitive and easy to be quick, can remedy the defective of chromatogram analysis method.The present invention is to solving 2 of batch samples, and the residual on-site supervision technology of 4-D has important practical significance.
Three, summary of the invention
For solve present residues of pesticides instrument analytical method cost height, complicated operation, poor specificity, sensitivity is low and shortcoming such as testing result instability, the invention provides that a kind of to have a high specific, high sensitivity, pin-point accuracy, pinpoint accuracy, method of operating simple, and can be used for 2 of batch samples fast detecting, the colloid gold test paper that 4-D is residual.
2, the residual quick detection test paper of 4-D comprises in box body, colloidal gold strip inner wrapping, sponge bracket and the support and includes concentrating sample extract, operation instructions.
The technical solution adopted for the present invention to solve the technical problems is:
(1) at first utilizes active ester method with 2, prepare artificial immunogen after 4-D and bovine serum albumin(BSA) (BSA) coupling, prepare 2 with this immunogen immune new zealand white rabbit, the 4-D polyclonal antibody.
(2) with mixed anhydride method with 2,4-D and ovalbumin (OVA) coupling, their compound is fixed in cellulose acetate film (NC) as detection line (T line) as envelope antigen.
(3) be fixed in cellulose acetate film (NC) as control line line (C line) with goat anti-rabbit antibody
(4) the sodium citrate improved method prepares collaurum, and the rabbit for preparing with tense marker resists 2,4-D antibody, and be fixed on the collaurum pad
(5) assemble test strips by Fig. 1
(6) detect with judge will be to be measured 2, the 4-D sample extracting solution drips on sample pad, after room temperature is placed 10min, and observation.As T line colour developing then negative (be in the sample 2,4-D content is less than controlled quentity controlled variable), on the contrary positive, i.e. T line do not develop the color (in the sample 2,4-D content is higher than controlled quentity controlled variable).Shown in the plate 1.
Advantage of the present invention is accurately to detect in water and fruits and vegetables and the grain 2 delicately, and 4-D is residual, and the pre-treatment process of sample is simple, and is consuming time few, a large amount of sample of fast detecting simultaneously, and the sample detection cost is far below traditional instrument detecting method.The storage life of reagent strip at least 6 months.
Four, description of drawings
Five, embodiment
(1) use of test strips: it is standby that sample extracting solution in the box is diluted to 500ml.Get working sample 1.0g, add 10ml extract milling and extracting, get supernatant 100 μ l and drop on the sample pad, room temperature is carried out interpretation after placing 10min.
(2) as negative, then in the sample 2,4-D content＜17ng/mL, otherwise 17ng/mL
Embodiment 12,4-D and carrier protein couplet:
(1) the active ester method coupling 2,4-D and bovine serum albumin(BSA) (BSA)
Take by weighing 250mg 2,4-D (about 270umol) is dissolved in (A liquid) in the 10mL anhydrous dimethyl base acid amides (DMF), take by weighing 500mgBSA and be dissolved among the 10mL 0.01mol/L pH7.4 PBS (B liquid), 250mg EDC is dissolved in an amount of PBS (C liquid), and the A drop is gone in the C liquid to react 1h; Then reactant liquor is dropwise splashed in the B liquid, and at stirring at room reaction 12h, then with reactant liquor dialysis, centrifugal, supernatant freeze drying, 4 ℃ of preservations.
(2) the mixed anhydride method coupling 2,4-D and oralbumin (0VA)
Accurately take by weighing 2,4-D 250mg adds the 10mLDMF dissolving, adds 50.0 μ L tri-n-butylamines and 30 μ L chloro-butyric acid isobutyl esters again in this solution successively, and the room temperature magnetic agitation is spent the night; Again this solution slowly is added drop-wise in the carbonic acid buffer of the 20mL 0.05mol/L pH9.6 that is dissolved with 450mgOVA stirring reaction 24h under stirring; This reactant liquor phosphate buffer to 0.01mol/LpH7.4 under 4 ℃ is dialysed, the centrifugal 10min of dislysate 3500rpmn, and the supernatant freeze drying gets synthetic antigen.
Embodiment 22, the preparation of 4-D polyclonal antibody:
With 2, the conjugate of 4-D and BSA is made immunogene, claim the 2mg immunogene to be dissolved in the 1mL stroke-physiological saline solution, mix with the 1mL complete Freund's adjuvant, new zealand white rabbit nape portion subcutaneous (6 point) is injected in fully emulsified back, later on every two all booster immunizations once, using incomplete Freund's adjuvant instead mixes with immunogene, the immunity position is identical, and from immunity for the third time, each immunity one week of back is detected serum titer from the rabbit ear vein blood sampling.Immunity is 5 times altogether, and whole blood was adopted from rabbit arteria carotis in one week in last immunity back, slightly carried rabbit anti-serum with sad-saturated ammonium sulfate method earlier, was further purified with the DE-52 anion exchange chromatography again, obtain purer 2, the 4-D polyclonal antibody.
The sodium citrate improved method prepares collaurum
(1) with new system deionized water preparation 100mL 0.01% gold chloride, place in the conical flask, electric furnace is heated to and boils.
(2) accurately draw a certain amount of 1.0% trisodium citrate, add rapidly in the conical flask, rock evenly after, put into electric furnace immediately and continue to boil, become red back until gold chloride and take out.Return to original volume with deionized water after being cooled to room temperature, sterile sealing is preserved.The maximum absorption band wavelength of 400～700nm UV scanning is 525nm as shown in Figure 2, and at the 237nm place less absorption peak is arranged, and bimodal is the feature of collaurum.Plate 2, the plate 3 big or small basically identical of observing colloid gold particle under the Electronic Speculum that is shown in do not have ellipse, polygonal gold grain, and particle diameter meets the colloid gold label requirement greatly about 20～30nm.
Embodiment 42, the colloid gold label of 4-D antibody
In the colloidal gold solution of 2mL volume, use 0.1mol/L K
2CO
3, 0.1mol/L HCl regulates pH value to 8.0, under magnetic agitation, antibody-solutions (84 μ g/mL) dropwise added (the about 5min of the protein of 1lmg adds) in the colloidal gold solution, stirring at room 15min.Adopt low temperature supercentrifugation purifying to remove wherein unlabelled antibody and the not collaurum of abundant mark and the various polymkeric substance that in labeling process, may form.Precipitation is dissolved in 1/10PB (the containing 1%BSA) damping fluid of original volume.4 ℃ of preservations are standby.
The assembling of embodiment 5 colloidal gold strips
(1) with an amount of envelope antigen and goat anti-rabbit antibody bag by in the NC film (on the 1cm * 2cm).
(2) an amount of gold mark 2, and 4-D antibody is fixed in glass fibre (on the 1cm * 1cm).
(3) get the plastic plate of making the test strips special use, tear the paper slip of pasting the nitrocellulose filter position earlier, the nitrocellulose filter that cuts respective length sticks on the plastic plate, the glass fibre of getting the good golden labeling antibody of absorption then is affixed on corresponding site, push down nitrocellulose filter, glue absorbent material again and do not have the all-glass paper of ADSORPTION OF GOLD labeling antibody.As shown in Figure 1
With 2, the stock solution of 4-D 2000.0ng/mL is mixed with 1000.0ng/mL, 500.0ng/mL, 100.0ng/mL, 50.0ng/mL, 20.0ng/mL, 10.0ng/mL, 5.0ng/mL and negative standard serial solution with the damping fluid that contains 5% methyl alcohol PBST.And be defined as under the positive concentration further accurately segmentation concentration, and the gold test strip bar is inserted in the above-mentioned standard serial solution behind 20～30s, take out the 10min observations.Result such as table 1
By table 1 and 2 as can be known, the test strips detection level more than 20.0ng/mL 2, during the 4-D titer, the result is positive.To this concentration further experiment, find at detectable concentration to be 2 of 17.0ng/mL, during 4-D, can observe sample test strips T line.Therefore, the detection sensitivity of test strips is defined as 17.0ng/mL.See plate 4, plate 5.
The mensuration (1000～5ng/mL) of table 1 sensitivity
Remarks: "+" "-" represents positive and negative respectively
The mensuration (20～10ng/mL) of table 2 sensitivity
Remarks: "+" "-" represents positive and negative respectively
Get methoxone (DMPC), (2-(4-Hydroxyphenoxy) propionic acid, Propionic) being mixed with concentration with damping fluid is that 200ng/mL and 100ng/mL test by test paper detecting method for 4-chloro-2-methylenedioxy phenoxy propionic acid (Mecoprop), 2-(4-hydroxyphenoxy) propionic acid.The results are shown in Table 3.
Table 3 test strips cross reaction test
*10 * dilution, "+" positive, "-" feminine gender
As seen from table, when interpolation concentration is 200.0ng/mL and 100.0ng/mL, 10 * dilution back test strips is to methoxone (DMPC), 4-chloro-2-methylenedioxy phenoxy propionic acid (Mecoprop), 2-(4-hydroxyphenoxy) propionic acid (2-(4-Hydroxyphenoxy) propionic acid, Propionic) 3 kinds of similar quality testing survey results are negative, when concentration is 200.0ng/mL, 10 * dilution back test strips is to 2, the 4-D testing result is positive, the test strips that shows this experiment development is to 2,4-D has higher specificity, only is applicable in the sample 2, and 4-D detects.
Get the test strips of different batches at random, to the negative sample of 10 * dilution, 2 of 20.0ng/mL, 4-D standard solution, 200.0ng/mL add concentration water sample 10 * dilution and carry out revision test.The results are shown in Table 4.
Table 4 test strips replica test
Annotate "+',, "-" represent that respectively the test strips testing result is positive and negative
The test strips of from 3 batches test strips, randomly drawing to 10 * dilute negative water sample, 20.0ng/mL 2,4-D titer, 200.0ng/mL add concentration water sample 10 * dilution testing result unanimity, illustrates that test strips repeatability is better between different batches.
Embodiment 9 test strips stability tests
(1) test strips of making is placed in 4 ℃ the refrigerator, respectively 1,2,3,4,5, get test strips after June and carry out that concentration is 0.0,10.0,20.0ng/mL 2, the test of 4-D standard specimen solution, stability test result (the having or not of detection line and nature controlling line, the sharpness of band and the degree that glass fibre discharges golden labelled antibody) is observed in each 2 repetitions.
(2) 37 ℃ of accelerated stability tests, 7d.The test strips of making placed in 37 ℃ the drying box, get test strips every day and carry out 2,4-D standard specimen solution (0.0,10.0,20.0ng/mL) test, stability test result (the having or not of detection line and nature controlling line, the sharpness of band and the degree that glass fibre discharges golden labelled antibody) is observed in 3 repetitions of every processing.
Airtight be stored in 4 ℃ of test strips under the condition 6 times 12 groups detect in the test, detection line is clear when detecting negative sample, background white goes up golden labeling antibody and discharges complete in conjunction with discharging pad; When detecting the 20.0ng/mL standard items, control stripes bar detection line is clear, and test sample test strips detection line complete obiteration (feminine gender) meets requirement of experiment.Test strips is saved in 6d under 37 ℃ of conditions, the detection line color begins to shoal when detecting negative sample, and oneself is very shallow, smudgy for 7d control stripes bar detection line color.The preservation condition of determining test strips at last is to place aluminium foil bag to vacuumize, add the airtight preservation of drying agent room temperature, and the term of validity is 6 months at least.
Claims (7)
1. one kind is used for 2, and the residual fast detecting colloidal gold strip of 4-D is characterized in that by the concentrating sample extract that comprises in box body, test strips, sponge bracket and the support.
2. a kind ofly be used for 2 according to claim 1 is described, the residual fast detecting colloidal gold strip of 4-D has in the box vacuum-packedly to be used for 2, the colloidal gold strip that 4-D detects.Envelope antigen and the antibody and the golden labeling antibody of absorption same amount on each test strips.
3. envelope antigen according to claim 2 and antibody, it is characterized by on the cellulose acetate film (NC) has bag by 2 of same amount, the coupled complex and the goat anti-rabbit antibody of 4-D and ovalbumin (OVA).The rabbit of the colloid gold label of absorption same amount resists 2, the 4-D polyclonal antibody on the glass fibre membrane.
4. resist 2 according to the described rabbit of claim 3, the 4-D preparation method of polyclonal antibody is as follows: with 2, the coupled complex of 4-D and bovine serum albumin (BSA) obtains 2, the 4-D polyclonal antibody as the female White Rabbit of immunogen immune New Zealand.
5. resist 2 according to right 3 described colloid gold label rabbits, the 4-D preparation method of polyclonal antibody is as follows: adopt sodium citrate reduction gold chloride to prepare collaurum; Drip rabbit at pH8.0 and resist 2,4-D polyclonal antibody, centrifugal purification make golden labeling antibody.
6. according to the described concentrating sample extract preparation of claim 1 (diluting 20 times of uses): Na
2HPO
42H
2O 288.4mg, NaH
2PO
42H
2O 5.9mg, Tween-20 100 μ L, methyl alcohol 4mL, distilled water 10mL.
According to the described test strips of claim 1 by forming with the lower part: the plastic plate of test strips special use (1cm * 8cm), plastic plate is pasted thieving paper (1cm * 3cm) from top to bottom successively, nitrocellulose filter (1cm * 2cm), collaurum absorption layer (glass fibre, 1cm * 1cm), sample pad (glass fibre, 1cm * 2cm).
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008102469600A CN101451997A (en) | 2008-12-31 | 2008-12-31 | Reagent strip for rapidly detecting colloidal gold for 2,4-D residual |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008102469600A CN101451997A (en) | 2008-12-31 | 2008-12-31 | Reagent strip for rapidly detecting colloidal gold for 2,4-D residual |
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| CN101451997A true CN101451997A (en) | 2009-06-10 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102539732A (en) * | 2010-12-20 | 2012-07-04 | 科顿集水共同体Crc有限公司 | Transverse flowing device |
| CN102707047A (en) * | 2012-06-05 | 2012-10-03 | 南通市产品质量监督检验所 | Colloidal gold test card for quickly testing ergot alkaloid and preparation method thereof |
| CN103048455A (en) * | 2012-09-27 | 2013-04-17 | 江苏维赛科技生物发展有限公司 | Herbicide 2, 4-D and pesticide CHI bigeminy detection card and preparation method thereof |
| CN104101707A (en) * | 2013-04-09 | 2014-10-15 | 河南工业大学 | 2, 4-dichlorphenoxyacetic acid residue rapid detection test paper strip |
-
2008
- 2008-12-31 CN CNA2008102469600A patent/CN101451997A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102539732A (en) * | 2010-12-20 | 2012-07-04 | 科顿集水共同体Crc有限公司 | Transverse flowing device |
| CN102539732B (en) * | 2010-12-20 | 2016-08-17 | 快速测试技术有限公司 | Lateral flow device |
| CN102707047A (en) * | 2012-06-05 | 2012-10-03 | 南通市产品质量监督检验所 | Colloidal gold test card for quickly testing ergot alkaloid and preparation method thereof |
| CN103048455A (en) * | 2012-09-27 | 2013-04-17 | 江苏维赛科技生物发展有限公司 | Herbicide 2, 4-D and pesticide CHI bigeminy detection card and preparation method thereof |
| CN103048455B (en) * | 2012-09-27 | 2015-03-04 | 江苏维赛科技生物发展有限公司 | Herbicide 2, 4-D and pesticide CHI bigeminy detection card and preparation method thereof |
| CN104101707A (en) * | 2013-04-09 | 2014-10-15 | 河南工业大学 | 2, 4-dichlorphenoxyacetic acid residue rapid detection test paper strip |
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Application publication date: 20090610 |