CN106568904A - Immunity colloid gold detection card of paraquat and preparation method thereof - Google Patents
Immunity colloid gold detection card of paraquat and preparation method thereof Download PDFInfo
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- CN106568904A CN106568904A CN201510655943.2A CN201510655943A CN106568904A CN 106568904 A CN106568904 A CN 106568904A CN 201510655943 A CN201510655943 A CN 201510655943A CN 106568904 A CN106568904 A CN 106568904A
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Abstract
The invention discloses an immunity colloid gold detection card of paraquat and a preparation method thereof, and relates to the technical field of detection of veterinary drug residues in animal-derived food. The detection card comprises a test strip in the shell of the detection card, and the test strip is composed of a PVC rubber sheet, a sample pad, a colloid gold combined pad, a coated film, and a water absorption pad. The colloid gold membrane is a glass cellulose membrane containing a monoclonal antibody of paraquat. The coated film is a cellulose nitrate film, which is provided with a T line and a C line. A paraquat-protein conjugate is coated in the T line, and a goat-anti-mouse IgG antibody is coated in the C line. The provided detection card is capable of rapidly detecting paraquat and is convenient and fast, and the results are accurate.
Description
Technical field
The present invention relates to immune colloid gold detection card of cereal foods Pesticide Residues detection technique field, more particularly to N,N'-dimethyl-.gamma..gamma.'-dipyridylium and preparation method thereof.
Background technology
N,N'-dimethyl-.gamma..gamma.'-dipyridylium, chemical name are 1-1- dimethyl -4-4- bipyridine cation salt, are a kind of quick steriland herbicides, with action of contace poison and certain systemic action.Can be absorbed by plant green tissues rapidly so as to withered.To non-green tissue without effect.Combined with soil in soil rapidly and be passivated, it is invalid to plant root and perennial subterraneous stem and perennial root.But N,N'-dimethyl-.gamma..gamma.'-dipyridylium is very big to people's toxicity, and without specially good effect antidote, oral ingestion mortality rate has been forbidden or has strictly limited using by more than 20 countries up to more than 90% at present.From 1 day July in 2014, the registration of revocation N,N'-dimethyl-.gamma..gamma.'-dipyridylium water preparation and production permit, stopping were produced for China；But retaining the outlet of Mu Yao manufacturing enterprises water preparations and overseas outlet production being specialized in using registering, allow, on July 1st, 2016 stops water preparation and sells at home and use.Therefore periodically N,N'-dimethyl-.gamma..gamma.'-dipyridylium residual is carried out detecting the necessary means of the monitoring for becoming many countries.
Prior art is mainly the detection methods such as gas chromatography-mass spectrography, high performance liquid chromatography for the detection of N,N'-dimethyl-.gamma..gamma.'-dipyridylium, but the defect of these technologies is obvious：Apparatus expensive, complex operation, it is difficult to promote.So, set up it is a kind of it is simple, effectively, be adapted to basic unit using assay method be extremely necessary.The purpose of the present invention is to develop a kind of enterprise that is more suitable for carry out Site Detection and fast and simple, with low cost qualitative checking method.
The content of the invention
For case above, the purpose of the present invention is exactly to overcome the prior art defect for existing and immune colloid gold detection card for providing a kind of N,N'-dimethyl-.gamma..gamma.'-dipyridylium and preparation method thereof, can effectively solving can quick, easily detect the problem of N,N'-dimethyl-.gamma..gamma.'-dipyridylium.
The N,N'-dimethyl-.gamma..gamma.'-dipyridylium colloidal-gold detecting-card of the present invention, constitute including the gold conjugation pad, the nitrocellulose filter, sample pad, adsorptive pads, PVC offset plates and the mould of plastics that have been coated with N,N'-dimethyl-.gamma..gamma.'-dipyridylium-BSA and sheep anti-mouse igg for being coated with monoclonal antibody colloid gold label thing, sample pad, pad are adhered to successively in one end of PVC offset plates, paste nitrocellulose filter, other end adhesion adsorptive pads in centre.
The preparation method of the N,N'-dimethyl-.gamma..gamma.'-dipyridylium colloidal-gold detecting-card of the present invention, is realized by following steps：
(1) prepared by colloidal gold solution：1 L conical flasks 1 are taken, 495 mL of ultra-pure water is added, is then added 1% gold chloride (HAuCl4·3H2O) 5 mL, is configured to 500 mL, 0.01% aqueous solution of chloraurate, adds 1% trisodium citrate (Na in the case where persistently stirring after heated and boiled3C6H5O7·2H2O) solution 5-7 mL, continue agitating heating, when the color of solution is changed into transparent aubergine completely, stop heating after maintaining 5 min, and moisturizing is cooled to room temperature to original volume, and 2-8 DEG C saves backup；
(2) pretreatment of antibody：Will labelling N,N'-dimethyl-.gamma..gamma.'-dipyridylium antibody in 1000 r/min, under the conditions of 4 DEG C, be centrifuged 20 min, take supernatant, be diluted to 1 mg/mL with 0.01 mol/L PBS；Or 1 mg/ml is diluted to 0.01 mol/L PBS, cross 0.22 m filter membranes；
(3) preparation of colloid gold label thing：40 mL of colloidal gold solution in step (1) being taken, colloidal gold solution pH to 8.5 being adjusted with 0.25 mol/L K2CO3,250 r/min of magnetic stirrer stirrings are added dropwise over 4 mL
Protein solution containing 0.3 mg antibody proteins, reacts 10 min；It is added dropwise over 4 mL
10% BSA, continues 10 min of stirring reaction；Gold labeling antibody solution room temperature low speed（1500 r/min）10 min are centrifuged, the precipitation formed by the gold grain for condensing is discarded；4 DEG C of red supernatant solution, 12000 r/min are centrifuged 20 min, abandon supernatant, collect precipitation, precipitation gold labeling antibody diluent is settled to 1 mL, N,N'-dimethyl-.gamma..gamma.'-dipyridylium monoclonal antibody colloid gold label thing is prepared into；
(4) preparation of colloidal gold film：Step (3) N,N'-dimethyl-.gamma..gamma.'-dipyridylium protein conjugate monoclonal antibody colloid gold label thing is sprayed on carrier glass cellulose membrane with the even concentration of 5 L/cm with film instrument is drawn, room temperature is dried or 37 DEG C of 3 h of drying naturally, makes the colloidal gold film of the monoclonal antibody colloid gold label thing of protein conjugate containing N,N'-dimethyl-.gamma..gamma.'-dipyridylium；
(5) it is coated with film preparation：Sheep anti-mouse igg antibody, N,N'-dimethyl-.gamma..gamma.'-dipyridylium protein conjugate are diluted to into 1 mg/mL, are sprayed on nitrocellulose filter with the concentration of 1 L/cm with film instrument is drawn successively, be prepared into coated film, after 37 DEG C of 2 h of coating, room temperature is dried or 37 DEG C of drying naturally；
(6) sample pad pre-treatment：Sample pad treatment fluid is uniformly coated on glass fibre element film, under conditions of air humidity is less than 60%, room temperature is dried naturally；
(7) assembling of detection card：PVC offset plates paste sample pad, colloidal gold film, coated film and absorbent cotton from top to bottom successively, are assembled into test strips, cut into the strip of one fixed width, then test strips are arranged in the detection card shell of the flat shelly of strip.
In the step (5) coated detection line T be located at the lower section of nature controlling line C；
Sample pad treatment fluid in the step (6) is, by 1 g bovin serum albumin (BSA) and 0.8 g Sodium Chloride (NaCl), to be settled to 100 mL with the 0.01 mol/L PBS containing 0.5%TRITON-100；
The present invention can be effectively used for determining N,N'-dimethyl-.gamma..gamma.'-dipyridylium, and method is simple, convenient, fast, as a result accurately.
Description of the drawings
Fig. 1 is the structure chart of the immune colloid gold detection card of N,N'-dimethyl-.gamma..gamma.'-dipyridylium of the present invention：The detection of 1. well, 2. detection line, 3. nature controlling line 4. 5. test strips of hole 6. detection card shell in figure.
Fig. 2 be N,N'-dimethyl-.gamma..gamma.'-dipyridylium of the present invention immune colloid gold detection card in test strips sectional structure chart, 7. sample pad, 8. 10. nitrocellulose filter of gold conjugation pad 9.PVC offset plates, 11. adsorptive pads in figure.
Specific embodiment
Embodiment 1
Embodiment shown in Fig. 1, Fig. 2：In figure, 9 is PVC offset plates；7 is sample pad；8 is gold conjugation pad, and monoclonal antibody colloid gold label thing has been coated with the gold conjugation pad；10 is coated film, i.e. nitrocellulose filter, and N,N'-dimethyl-.gamma..gamma.'-dipyridylium-BSA and sheep anti-mouse igg have been coated with the nitrocellulose filter；11 is adsorptive pads, is made up of absorbent material such as filter paper.
(sample end) adhesion sample pad 7, pad 8 on one end of PVC offset plates 9, sample pad 7 and pad 8 are side by side configuration.
In the intermediate adhesion nitrocellulose filter 10 of PVC offset plates 9.Sheep anti-mouse igg nature controlling line 3 and N,N'-dimethyl-.gamma..gamma.'-dipyridylium-BSA detection lines 2 are provided with nitrocellulose filter 10.
In the other end adhesion adsorptive pads 11 of PVC offset plates 9.One end of nitrocellulose filter 10 is slightly intersected with pad 8, and the other end is slightly intersected with adsorptive pads 11.The test strips 5 can be incorporated with detecting card shell 6 made by mould of plastics, make detection card, and well 1 and detection hole 4 are provided with the upper lid of detection card shell 6, and, just to well 1, nitrocellulose filter 10 is just to detecting hole 4 for sample pad 7.
Embodiment 2
Prepared by N,N'-dimethyl-.gamma..gamma.'-dipyridylium colloidal-gold detecting-card, implemented by following steps：
It is prepared by colloidal gold solution：1 L conical flasks 1 are taken, 495 mL of ultra-pure water is added, is then added 1% gold chloride (HAuCl4·3H2O) 5 mL, is configured to 500 mL, 0.01% aqueous solution of chloraurate, adds 1% trisodium citrate (Na in the case where persistently stirring after heated and boiled3C6H5O7·2H2O) solution 5-7 mL, continue agitating heating, when the color of solution is changed into transparent aubergine completely, stop heating after maintaining 5 min, and moisturizing is cooled to room temperature to original volume, and 2-8 DEG C saves backup；
The pretreatment of antibody：Will labelling N,N'-dimethyl-.gamma..gamma.'-dipyridylium antibody in 1000 r/min, under the conditions of 4 DEG C, be centrifuged 20 min, take supernatant, be diluted to 1 mg/mL with 0.01 mol/L PBS；Or 1 mg/ml is diluted to 0.01 mol/L PBS, cross 0.22 m filter membranes；
The preparation of colloid gold label thing：40 mL of colloidal gold solution in step (1) being taken, colloidal gold solution pH to 8.5 being adjusted with 0.25 mol/L K2CO3,250 r/min of magnetic stirrer stirrings are added dropwise over 4 mL
Protein solution containing 0.3 mg antibody proteins, reacts 10 min；It is added dropwise over 4 mL
10% BSA, continues 10 min of stirring reaction；Gold labeling antibody solution room temperature low speed（1500 r/min）10 min are centrifuged, the precipitation formed by the gold grain for condensing is discarded；4 DEG C of red supernatant solution, 12000 r/min are centrifuged 20 min, abandon supernatant, collect precipitation, precipitation gold labeling antibody diluent is settled to 1 mL, N,N'-dimethyl-.gamma..gamma.'-dipyridylium monoclonal antibody colloid gold label thing is prepared into；
The preparation of colloidal gold film：Step (3) N,N'-dimethyl-.gamma..gamma.'-dipyridylium protein conjugate monoclonal antibody colloid gold label thing is sprayed on carrier glass cellulose membrane with the even concentration of 5 L/cm with film instrument is drawn, room temperature is dried or 37 DEG C of 3 h of drying naturally, makes the colloidal gold film of the monoclonal antibody colloid gold label thing of protein conjugate containing N,N'-dimethyl-.gamma..gamma.'-dipyridylium；
Coating film preparation：Sheep anti-mouse igg antibody, N,N'-dimethyl-.gamma..gamma.'-dipyridylium protein conjugate are diluted to into 1 mg/mL, are sprayed on nitrocellulose filter with the concentration of 1 L/cm with film instrument is drawn successively, be prepared into coated film, after 37 DEG C of 2 h of coating, room temperature is dried or 37 DEG C of drying naturally；
Sample pad pre-treatment：Sample pad treatment fluid is uniformly coated on glass fibre element film, under conditions of air humidity is less than 60%, room temperature is dried naturally；
The assembling of detection card：PVC offset plates paste sample pad, colloidal gold film, coated film and absorbent cotton from top to bottom successively, are assembled into test strips, cut into one fixed width strip, then test strips are arranged in the detection card shell of the flat shelly of strip.
Claims (2)
1. N,N'-dimethyl-.gamma..gamma.'-dipyridylium colloidal-gold detecting-card, it is characterised in that prepare as steps described below：
(1) prepared by colloidal gold solution：1 L conical flasks 1 are taken, 495 mL of ultra-pure water is added, is then added 1% gold chloride HAuCl4·3H2O) 5 mL, is configured to 500 mL, 0.01% aqueous solution of chloraurate, adds 1% trisodium citrate Na in the case where persistently stirring after heated and boiled3C6H5O7·2H2O solution 5-7 mL, continue agitating heating, when the color of solution is changed into transparent aubergine completely, stop heating after maintaining 5 min, and moisturizing is cooled to room temperature to original volume, and 2-8 DEG C saves backup；
(2) pretreatment of antibody：Will labelling N,N'-dimethyl-.gamma..gamma.'-dipyridylium antibody 1000
R/min, under the conditions of 4 DEG C, is centrifuged 20 min, takes supernatant, be diluted to 1 mg/mL with 0.01 mol/L PBS；Or 1 mg/ml is diluted to 0.01 mol/L PBS, cross 0.22 m filter membranes；
(3) preparation of colloid gold label thing：40 mL of colloidal gold solution in step (1) is taken, 0.25 mol/L is used
K2CO3Colloidal gold solution pH to 8.5 is adjusted, 250 r/min of magnetic stirrer stirrings are added dropwise over protein solutions of 4 mL containing 0.3 mg antibody proteins, react 10 min；It is added dropwise over 4 mL
10% BSA, continues 10 min of stirring reaction；Gold labeling antibody solution room temperature 1500
R/min is centrifuged 10 min, discards the precipitation formed by the gold grain for condensing；4 DEG C of red supernatant solution, 12000 r/min centrifugations 20
Min, abandons supernatant, collects precipitation, precipitation gold labeling antibody diluent is settled to 1 mL, N,N'-dimethyl-.gamma..gamma.'-dipyridylium monoclonal antibody colloid gold label thing is prepared into；
(4) preparation of colloidal gold film：Step (3) N,N'-dimethyl-.gamma..gamma.'-dipyridylium protein conjugate monoclonal antibody colloid gold label thing is sprayed on carrier glass cellulose membrane with the even concentration of 5 L/cm with film instrument is drawn, room temperature is dried or 37 DEG C of 3 h of drying naturally, makes the colloidal gold film of the monoclonal antibody colloid gold label thing of protein conjugate containing N,N'-dimethyl-.gamma..gamma.'-dipyridylium；
(5) it is coated with film preparation：Sheep anti-mouse igg antibody, N,N'-dimethyl-.gamma..gamma.'-dipyridylium protein conjugate are diluted to into 1 mg/mL, are sprayed on nitrocellulose filter with the concentration of 1 L/cm with film instrument is drawn successively, be prepared into coated film, after 37 DEG C of 2 h of coating, room temperature is dried or 37 DEG C of drying naturally；
(6) sample pad pre-treatment：Sample pad treatment fluid is uniformly coated on glass fibre element film, under conditions of air humidity is less than 60%, room temperature is dried naturally；
(7) assembling of detection card：PVC offset plates paste sample pad, colloidal gold film, coated film and absorbent cotton from top to bottom successively, are assembled into test strips, cut into the strip of one fixed width, then test strips are arranged in the detection card shell of the flat shelly of strip.
2. N,N'-dimethyl-.gamma..gamma.'-dipyridylium colloidal-gold detecting-card according to claim 1, it is characterised in that in the step (5) coated detection line T be located at the lower section of nature controlling line C；
Sample pad treatment fluid in the step (6) is, by 1 g bovin serum albumin (BSA) and 0.8 g Sodium Chloride (NaCl), to be settled to 100 mL with the 0.01 mol/L PBS containing 0.5%TRITON-100.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510655943.2A CN106568904A (en) | 2015-10-13 | 2015-10-13 | Immunity colloid gold detection card of paraquat and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510655943.2A CN106568904A (en) | 2015-10-13 | 2015-10-13 | Immunity colloid gold detection card of paraquat and preparation method thereof |
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| CN106568904A true CN106568904A (en) | 2017-04-19 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108251381A (en) * | 2018-04-04 | 2018-07-06 | 江南大学 | A kind of paraquat monoclonal antibody hybridoma cell strain and its application |
| CN110554193A (en) * | 2019-09-30 | 2019-12-10 | 重庆市职业病防治院（重庆市第六人民医院） | Preparation method of colloidal gold immunochromatographic test strip for paraquat in urine |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104655835A (en) * | 2013-11-20 | 2015-05-27 | 南京亿特生物科技有限公司 | Immune colloidal gold detect card of chlorpromazine, and preparation method thereof |
-
2015
- 2015-10-13 CN CN201510655943.2A patent/CN106568904A/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104655835A (en) * | 2013-11-20 | 2015-05-27 | 南京亿特生物科技有限公司 | Immune colloidal gold detect card of chlorpromazine, and preparation method thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108251381A (en) * | 2018-04-04 | 2018-07-06 | 江南大学 | A kind of paraquat monoclonal antibody hybridoma cell strain and its application |
| CN108251381B (en) * | 2018-04-04 | 2020-09-04 | 江南大学 | Paraquat monoclonal antibody hybridoma cell strain and application thereof |
| CN110554193A (en) * | 2019-09-30 | 2019-12-10 | 重庆市职业病防治院（重庆市第六人民医院） | Preparation method of colloidal gold immunochromatographic test strip for paraquat in urine |
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Application publication date: 20170419 |
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