CN105785021A - Quick detecting card for immunochromatography of organophosphorus and carbamate pesticide multiresidue cholinesterase - Google Patents
Quick detecting card for immunochromatography of organophosphorus and carbamate pesticide multiresidue cholinesterase Download PDFInfo
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- CN105785021A CN105785021A CN201610215012.5A CN201610215012A CN105785021A CN 105785021 A CN105785021 A CN 105785021A CN 201610215012 A CN201610215012 A CN 201610215012A CN 105785021 A CN105785021 A CN 105785021A
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention discloses a quick detecting card for immunochromatography of organophosphorus and carbamate pesticide multiresidue cholinesterase.The quick detecting card comprises a bottom board and a covering board.A plurality sets of buffering pads are arranged on the bottom board, a plurality of sample pads are crossed on each buffering pad, and enzyme pads, detection films and water absorbing pads are sequentially arranged and pasted in the mode of being close to the sample pads.A plurality of feeding holes and a plurality of windows are formed in the covering board.The upper surfaces of the detection films are wrapped by organophosphorus and carbamate pesticide protein conjugate and anti-bio-enzyme antibodies IgG in parallel.The quick detecting card is rapid and easy to operate; various kinds of organophosphorus and carbamate pesticide in samples can be rapidly detected at the same time, the sensitivity is 0.008 mg/kg-0.987 mg/kg, the result can be observed in 3 minutes to 5 minutes, and the quick detecting card is suitable for quickly detecting organophosphorus and carbamate pesticides in vegetables, fruits and environment water samples.
Description
Technical field
The invention belongs to Pesticides Testing technical field.More particularly, to a kind of organophosphorus and N-methylcarbamate pesticides many residuals acetylcholine esterase immunochromatography quick measuring card.
Background technology
Pesticide is for preventing and treating the Important Agricultural means of production of disease pest harm in agricultural production, is widely used in the production of fruit, vegetable and grain.Pesticide has played highly important effect in ensureing the good harvest of agricultural byproducts.But, indiscriminate use of pesticide in recent years brings huge threat also to environment and the healthy of the mankind.Increasing country pays much attention to indiscriminate use of pesticide gradually.Owing to pesticide variety is various and composite use phenomenon is general, set up the multi-residue determination method that can simultaneously detect multiple object very necessary.Instrument analytical method can realize multi-residue determination, however it is necessary that the instrument and equipment of costliness and professional operator, and sample pre-treatments loaded down with trivial details time-consuming, testing cost is high, it is difficult to meet high flux, quickly, the needs of on-line checking.
At present, develop quick multi-residue determination method and become the important means of the deficiency making up instrumental method.From 80 years 20th century, suppress cholinesterase activity principle to set up the rapid screening of " inhibiting AChE " the Organophosphorus and carbamate pesticides class pesticide that starts to be widely used in agricultural product based on Organophosphorus and carbamate pesticides class pesticide, rapidly be extremely be widely applied due to its simple and rapid characteristic.Progressively withdrawing from the market yet with its sensitivity and less stable, only the minority developing country such as Korea S is still using at present.Immunoassay based on antigen and antibody specific association reaction also is able to the detection for part pesticide, but due to antibody preparation difficulty, somewhat expensive, and instability, can only a certain or a group pesticide can be detected at present.The method for quick that development has more broad spectrum activity and stability is extremely urgent.
Summary of the invention
The technical problem to be solved in the present invention is the defect and the deficiency that overcome prior art, a kind of pesticide multi-residues biological enzyme quick measuring card is provided, the organophosphorus and N-methylcarbamate pesticides simultaneously, simply and quickly detecting in food and environment can be realized, the quick detection of Multiple Pesticides residual in water fruits and vegetables and environmental water sample can be applied.
It is an object of the invention to provide a kind of organophosphorus and N-methylcarbamate pesticides many residuals acetylcholine esterase immunochromatography quick measuring card.
Another object of the present invention is to provide the application in detection organophosphorus and N-methylcarbamate pesticides many residuals of the described quick measuring card.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of organophosphorus and N-methylcarbamate pesticides many residuals acetylcholine esterase immunochromatography quick measuring card, described quick measuring card includes base plate 1 and capping plate 9；Described base plate 1 includes some groups of cushion pads 2, each cushion pad 2 is pasted onto on base plate 1 across cushion pad 2 one side described in several sample pad 3(, cushion pad 2 another side pastes sample pad 3), and then sample pad 3 is also arranged in sequence with enzyme pad 4, detection membrane 5 and adsorptive pads 8；Described sample pad 3, enzyme pad 4, detection membrane 5 and adsorptive pads 8 are pasted onto on base plate 1 successively；Described capping plate 9 includes several well 10 and several visual windows 11；Wherein, described detection membrane 5 upper surface is coated organophosphor and carbamate chemicals for agriculture protein conjugate (detection line 6) and antibiont enzyme antibody IgG(nature controlling line 7 side by side).
Further, described detection membrane 5 is coated organophosphor with carbamate chemicals for agriculture protein conjugate as detection line 6, it is coated 1 antibiont enzyme antibody IgG line as nature controlling line 7, organophosphor both can be combined with acetylcholine esterase with carbamate chemicals for agriculture protein conjugate, it is also possible to is identified by antibiont enzyme antibody IgG.
Wherein it is preferred to, described organophosphor is as follows with the structure of carbamate chemicals for agriculture protein conjugate:
Wherein it is preferred to, described coupling protein is bovine serum albumin (BSA) or ovalbumin (OVA).
Preferably, the enzyme in described antibiont enzyme antibody IgG is derive from silkworm and by the restructuring acetylcholinesterase of genetic modification.
Concrete it is highly preferred that the preparation method of described enzyme is as follows:
The present invention aminoacid sequence according to known silkworm acetylcholinesterase, is suitably changed, and has maximally utilised the codon of escherichia coli preference, has designed and synthesized new acetylcholinesterasegene genebmaceFragment.Specifically, the new acetylcholinesterasegene gene of designbmaceThe sequence of fragment is as follows:
ATGATCAACTACTGATCCTAGGCTGATCCTAATAGATTCTGATCCTATTGATCCTAAGGTACCCTATACCTATACTAGGTGATCCTATGATCCTATGATCCTAGGTGATCCTATGATCCTATGATCCTATCCTATACTAGGTGATCCTATGATCCTATGATCACTAGGTGATCCTATGATCCTATGATCCCCTATACTAGCCTATACTAGGTGATCCTATGATCCTATGATCGTGATCCTATGATCCTATGATCGATCTTGATCCCTATACTAGGTGATCCTATGATCCTATGATCCTAAGGATGATCCTACTTACGTAGCTATTGATCCTAGGATGATCCTATATGACCTATACTAGGTGATCCTATGATCCTATGATCTTGATCCTATGATGATCCTATCCTACCTAGGCCGTATGACGTAGCTAGTACGTGATCCTAACCTATACTAGGTGATCCTATGATCCTATGATCTCGATCTGATCCCTACCTATACTAGGTGATCCTATGATCCTATGATCTCCTATACTAGGTGATCCTATGACCTATACTAGGTGATCCTATGATCCTATGATCTCCTATGATCACCTATACTAGGTGATCCTATGACCTATACTAGGTGATCCTATGATCCTATGATCTCCTATGATCCTAGGTGATCCTATGATCCTATGATCTGATCCTACTAGATTGATCCTACGACCTATACTAGGTGATCCTATGATCCTATGATCCCTATACTAGGTGATCCTATGATCCTATGATCCCTATACTAGGTGATCCTATGATCCTATGATCCCTATACTAGGTGATCCTATGATCCTATGATCTCCCTATACTAGGTGATCCTATGATCCTATGATCGTAGTAGCTGATCCTACTAGCTAGCTAGCTTCCCTTGATCCTATAGCTGATTGATCCTACTATAGTGATCCTACCTATACTAGGTGATCCTATGATCCTATGATCCCTATACTAGGTGATCCTATGATCCTATGATCGATCTGATCCTGATCCTATGATCCTATACTAGGTGATCCTATGATCCTATGATCCTATGATCCTATATTGCTTTG。
Then by the new acetylcholinesterasegene gene of this designbmaceRestructuring is to fusion expression vector (preferably, fusion expression vector used can be pET28 (a), pUC118 or pEZZ318 etc.) in, and it is transformed in escherichia coli, obtained by colibacillary cultivation and there is bioactive expression product identical with natural acetylcholinesterase restructuring acetylcholinesterase.
More specifically, the preparation process of above-mentioned restructuring acetylcholinesterase is as follows:
(1) sequential design: as it has been described above, design new acetylcholinesterasegene genebmaceFragment；
(2) recombinant expression carrier is built: with Nde I, BamH I by new acetylcholinesterasegene genebmaceFragment cuts from recombiant plasmid pUC118-CM, is inserted in carrier pET28 (a) with Nde I, BamH I digestion, converts e. coli bl21, recombiant plasmid called after pET28 (a)-CM obtained；
(3) convert escherichia coli: by the effect of Nde I, BamH I digested vector pET28 (a), recombiant plasmid pET28 (a)-CM is converted e. coli bl21；
(4) cultivate escherichia coli and obtain restructuring acetylcholinesterase: after recombiant plasmid pET28 (a)-CM converts e. coli bl21, acetylcholinesterase is under T7 promoter controls, with a molecular weight only 2000 little peptide amalgamation and expression, final obtain restructuring acetylcholinesterase.
Furthermore it is preferred that described antibiont enzyme antibody IgG is rabbit polyclonal antibody, its preparation method is by immunity new zealand white rabbit after enzyme emulsifying, through the rabbit anti-serum that screening obtains.
Furthermore it is preferred that described base plate 1 is one side scribbles adhesive sticker and the plastic plate not absorbed water, plays the fixing effect supporting other ingredients of quick measuring card and be merged into quick measuring card with capping plate 9.
Preferably, the preparation method of described cushion pad 2 and sample pad 3 is: all-glass paper or filter paper are immersed in the Tris-HCl of pH6.4～7.2 or 0.1mol/L, pH6.4～PBS of 7.2 in, process the preferred 5min of 3～10min(), take out dried and get final product.
Preferably, described dry as drying in 50～70 DEG C (preferably 60 DEG C), vacuum drying or other modes dry.
Described cushion pad 2 and sample pad 3 act, when detection, the effect absorbing sample solution, and sample solution is on average assigned in two sample pad 3 by cushion pad 2, and formation moves up.
Preferably, the preparation method of described enzyme pad 4 is: adopt filter paper as material, filter paper is immersed in the solution of acetylcholine esterase and stabilizer, the preferred 40min of immersion 25～50min(is stirred) under 15～25 DEG C (preferably 20 DEG C), then dry, in vacuum desiccator, be dried overnight the preferred 12h of 10～15h().
Preferably, described stabilizer is mass fraction is the sucrose solution of 60%.
Preferably, described acetylcholine esterase zymolyte, gold colloidal or quantum dot etc. carry out labelling.
Described enzyme pad 4 plays effect that is fixing and that adhere to acetylcholine esterase.Described detection membrane 5 is pasted onto on enzyme pad.
Additionally on above-mentioned quick measuring card, described well 10 is identical with the quantity of cushion pad 2, and visual window 11 is identical with the quantity of detection membrane 5；After capping plate 9 is combined formation detection card with base plate 1, well 10 faces the middle of cushion pad 2, and visual window 11 faces detection membrane 5 respectively.
Preferably, described adsorptive pads 8 is the all-glass paper of drying at room temperature, filter paper or absorbent paper.The Main Function of adsorptive pads 8 is in that to absorb the mobile sample solution come up.
Preferably, described capping plate 9 is plastic plate.
As one preferably can embodiment, the quick measuring card of the present invention include a base plate 1 and one capping plate 9, described base plate 1 includes a cushion pad 2, across two sample pad 3 on cushion pad 2, enzyme pad 4, detection membrane 5 and adsorptive pads 8 are respectively arranged with two.Namely as shown in Figure 1, so that base plate 1 to include a cushion pad 2, cushion pad 2 illustrates for example across two sample pad 3: described quick measuring card includes base plate 1 and capping plate 9；Described base plate 1 includes a cushion pad 2, cushion pad 2 is pasted onto on base plate 1 across cushion pad 2 one side described in two sample pad 3(, cushion pad 2 another side pastes sample pad 3), and then each sample pad 3 is also arranged in sequence with enzyme pad 4, detection membrane 5 and adsorptive pads 8；Described sample pad 3, enzyme pad 4, detection membrane 5 and adsorptive pads 8 are pasted onto on base plate 1 successively；Described capping plate 9 includes a well 10 and two visual windows 11；Wherein, described detection membrane 5 upper surface is coated organophosphor and carbamate chemicals for agriculture protein conjugate (detection line 6) and antibiont enzyme antibody IgG(nature controlling line 7 side by side).
The assemble method of pesticide quick measuring card of the present invention following (to comprise a cushion pad 2):
Pasting two groups of sample pad 3, enzyme pad 4, detection membrane 5 and adsorptive pads 8 on base plate 1 successively, by cushion pad 2 across in two sample pad 3, then covered by capping plate 9, well 10 is facing to cushion pad 2 middle, and two visual windows 11 are respectively toward to two detection membrane 5.
Quick measuring card application in detection organophosphorus and N-methylcarbamate pesticides many residuals is stated, also within protection scope of the present invention it addition, above-mentioned.
Preferably, described organophosphorus and N-methylcarbamate pesticides is dichlorvos, coumafos, Bassa, parathion, Furadan, phoxim, methidathion, O-2,4-dichlorophenyl O,O-diethyl phosphorothioate, quintiofos, triazophos, triazotion, Phosalone, Menite, omethoate, Di-captan, Ficam, cynock, sevin, parathion-methyl, Malathion, fenifrothion, fenthion, famphur, C-9491, chlorpyrifos-methyl or disulfoton etc..
It addition, the quick measuring card of the present invention is in use, be applicable to arbitrary sample.
Preferably, when detecting sample and being water sample, the judgement of detection method and result is: directly draws environmental water sample and is added drop-wise in well 10, drip several (preferably 4), after standing keeps flat 3～5min, if all there are lines in two detection membrane, it was shown that sample is negative, do not contain organophosphor or carbamate chemicals for agriculture, be otherwise positive.No matter sample is negative or positive, and lines should occur in nature controlling line, if nature controlling line is without lines, it was shown that reagent paper lost efficacy.
When detecting sample and being fruit and vegetable, the judgement of detection method and result is: fruit (bark fetching part) or vegetable are cut into small pieces, weigh a small amount of (preferred 1g) and extract the preferred 10min of 8～15min(by a small amount of (preferred 1mL) 0.05mol/L PBS solution containing 10% methanol), the centrifugal 5min of the centrifugal preferred 5000rpm of 3～10min(of 3000～5500rpm), take supernatant directly to detect, method and the same water sample of judged result.
The Cleaning Principle of quick measuring card of the present invention is:
Adding sample by well, sample arrives sample pad by cushion pad, moves on to enzyme pad on then.If containing organophosphor or carbamate chemicals for agriculture in sample, can be combined in continuation and move on to detection line by acetylcholine esterase on enzyme pad, because the acetylcholine esterase only one of which binding site on enzyme pad, after many residual pesticide in sample are in combination, organophosphorus and N-methylcarbamate pesticides protein conjugate on detection line cannot be combined with acetylcholine esterase, then detects that line (T) is wireless to be manifested；When not having Organophosphorus and carbamate pesticides class pesticide in sample, acetylcholine esterase will be caught by organophosphor or carbamate chemicals for agriculture protein conjugate when arriving detection line (T), forms macroscopic lines.Whether sample contains organophosphor or lines all can occur in aminomethyl propylhomoserin ester pesticides nature controlling line (C).Therefore, if containing only there being organophosphorus pesticide in sample, above detection membrane only occurs a line；If containing only carbamate chemicals for agriculture in sample, below detection membrane only occurs a line, if two class pesticide are all sometimes, above detection membrane and following detection membrane one line all can occur.
Principle, the present invention has surmounted and original utilizes Organophosphorus and carbamate pesticides class pesticide that this single characteristic of cholinesterase activity can be suppressed to detect pesticide roughly, combine immunologic principle on this basis, chromatographic film is coated with corresponding organophosphor and carbamates pesticide protein conjugate and antibiont enzyme antibody IgG, in conjunction with physics chromatography, utilize the structural models of the detection of immune colloid gold card, acetylcholine esterase instead of the antibody of original detection object, use the principle that acetylcholine esterase itself can be combined with antibiont enzyme antibody IgG as a kind of antigen to detect harmful object again, this may be considered the colloidal-gold detecting-card a kind of combination with inhibiting AChE.
From the structure of detection card, the present invention detect card with in prior art indirect competitive immune colloid gold detection card structure similar, but coated in prior art be antibody, the present invention is coated is acetylcholine esterase, in prior art, coated two resist, and the present invention is coated is antibiont enzyme antibody IgG；And it is coated the material used by acetylcholine esterase and is better than the material used by inhibiting AChE to a certain extent, therefore will be better in stability.
The effect of detection, the present invention is more accurate than inhibiting AChE of the prior art, more stable.Existing inhibiting AChE all can only detect the content of pesticide roughly, and present invention incorporates the advantage of immune detection card, has relatively low detection to limit in a lot of pesticide, and very sensitive, has been simultaneously introduced acetylcholine esterase, has made Detection results sensitiveer.
The above-mentioned quick measuring card of the present invention can detect Organophosphorus and carbamate pesticides class pesticide multi-residues simultaneously, this quick measuring card include base plate and capping plate, and at least two groups paste successively cushion pad, sample pad, enzyme pad, detection membrane, adsorptive pads；Described cushion pad one side is pasted onto on base plate, and another side pastes sample pad, and across the sample pad pasting at least two on cushion pad, and then sample pad pastes enzyme pad, detection membrane, adsorptive pads successively；Described capping plate is provided with well and visual window, and described well faces toward cushion pad, and described visual window is respectively toward to detection membrane；Described detection membrane upper surface is coated pesticide protein conjugate and antibiont enzyme antibody IgG side by side, forms detection line and nature controlling line respectively.The principle of this quick measuring card is the physics immunochromatographiassays assays method utilizing acetylcholine esterase to substitute gold labeling antibody, have highly sensitive, it is suitable for scene screening, simply, quickly, the advantage such as cost is low, sample aequum is few, pre-treatment is simple and convenient, the Pesticides Testing for ultramicron has extremely wide market prospect.Acetylcholine esterase physics immunochromatography is the characteristic that the acetylcholine esterase utilizing and organophosphorus and N-methylcarbamate pesticides having high susceptibility can directly combine with detection target molecule, the method that then recycling immunochromatography principle is measured.It is easy and simple to handle quickly, namely a few minutes are it can be seen that result, cost is also very cheap, it is adaptable to the detection of all of Organophosphorus and carbamate pesticides class pesticide, has filled up the pesticide multi-residues quick measuring card blank at home and abroad utilizing acetylcholine esterase together with immune chromatography method in prior art.
The method have the advantages that
(1) simple to operation: the quick measuring card of the present invention is easy and simple to handle, rapid and convenient, result are accurate, can detect after water fruits and vegetables and Environmental Water sample are carried out simple process, it is only necessary to by distinguishing that lines situation occur it is determined that pesticide residues situation in sample.This quick measuring card does not need any instrument and equipment, and common layman is operable, with the naked eye gets final product interpretation.
(2) the enzyme pad of quick measuring card of the present invention adopts special stabilizer to keep the stability of its Chinese medicine so that it is have very strong capacity of resisting disturbance, and can stay active for long periods.
(3) high flux: can realize detecting while Organophosphorus and carbamate pesticides class pesticide；One quick measuring card just can realize the Rapid Detection of two big class totally tens kinds of toxic pesticides minimal residues.
(4) high sensitivity: the detection sensitivity of quick measuring card of the present invention is 0.008～0.987mg/kg, gets final product observed result in 3～5 minutes, is suitable for the organophosphorus and N-methylcarbamate pesticides in veterinary antibiotics and environmental water sample is used for quickly detecting.
(5) environmental protection: making of card adopts the biomaterial being prone to degraded, nontoxic, safe and reliable, environmentally friendly.
Accompanying drawing explanation
Fig. 1 is the structural representation of quick measuring card of the present invention.
Detailed description of the invention
Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but the present invention is not limited in any form by embodiment.Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.
Unless stated otherwise, following example agents useful for same and material are commercial.
The structure of embodiment 1 quick measuring card of the present invention
1, quick measuring card of the present invention includes base plate and capping plate, and base plate includes cushion pad that at least two groups paste successively, sample pad, enzyme pad, detection membrane, adsorptive pads；Described cushion pad one side is pasted onto on base plate, and cushion pad another side pastes sample pad, and and then sample pad pastes enzyme pad, detection membrane, adsorptive pads successively；Paste described capping plate on bonnet successively and namely obtain described quick measuring card；Described capping plate is provided with well and visual window, and described well faces toward cushion pad, and described visual window is respectively toward to detection membrane；Described detection membrane upper surface is coated organophosphor and carbamates pesticide protein conjugate and antibiont enzyme antibody IgG side by side, forms detection line and nature controlling line respectively.
Further, described detection membrane 5 is coated organophosphor with carbamate chemicals for agriculture protein conjugate as detection line 6, it is coated 1 antibiont enzyme antibody IgG line as nature controlling line 7, organophosphor both can be combined with acetylcholine esterase with carbamate chemicals for agriculture protein conjugate, it is also possible to is identified by antibiont enzyme antibody IgG.
2, wherein, described organophosphor is as follows with the structure of carbamate chemicals for agriculture protein conjugate:
Wherein, described coupling protein is bovine serum albumin (BSA) or ovalbumin (OVA).
3, the enzyme in described antibiont enzyme antibody IgG is derive from silkworm and by the restructuring acetylcholinesterase of genetic modification.The preparation method of described enzyme is as follows:
The present invention aminoacid sequence according to known silkworm acetylcholinesterase, is suitably changed, and has maximally utilised the codon of escherichia coli preference, has designed and synthesized new acetylcholinesterasegene gene fragment.Specifically, the new acetylcholinesterasegene gene of designbmaceThe sequence of fragment is as follows:
ATGATCAACTACTGATCCTAGGCTGATCCTAATAGATTCTGATCCTATTGATCCTAAGGTACCCTATACCTATACTAGGTGATCCTATGATCCTATGATCCTAGGTGATCCTATGATCCTATGATCCTATCCTATACTAGGTGATCCTATGATCCTATGATCACTAGGTGATCCTATGATCCTATGATCCCCTATACTAGCCTATACTAGGTGATCCTATGATCCTATGATCGTGATCCTATGATCCTATGATCGATCTTGATCCCTATACTAGGTGATCCTATGATCCTATGATCCTAAGGATGATCCTACTTACGTAGCTATTGATCCTAGGATGATCCTATATGACCTATACTAGGTGATCCTATGATCCTATGATCTTGATCCTATGATGATCCTATCCTACCTAGGCCGTATGACGTAGCTAGTACGTGATCCTAACCTATACTAGGTGATCCTATGATCCTATGATCTCGATCTGATCCCTACCTATACTAGGTGATCCTATGATCCTATGATCTCCTATACTAGGTGATCCTATGACCTATACTAGGTGATCCTATGATCCTATGATCTCCTATGATCACCTATACTAGGTGATCCTATGACCTATACTAGGTGATCCTATGATCCTATGATCTCCTATGATCCTAGGTGATCCTATGATCCTATGATCTGATCCTACTAGATTGATCCTACGACCTATACTAGGTGATCCTATGATCCTATGATCCCTATACTAGGTGATCCTATGATCCTATGATCCCTATACTAGGTGATCCTATGATCCTATGATCCCTATACTAGGTGATCCTATGATCCTATGATCTCCCTATACTAGGTGATCCTATGATCCTATGATCGTAGTAGCTGATCCTACTAGCTAGCTAGCTTCCCTTGATCCTATAGCTGATTGATCCTACTATAGTGATCCTACCTATACTAGGTGATCCTATGATCCTATGATCCCTATACTAGGTGATCCTATGATCCTATGATCGATCTGATCCTGATCCTATGATCCTATACTAGGTGATCCTATGATCCTATGATCCTATGATCCTATATTGCTTTG。
Then by the new acetylcholinesterasegene gene of this designbmaceRestructuring is to fusion expression vector (preferably, fusion expression vector used can be pET28 (a), pUC118 or pEZZ318 etc.) in, and it is transformed in escherichia coli, obtained by colibacillary cultivation and there is bioactive expression product identical with natural acetylcholinesterase restructuring acetylcholinesterase.
More specifically, the preparation process of above-mentioned restructuring acetylcholinesterase is as follows:
(1) sequential design: as it has been described above, design new acetylcholinesterasegene genebmaceFragment；
(2) recombinant expression carrier is built: with Nde I, BamH I by new acetylcholinesterasegene genebmaceFragment cuts from recombiant plasmid pUC118-CM, is inserted in carrier pET28 (a) with Nde I, BamH I digestion, converts e. coli bl21, recombiant plasmid called after pET28 (a)-CM obtained；
(3) convert escherichia coli: by the effect of Nde I, BamH I digested vector pET28 (a), recombiant plasmid pET28 (a)-CM is converted e. coli bl21；
(4) cultivate escherichia coli and obtain restructuring acetylcholinesterase: after recombiant plasmid pET28 (a)-CM converts e. coli bl21, acetylcholinesterase is under T7 promoter controls, with a molecular weight only 2000 little peptide amalgamation and expression, finally obtain expression product.
Single from the SDS-PAGE behavior of expression product, containing recombiant plasmid and the thalline expressing protein band zero difference (because the two has a band of expression at molecular weight about 6000) containing empty plasmid.In order to identify expression product further, also expression product is carried out determination of activity, through determination of activity, it is thus achieved that restructuring acetylcholinesterase.
4, described antibiont enzyme antibody IgG is rabbit polyclonal antibody, and its preparation method is by immunity new zealand white rabbit after enzyme emulsifying, through the rabbit anti-serum that screening obtains.
The structure of embodiment 2 quick measuring card of the present invention
1, as shown in Figure 1, quick measuring card of the present invention includes base plate 1 and capping plate 9.
Described base plate 1 includes a cushion pad 2, and two groups of sample pad 3, enzyme pad 4, detection membrane 5 and adsorptive pads 8.
Wherein, described cushion pad 2 is as a whole, and in two sample pad 3, described two groups of sample pad 3, enzyme pad 4, detection membrane 5 and adsorptive pads 8 are pasted onto on base plate 1 successively；Described detection membrane 5 upper surface is coated organophosphor and carbamate chemicals for agriculture protein conjugate (detection line 6) and antibiont enzyme antibody IgG(nature controlling line 7 side by side).
Described capping plate 9 includes well 10 and visual window 11.
2, the function of the present invention above-mentioned quick measuring card each several part describes as follows with preparation method:
(1) base plate 1: scribble adhesive sticker and the plastic plate not absorbed water for one side, plays the fixing effect supporting other ingredients of quick measuring card and is merged into quick measuring card with capping plate 9.
(2) cushion pad 2 and sample pad 3: all-glass paper immerses 5min in the Tris-HCl of pH6.4～7.2, takes out, vacuum drying, obtains cushion pad 2 and sample pad 3.Cushion pad 2 and sample pad 3 act, when detection, the effect absorbing sample solution, and sample solution is on average assigned in two sample pad 3 by cushion pad 2, and formation moves up.
(3) enzyme pad 4: play effect that is fixing and that adhere to acetylcholine esterase.
Enzyme pad processes: be immersed in by filter paper in the solution of acetylcholine esterase and stabilizer respectively, selects stirring at 20 DEG C to soak 40min, then dries, be placed in vacuum desiccator and be dried overnight 12h.
(4) detection membrane 5: be coated organophosphor in detection membrane 5 with carbamate chemicals for agriculture protein conjugate as detection line 6, it is coated 1 antibiont enzyme antibody IgG line as nature controlling line 7, organophosphor both can be combined with acetylcholine esterase with carbamate chemicals for agriculture protein conjugate, it is also possible to is identified by antibiont enzyme antibody IgG.
(5) adsorptive pads 8: adopt the all-glass paper after drying at room temperature, filter paper or absorbent paper as adsorptive pads.Adsorptive pads 8 Main Function is in that to absorb the mobile sample solution come up.
(6) capping plate 9: having well 10 and visual window 11 above plastic plate, well 10 is facing to cushion pad middle, and two visual windows 11 are respectively toward to two detection membrane 5；Capping plate 9 can be combined formation detection card with base plate 1.
3, the assembling of pesticide quick measuring card of the present invention
As shown in Figure 1, base plate 1 is pasted two groups of sample pad 3, enzyme pad 4, detection membrane 5 and adsorptive pads 8 successively, by cushion pad 2 across in two sample pad 3, then capping plate 9 is covered, well 10 is facing to cushion pad 2 middle, and two visual windows 11 are respectively toward to two detection membrane 5.
The application of embodiment 3 quick measuring card of the present invention
1, in the quick measuring card of the present embodiment, enzyme pad 4 is the acetylcholine esterase having adsorbed zymolyte labelling, this be the present embodiment quick measuring card to put place, other parts structure is identical with composition and embodiment 1.
2, this quick measuring card is utilized, according to detection method provided by the invention, the lowest detectable limit of organophosphorus and N-methylcarbamate pesticides in several frequently seen Folium Camelliae sinensis is as shown in table 1 below:
The table 1 acetylcholine esterase immunochromatography quick measuring card lowest detectable limit (mg/kg) to several frequently seen Pesticides in Tea medicine
The application of embodiment 4 quick measuring card of the present invention
1, the detection of environmental water sample: directly draw environmental water sample and be added drop-wise in well 10, drip 4, stand after keeping flat 3～5min.If all there are lines in two detection membrane, it was shown that sample is negative, does not contain organophosphor or carbamate chemicals for agriculture, is otherwise positive.No matter sample is negative or positive, and lines should occur in nature controlling line, if nature controlling line is without lines, it was shown that reagent paper lost efficacy.
2, the detection of fruit and vegetable sample: fruit (bark fetching part) or vegetable are cut into small pieces, weighs the 1g 1mL0.05mol/L PBS solution containing 10% methanol and extracts the centrifugal 5min of 10min, 5000rpm, take supernatant and directly detect, method and the same environmental water sample of judged result.
3, the detection of organophosphorus and N-methylcarbamate pesticides is limit as shown in table 2 by the present invention.
Table 2 is this quick measuring card detection limit (mg/kg) to organophosphorus and N-methylcarbamate pesticides
The contrast that embodiment 5 quick measuring card of the present invention blocks with commercially available detection
1, choosing 7 blank samples (wherein 4 is pond water sample, and 3 is food and vegetable sample) and add appropriate omethoate, adopt this quick measuring card and commercially available speed to survey the scraps of paper simultaneously and be measured, and adopt HPLC-MS/MS method to confirm simultaneously, result is in Table 3.
3 quick measuring cards of table survey the scraps of paper to sample determination comparing result with commercially available speed
From result shown in table 3 it can be seen that this quick measuring card and commercially available speed are surveyed the scraps of paper and can be measured negative sample preferably.Illustrate that quick measuring card of the present invention has better sensitivity, be better than traditional speed and survey the scraps of paper.
Claims (10)
1. organophosphorus and N-methylcarbamate pesticides many residuals acetylcholine esterase immunochromatography quick measuring card, it is characterised in that described quick measuring card includes base plate 1 and capping plate 9；Including some groups of cushion pads 2 on described base plate 1, across several sample pad 3 on each cushion pad 2, and then sample pad 3 is also arranged in sequence with enzyme pad 4, detection membrane 5 and adsorptive pads 8；Described sample pad 3, enzyme pad 4, detection membrane 5 and adsorptive pads 8 are pasted onto on base plate 1 successively；Described capping plate 9 includes several well 10 and several visual windows 11；Wherein, described detection membrane 5 upper surface is coated organophosphor and carbamate chemicals for agriculture protein conjugate and antibiont enzyme antibody IgG side by side.
2. organophosphorus and N-methylcarbamate pesticides many residuals acetylcholine esterase immunochromatography quick measuring card according to claim 1, it is characterized in that, described detection membrane 5 is coated organophosphor with carbamate chemicals for agriculture protein conjugate as detection line 6, is coated 1 antibiont enzyme antibody IgG line as nature controlling line 7.
3. organophosphorus and N-methylcarbamate pesticides many residuals acetylcholine esterase immunochromatography quick measuring card according to claim 1, it is characterised in that described organophosphor is as follows with the structure of carbamate chemicals for agriculture protein conjugate:
。
4. organophosphorus and N-methylcarbamate pesticides many residuals acetylcholine esterase immunochromatography quick measuring card according to claim 1, it is characterised in that the enzyme in described antibiont enzyme antibody IgG is derive from silkworm and by the restructuring acetylcholinesterase of genetic modification.
5. organophosphorus and N-methylcarbamate pesticides many residuals acetylcholine esterase immunochromatography quick measuring card according to claim 1, it is characterized in that, described antibiont enzyme antibody IgG is rabbit polyclonal antibody, its preparation method is by immunity new zealand white rabbit after enzyme emulsifying, through the rabbit anti-serum that screening obtains.
6. organophosphorus and N-methylcarbamate pesticides many residuals acetylcholine esterase immunochromatography quick measuring card according to claim 1, it is characterized in that, the preparation method of described cushion pad 2 and sample pad 3 is: all-glass paper or filter paper are immersed in the Tris-HCl of pH6.4～7.2 or 0.1mol/L, pH6.4～PBS of 7.2 in, process 3～10min, take out dried and get final product；
The preparation method of described enzyme pad 4 is: adopts filter paper as material, is immersed in by filter paper in the solution of acetylcholine esterase and stabilizer, stirs immersion 25～50min, then dry at 15～25 DEG C, dry 10～15h.
7. organophosphorus and N-methylcarbamate pesticides many residuals acetylcholine esterase immunochromatography quick measuring card according to claim 6, it is characterised in that described acetylcholine esterase zymolyte, gold colloidal or quantum dot carry out labelling.
8. organophosphorus and N-methylcarbamate pesticides many residuals acetylcholine esterase immunochromatography quick measuring card according to claim 1, it is characterised in that described well 10 is identical with the quantity of cushion pad 2, and visual window 11 is identical with the quantity of detection membrane 5；After capping plate 9 is combined formation detection card with base plate 1, well 10 faces the middle of cushion pad 2, and visual window 11 faces detection membrane 5 respectively.
9. the application in detection organophosphorus and N-methylcarbamate pesticides many residuals of the quick measuring card described in claim 1.
10. apply according to claim 9, it is characterized in that, described organophosphorus and N-methylcarbamate pesticides is dichlorvos, coumafos, Bassa, parathion, Furadan, phoxim, methidathion, O-2,4-dichlorophenyl O,O-diethyl phosphorothioate, quintiofos, triazophos, triazotion, Phosalone, Menite, omethoate, Di-captan, Ficam, cynock, sevin, parathion-methyl, Malathion, fenifrothion, fenthion, famphur, C-9491, chlorpyrifos-methyl or disulfoton.
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