CN109324182A - A kind of fluorescent micro-ball immune chromatography test paper strip and its preparation method and application detecting pendimethalin - Google Patents
A kind of fluorescent micro-ball immune chromatography test paper strip and its preparation method and application detecting pendimethalin Download PDFInfo
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- CN109324182A CN109324182A CN201811104582.2A CN201811104582A CN109324182A CN 109324182 A CN109324182 A CN 109324182A CN 201811104582 A CN201811104582 A CN 201811104582A CN 109324182 A CN109324182 A CN 109324182A
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Abstract
The invention discloses remaining fluorescent micro-ball immune chromatography test paper strips of a kind of detection pendimethalin and its preparation method and application.The test strips include bottom plate and successively overlap the sample bonding pad of stickup on bottom plate, nitrocellulose filter and water absorption pad, the pendimethalin monoclonal antibody of fluorescent microsphere label is embedded on sample bonding pad, detection zone and quality control region are fixed on nitrocellulose filter, detection zone is coated with pendimethalin hapten-carrier protein conjugate, quality control region is coated with sheep anti mouse antiantibody, pendimethalin monoclonal antibody is prepared using pendimethalin hapten-carrier protein conjugate as immunogene, pendimethalin haptens is by 3- (the third ammonia of 1- ethyl) -6- methyl -2, 4- dinitrobenzal-dehyde reacts to obtain with 3- hydrazinepropionic acid.The present invention also provides the preparation method and application of the test strips test strips to detect the remaining method of pendimethalin.Test strips of the invention have many advantages, such as that high sensitivity, detection are quick, easy to operate, economical and practical.
Description
Technical field
The invention belongs to Detecting Pesticide fields, and in particular to a kind of remaining fluorescent microsphere of detection pendimethalin is immune
Chromatograph test strip and its preparation method and application, it is especially suitable for pendimethalins in agricultural product, tobacco and tobacco product to remain
Detection.
Background technique
Pendimethalin is a kind of dry crop selective herbicide, can be widely applied to corn and soybean, peanut, cotton,
The field weeding of the various crops such as direct-seeding dry rice, potato, tobacco, vegetables.Currently, pendimethalin is the big herbicide in third place in the world, pin
The volume of selling is only second to steriland herbicide glyphosate, paraquat, and the maximum selective herbicide of sales volume in the world, in China
It is very widely used in agricultural production.Therefore, residue problem of the pendimethalin in crops also receives highest attention.GB
In 2763-2016 " national food safety standard Pesticide maximum residue limit " regulation pendimethalin paddy, leek,
Cabbage, common Chinese cabbage, spinach, celery, the maximum residue limit (MRL) in Chinese cabbage are 0.2 mg/kg, in brown rice, jade
Rice, cottonseed, garlic, the MRL in lettuce are 0.1 mg/kg；The U.S. provides that MRL of the pendimethalin in beans, tealeaves, garlic is
0.1 mg/kg, the MRL in leek are 0.2 mg/kg, and the MRL in carrot is 0.5 mg/kg；European Union provides pendimethalin
MRL in beans, carrot is 0.2 mg/kg, and the MRL in celery is 0.1 mg/kg.International tobacco scientific research cooperation
Center (CORESTA) provides that the guiding residue limits of pendimethalin in tobacco are 5 mg/kg, and China not yet formulates two in tobacco
The maximum residue limit of penta spirit of first.
The detection method of pendimethalin is mainly instrument detection method at present, such as gas chromatography, gas chromatography-mass spectrum
Method, gas-chromatography tandem mass spectrometry, etc..But since these analysis methods need the inspection of expensive large-scale instrument and equipment and profession
Survey personnel, pretreatment process is complicated, cumbersome, testing cost is high, analysis speed is slow, it is difficult to meet field monitoring and a large amount of samples
The needs of persticide residue rapid screening in this.Therefore, it develops a kind of not examined equipment limit and can be realized to large quantities of
The product and method that amount sample is used for quickly detecting become problem in the urgent need to address.
Fluorescent micro-ball immune chromatography technology is grown up in fluorochrome label technology, is examined as a kind of immunology
Survey method, it is the combination of affine in immunity technology, immunolabelling technique, immunochromatography technique, is had quick, easy to operate etc. excellent
Point.Compared to conventional tag object, the luminous intensity of fluorescent microsphere can enhance with the enhanced strength of exciting light, so fluorescent microsphere
Label is expected to improve the detection limit of immunochromatography technique；And under the action of microballoon shell structure, fluorescent microsphere has relatively stable
Morphosis, homogeneous grain diameter, monodispersity are good, stability is good, luminous efficiency is high, reproducible, there is preferable bio-compatible
Property；And dyestuff fluorescent quenching greatly reduces after forming microballoon, transmitting is strong and stablizes, and substantially not by external environment media variations
It influences.Therefore, fluorescent micro-ball immune chromatography technology has the advantages that detection sensitivity is high, easy to operate, stability is good simultaneously.It cuts
To currently, there has been no the appearance of pendimethalin fluorescent micro-ball immune chromatography test paper strip in the market.
Summary of the invention
An object of the present invention is to provide a kind of high sensitivity, the diformazan penta that easy to operate, at low cost, detection time is short
Clever residue detection fluorescent micro-ball immune chromatography test paper strip；It is a further object to provide the preparation sides of above-mentioned test strips
Method；It is also another object of the present invention to provide application of the above-mentioned test strips in detection pendimethalin.
To achieve the goals above, the technical solution that the present invention takes is:
There is provided a kind of detection pendimethalin remaining fluorescent micro-ball immune chromatography test paper strip, it include bottom plate and on bottom plate successively
Sample bonding pad, nitrocellulose filter and the water absorption pad pasted are overlapped, fluorescent microsphere label is embedded on the sample bonding pad
Pendimethalin monoclonal antibody, be fixed with detection zone and quality control region on the nitrocellulose filter, detection zone is coated with diformazan
Penta clever hapten-carrier protein conjugate, quality control region are coated with sheep anti mouse antiantibody, the pendimethalin monoclonal antibody be with
Pendimethalin hapten-carrier protein conjugate is prepared as immunogene, and the pendimethalin hapten-carrier albumen is even
Joining object is to be obtained by pendimethalin haptens with carrier protein couplet, and the pendimethalin haptens is by 3- (1- ethyl third
Ammonia) -6- methyl -2,4- dinitrobenzal-dehyde reacts to obtain with 3- hydrazinepropionic acid, molecular structural formula are as follows:
。
The pendimethalin haptens the preparation method is as follows:
0.60 g 3- (the third ammonia of 1- ethyl) -6- methyl -2,4- dinitrobenzal-dehyde is taken, 50 mL acetonitriles is added to dissolve, after clarification, drop
5 mL of methanol solution of 0.32 g 3- hydrazinepropionic acid of solubilization solution, is stirred at room temperature 3 h；Stopping reaction, revolving removes organic solvent,
Add water, 0.45 g of adding sodium hydroxide adds 80 mL ethyl acetate to extract, divides and go organic phase, water phase adds dilute hydrochloric acid after dissolution clarification
PH=4 are adjusted, 50 mL chloroforms is added to extract, are washed, concentration is recrystallized with ethyl alcohol/petroleum ether of volume ratio 1:1, obtains haptens
0.70 g of product.
The pendimethalin hapten-carrier protein conjugate is to be obtained by pendimethalin haptens with carrier protein couplet
It arrives, the carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
The pendimethalin monoclonal antibody is to be secreted to obtain by pendimethalin monoclonal antibody hybridoma cell strain；It is described
Sheep anti mouse antiantibody is to obtain source of mouse antibody mediated immunity sheep.
The fluorescent microsphere is the microballoon that fluorescent material is wrapped up with polystyrene that diameter is 100 ~ 300 nm, and surface connects
It is connected to-COOH group, the fluorescent material is fluorescein isothiocynate.
Another technical solution that the present invention takes is to provide that a kind of to prepare the above-mentioned remaining fluorescence of detection pendimethalin micro-
The method of ball immuno-chromatographic test paper strip, it includes the following steps:
1) preparation of sample bonding pad: pendimethalin monoclonal antibody is marked with commercially available fluorescent microsphere, and by it with specific slow
After rushing system dilution, sample bonding pad is soaked in dilution buffer, is prepared after vacuum freeze drying；
2) preparation of nitrocellulose filter: pendimethalin hapten-carrier protein conjugate is sprayed on nitrocellulose filter
Detection zone range, detection zone is made；Sheep anti mouse antiantibody is sprayed into the quality control region range on nitrocellulose filter, matter is made
Control area；
3) assemble and shear: successively overlap joint pastes the sample for being embedded with fluorescent microsphere label pendimethalin monoclonal antibody on bottom plate
Product bonding pad, the nitrocellulose filter and water absorption pad for being fixed with detection zone and quality control region, and it is as glimmering to cut into required width
Light micro-ball immune chromatography test paper strip.
Specifically, step includes:
1) prepared by haptens: 3- (the third ammonia of 1- ethyl) -6- methyl -2,4- dinitrobenzal-dehyde and 3- hydrazinepropionic acid are passed through a system
Column reaction obtains pendimethalin haptens product；
2) by pendimethalin haptens and carrier protein couplet, pendimethalin hapten-carrier protein conjugate is obtained；
3) mouse is immunized with pendimethalin hapten-carrier protein conjugate, by mouse boosting cell and myeloma cell by melting
It closes, screening, obtains pendimethalin monoclonal hybridoma strain；
4) mouse IgG immune health goat is extracted, sheep anti mouse antiantibody is obtained；
5) pendimethalin hapten-carrier protein conjugate and sheep anti mouse antiantibody are sprayed to the inspection of nitrocellulose filter respectively
Coverage of survey area (T) and quality control region range (C)；
6) by sample bonding pad (final concentration of 0.5% volume of bovine serum albumin(BSA) in buffer system containing bovine serum albumin(BSA)
Percentage composition), pH value 7.2,0.1 mol/L phosphate buffer uniformly impregnate 2 h, 2 h are dried at 37 DEG C；
7) with commercially available fluorescent microsphere mark pendimethalin monoclonal antibody, and by its with specific buffer system dilution after, will 6)
Processed sample bonding pad is soaked in dilution buffer, spare after vacuum freeze drying；
8) successively overlap joint stickup is embedded with the sample bonding pad of fluorescent microsphere label pendimethalin monoclonal antibody, consolidates on bottom plate
Surely there are the nitrocellulose filter and water absorption pad of detection zone and quality control region, and cutting into required width is that layer is immunized in fluorescent microsphere
Analyse test strips.
Another technical solution that the present invention takes is to provide a kind of above-mentioned remaining fluorescent microsphere of detection pendimethalin and exempts from
Application of the epidemic disease chromatograph test strip in detection pendimethalin, it includes the following steps:
1) sample pre-treatments；
2) it is detected with the remaining fluorescent micro-ball immune chromatography test paper strip of detection pendimethalin；
3) fluorescence detector analysis detection result is used.
Compared with prior art, the invention has the following advantages:
(1) high specificity, high sensitivity: this test strips uses and is embedded in the pendimethalin monoclonal antibody that fluorescent microsphere marks
On sample bonding pad, have hydrophily it is good, can large capacity absorption antibody coupling matter, rapidly again moisten, antibody conjugates release fill
Point, performance is good, release is fast, form is good etc., and advantages reduce cost to reduce error, increase the reaction sensitivity of whole system；
(2) polystyrene has been wrapped up on fluorescent microsphere surface, realizes the protection to fluorescent material fluorescein isothiocynate, reduces
The interference of external environment increases the stability and fluorescence lifetime of fluorescent microsphere；
(3) fluorescent microsphere surface modified active group-COOH, using the method for chemical coupling come labelled antibody, formed antibody with
The stable bond of microballoon.
There is no at present for detecting agricultural product, in tobacco and tobacco product pendimethalin fluorescent micro-ball immune chromatography test paper
Item, the present invention have filled up the blank.Haptens of the invention has appropriate terminal reactive group, decorating site and spacer length
Selection is suitable, and can utmostly simulate the molecular structure of pendimethalin, and the test strips developed based on this haptens have
The characteristics of high sensitivity, high specificity.Test strips of the invention simultaneously are at low cost, easy to operate, detection time is short, suitable
The advantages of various units use, storage is simple, long shelf-life.To sum up, the remaining side of pendimethalin is detected with test strips of the present invention
Easy, quick, intuitive, accurate, applied widely, at low cost, the easy popularization and use of method.
Detailed description of the invention
Fig. 1 is fluorescent micro-ball immune chromatography test paper strip the schematic diagram of the section structure, in figure: 1, sample bonding pad；2, nitric acid is fine
Tie up plain film；3, water absorption pad；4, detection zone；5, quality control region；6, bottom plate；
Fig. 2 is fluorescent micro-ball immune chromatography test paper strip top view；
Fig. 3 is pendimethalin hapten synthesis figure (figure is as Figure of abstract).
Specific embodiment
Below with reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate this
Invention, and be not intended to limit the scope of the invention.
The composition of the detection remaining fluorescent micro-ball immune chromatography test paper strip of pendimethalin of embodiment 1
Referring to Fig. 1: the test strips are made of bottom plate, sample bonding pad, nitrocellulose filter and water absorption pad.
Successively overlap joint is pasted on bottom plate 6 in order for the sample bonding pad 1, nitrocellulose filter 2 and water absorption pad 3, sample
The end of product bonding pad is connected with the beginning of nitrocellulose filter, and the end of nitrocellulose filter is connected with the beginning of water absorption pad,
The beginning of sample bonding pad is aligned with the beginning of bottom plate, and the end of water absorption pad is aligned with the end of bottom plate.
Detection zone 4 and quality control region 5 are fixed on the nitrocellulose filter, detection zone is coated with pendimethalin haptens-
Carrier protein couplet object, quality control region are coated with sheep anti mouse antiantibody.
The bottom plate is PVC bottom plate；The sample bonding pad is mineral wool；The water absorption pad is blotting paper.
The preparation of the detection remaining fluorescent micro-ball immune chromatography test paper strip of pendimethalin of embodiment 2
The preparation method of the test strips mainly comprises the steps that
1) preparation of sample bonding pad: pendimethalin monoclonal antibody is marked with commercially available fluorescent microsphere, and by it with specific slow
After rushing system dilution, sample bonding pad is soaked in dilution buffer, is prepared after vacuum freeze drying；
2) preparation of nitrocellulose filter: pendimethalin hapten-carrier protein conjugate is sprayed on nitrocellulose filter
Detection zone range, detection zone is made；Sheep anti mouse antiantibody is sprayed into the quality control region range on nitrocellulose filter, matter is made
Control area；
3) assemble and shear: successively overlap joint pastes the sample for being embedded with fluorescent microsphere label pendimethalin monoclonal antibody on bottom plate
Product bonding pad, the nitrocellulose filter and water absorption pad for being fixed with detection zone and quality control region, and it is as glimmering to cut into required width
Light micro-ball immune chromatography test paper strip.
Substep narration in detail below:
(1) preparation of each component
1, the preparation of pendimethalin haptens
0.60 g 3- (the third ammonia of 1- ethyl) -6- methyl -2,4- dinitrobenzal-dehyde is taken, 50 mL acetonitriles is added to dissolve, after clarification, drop
5 mL of methanol solution of 0.32 g 3- hydrazinepropionic acid of solubilization solution, is stirred at room temperature 3 h；Stopping reaction, revolving removes organic solvent,
Add water, 0.45 g of adding sodium hydroxide adds 80 mL ethyl acetate to extract, divides and go organic phase, water phase adds dilute hydrochloric acid after dissolution clarification
PH=4 are adjusted, 50 mL chloroforms is added to extract, are washed, concentration is recrystallized with ethyl alcohol/petroleum ether of volume ratio 1:1, obtains haptens
0.70 g of product, yield 90.1%.
Above-mentioned haptens is taken to identify through nuclear magnetic resonance spectroscopy,1H-NMR(CDCl3, 300 MHZ) and δ: 8.505 (10,1H),
4.363 (11,1H, quint, J=6.864), 8.326 (13,1H), 2.247 (14,3H), 1.525 (16,2H, qd, J=
7.141, J=6.864), 1.525 (17,2H, qd, J=7.141, J=6.864), 0.822 (21,3H, t, J=7.141), 0.822
(22,3H, t, J=7.141), 3.680 (23,2H, t, J=6.869), 2.679 (24,2H, t, J=6.869).Chemical shift δ=
3.680,2.679 for the hydrogen on haptens spacerarm resonance absorbing peak, the presence at the two peaks and position show that haptens closes
At success.
2, the preparation of immunogene
Pendimethalin haptens and bovine serum albumin(BSA) (BSA) coupling obtain immunogene.
15 mg haptens are taken, are dissolved in 1 mL n,N-Dimethylformamide (DMF)；Add 8 mg carbodiimides
(EDC), 24 h are stirred at room temperature, obtain reaction solution A；30 mg of BSA is weighed, is allowed to be substantially dissolved in 4 mL, 0.1 mol/L phosphorus
In phthalate buffer (PB, pH value 7.0), reaction solution A is slowly dropped in protein solution dropwise, and stir 24 at room temperature
H changes 3 dialyzates with 0.01 mol/L phosphate buffer (PBS), 4 DEG C of 3 d of dialysis daily, unreacted small to remove
Molecular substance obtains immunogene, packing, -20 DEG C of preservations.
3, the preparation of coating antigen
Pendimethalin haptens and ovalbumin (OVA) coupling obtain coating antigen.
20 mg haptens are taken, are dissolved in 1 mL DMF；Add 10 mg dicyclohexylcarbodiimides (DCC), at room temperature
24 h are stirred, are filtered, insoluble solids is removed, obtains reaction solution A；30 mg of OVA is weighed, is allowed to be substantially dissolved in 6 mL 0.1
Mol/L PB(pH value be 7.0) in, reaction solution A is slowly dropped in protein solution dropwise, and stir 24 h at room temperature, use
0.01 4 DEG C of mol/L PBS dialysis 3d, changes 3 dialyzates daily and is coated with removing unreacted small-molecule substance
Original, packing, -20 DEG C of preservations.
4, the preparation of pendimethalin monoclonal antibody
(1) acquisition of hybridoma
First immunisation: pendimethalin haptens-BSA conjugate (immunogene) and the Freund's complete adjuvant of equivalent is fully emulsified,
The Balb/c mouse of 6 week old, every 0.2 mL is subcutaneously injected；
Booster immunization is twice: since first immunisation, booster immunization is primary every two weeks, replaces Freund complete with not formula Freund's incomplete adjuvant
Full adjuvant, method and the same first immunisation of dosage；
Potency and inhibition are surveyed in eyeground vein blood sampling to last time booster immunization after a week, have inhibition and potency reach 1:10000 with
Following final immunization was carried out when upper: 0.1 mL of immunogen solution of any adjuvant is not added in intraperitoneal injection, puts to death mouse after three days, takes
Its spleen is merged with myeloma cell；
Cell supernatant is measured using indirect competitive enzyme-linked immunosorbent analysis method, screens positive hole.Using limiting dilution assay to sun
Property hole carry out cloning, obtain and establish the hybridoma cell strain of stably excreting pendimethalin monoclonal antibody, take in logarithm
Cell suspension is made with frozen stock solution in the hybridoma in growth period, is sub-packed in cryopreservation tube, saves for a long time in liquid nitrogen.
(2) preparation of monoclonal antibody
Cell recovery: pendimethalin monoclonal antibody hybridoma cell strain cryopreservation tube is taken out, 37 DEG C of water-bath middling speeds are immediately placed in
Melt, after centrifugation removal frozen stock solution, moves into culture culture in glassware；
Preparation ascites and antibody purification: using method is induced in vivo, by Balb/c mouse (8 week old) Intraperitoneal injection sterilizing paraffin oil
Only, hybridoma 5 × 10 is injected intraperitoneally in 0.5 mL/ after 7 days5A/only, ascites is acquired after 7 days.With octanoic acid-saturated ammonium sulfate
Method is purified, and pendimethalin monoclonal antibody solution (- 20 DEG C of preservations) is obtained.
(3) measurement of antibody titer
Potency with indirect competitive ELISA method measurement antibody is 1:(300000 ~ 700000).
Indirect competitive ELISA method: using pendimethalin haptens-OVA conjugate coated elisa plate, and pendimethalin mark is added
The sheep anti mouse antiantibody solution of quasi- product solution, pendimethalin monoclonal antibody solution and horseradish peroxidase-labeled, 25 DEG C anti-
30 min are answered, liquid in hole is poured out, is washed 3 ~ 5 times with cleaning solution, is patted dry with blotting paper；Substrate developing solution, 25 DEG C of reactions are added
After 15 min, terminate liquid is added and terminates reaction；Setting microplate reader measures every hole absorbance value at 450 nm of wavelength.
(4) measurement of monoclonal antibody specificity
Antibody specificity refer to the ability of its homospecificity antigen binding compared with such antigen-analogues ability, often
Use cross reacting rate as evaluation criterion.Cross reaction is smaller, and the specificity of antibody is then higher.
The dinitroaniline herbicides such as pendimethalin, flumetralim, butralin, trefanocide, benfluralin are done in this experiment
It is serially diluted, carries out indirect competitive ELISA with monoclonal antibody respectively, make standard curve, analysis obtains IC50, then presses
Formula calculates cross reacting rate:
The cross reacting rate of each analog as the result is shown are as follows: pendimethalin 100%, flumetralim < 1%, butralin < 1%, trefanocide
< 1%, benfluralin < 1%.Antibody of the present invention is to other dinitroanilines such as flumetralim, butralin, trefanocide, benfluralin
Herbicide no cross reaction has specific binding just for pendimethalin.
5, the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, pathogen-free domestic sheep is immunized using source of mouse antibody as immunogene, obtains sheep anti mouse antiantibody.
6, the preparation of fluorescent microsphere label pendimethalin monoclonal antibody
(1) it activates: commercially available internal embedding fluorescent dye, surface modification being taken to have the 100 μ L of microsphere suspensions of carboxyl functional group to be suspended
In 900 μ L activation buffers, supernatant is abandoned after 4 DEG C of 10000 r/min is centrifuged 10 min, it is slow in 1 mL activation that microballoon is resuspended
It in fliud flushing, washs microballoon 2 times in this way, appropriate activator is added, shaken at room temperature activates 10 min after mixing；
(2) it is coupled: (1) described suspension being abandoned into supernatant after 4 DEG C of 10000 r/min is centrifuged 10 min, is resuspended in coupling buffering
It in liquid, washs microballoon 2 times in this way, 10 ~ 20 μ L pendimethalin monoclonal antibody solutions (1 mg/mL of protein concentration) is added, mix
Shaken at room temperature is coupled 120 min after even；
(3) it closes: (2) described suspension being abandoned into supernatant after 4 DEG C of 10000 r/min is centrifuged 10 min, is resuspended in closing buffering
It in liquid, washs microballoon 1 time in this way, 30 min of room temperature concussion closing after mixing；
(4) it stores: (3) described suspension being abandoned into supernatant after 4 DEG C of 10000 r/min is centrifuged 10 min, is resuspended in storage buffering
In liquid, washs microballoon 1 time, be kept in dark place after mixing in 4 DEG C in this way.
The activation buffer is 2- (N- morpholine) ethanesulfonic acid (MES) buffering that pH value is 5.5 ~ 6.5,0.05 mol/L
Liquid.
The activator is water-soluble carbodiimide, wherein molal weight ratio EDC: NHS: COOH=(1.5 ~ 3): (8 ~
20): 1, required concentration is diluted to activation buffer before use.
The coupling buffer is that the borate buffer solution that pH value is 7.5 ~ 8.5 0.05 mol/L (is avoided using in the presence of trip
Solvent from amine).
The Block buffer is containing 0.1 ~ 0.4 mol/L primary amine (hydroxylamine hydrochloride, ethanol amine or ethylaminoethanol), 1% ~ 10%
The PB buffer that the pH value of BSA is 7.4.
The storage buffer is containing 0.01% NaN3, 0.1% BSA pH value be 7.4 PB buffer.
7, the preparation of sample bonding pad
(1) by sample bonding pad use containing bovine serum albumin(BSA) (BSA in buffer system final concentration of 0.5%, volume basis contains
Amount), pH value 7.2,0.1 mol/L phosphate buffer uniformly impregnate 2 h, 2 h are dried at 37 DEG C；
(2) after diluting the pendimethalin monoclonal antibody that the fluorescent microsphere of storage marks with storage buffer, (1) is processed
Sample bonding pad be soaked in dilution buffer, it is spare after vacuum freeze drying.
8, the preparation of nitrocellulose (NC) film
Pendimethalin haptens-ovalbumin conjugate is diluted to 100 with 0.05 mol/L, the PBS buffer solution that pH value is 7.2
μ g/mL is sprayed at the detection zone (T) on NC film with Isoflow point film instrument, and spray film amount is 1.0 μ L/cm；With 0.01
Sheep anti mouse antiantibody is diluted to 200 μ g/mL by mol/L, the PBS buffer solution that pH value is 7.4, is sprayed with Isoflow point film instrument
The quality control region (C) being applied on NC film, spray film amount are 1.0 μ L/cm.Dry 2 h under the conditions of the NC film prepared is placed in 37 DEG C,
It is spare.
(2) assembling of test strips
By sample bonding pad, nitrocellulose filter, water absorption pad, successively overlap joint is pasted and fixed on bottom plate from left to right, and sample combines
The end of pad is connected with the beginning of nitrocellulose filter, and the end of nitrocellulose filter is connected with the beginning of water absorption pad, sample knot
The beginning for closing pad is aligned with the beginning of bottom plate, and the end of water absorption pad is aligned with the end of bottom plate, is then cut into 3.96 mm with machine
Wide small item forms test card in special plastics fabrication.Pendimethalin fluorescent micro-ball immune chromatography test paper is stuck in 2 ~ 8
DEG C cool place is protected from light kept dry, and validity period is 12 months.
The application of the detection remaining fluorescent micro-ball immune chromatography test paper strip of pendimethalin of embodiment 3
1, sample pre-treatments
Into 50 mL centrifuge tubes 10 mL acetonitriles are added, with vortex instrument whirling motion in sample to be tested after weighing 1.0 ± 0.05 g homogenate
1 min, room temperature (20 ~ 25 DEG C) 3000 rpm or more are centrifuged 5 min；Take 2 mL of supernatant into 10 mL centrifuge tubes, in 40-50
It is dried with nitrogen in DEG C water-bath, 500 μ L samples is added and redissolve liquid, with 2 min of vortex instrument whirling motion, mixed to be measured.
2, it is detected with test strips
It draws 100 μ L sample to be examined solution to be vertically added dropwise in test card well, liquid starts timing, reaction 10 when flowing
min；By in the carrier of test card insertion KFT-100A type fluorescence detector, project to be checked is selected by touch display screen, is pressed
Under " start to detect " key, fluorescence detector will be scanned test to test card automatically, by reading on the display screen of instrument
Take or print testing result.
3, Analysis of test results
After the completion of test, the power of the fluorescence signal obtained according to detection is calculated diformazan penta in tobacco leaf sample by instrument automatically
The concentration value of spirit, and yin and yang attribute judgement is provided according to preset threshold value.
Negative (-): it if being as the result is shown feminine gender on the display screen of fluorescence detector, indicates not containing diformazan in sample
Penta spirit or its concentration are lower than detection limit.
Positive (+): if being as the result is shown the positive on the display screen of fluorescence detector, pendimethalin concentration in sample is indicated
It is limited equal to or higher than detection.
It is invalid: if fluorescence signal intensity is not detected in quality control region, to show that incorrect operating process or test card have been failed.
The determination of the detection remaining fluorescent micro-ball immune chromatography test paper strip technical parameter of pendimethalin of embodiment 4
1, detection limit test
Added respectively into blank tobacco, tealeaves, spinach, celery sample pendimethalin standard items to final concentration of 0.05,0.1,
0.2 mg/kg, is detected with fluorescent micro-ball immune chromatography test paper strip, as a result are as follows: when pendimethalin concentration is 0.05 mg/kg,
Fluorescence detector is detected as feminine gender；When pendimethalin concentration is 0.1,0.2 mg/kg, fluorescence detector test positive shows
This test strips is limited to 0.1 mg/kg to the detection of pendimethalin in tobacco, tealeaves, spinach, celery.
2, false positive rate, false negative rate test
Known pendimethalin content is taken to be greater than the negative sample of the tobacco positive sample for detecting limit and content less than detection limit respectively
It each 20 parts, is detected respectively with the fluorescent micro-ball immune chromatography test paper strip that 3 batches produce, calculates its yin and yang attribute rate.As a result
It see the table below.
Table 1 detects positive, negative sample result
The result shows that: when detecting positive sample with the test strips of 3 batch productions, as a result it is all positive, it is known that positive coincidence rate
It is 100%, false negative rate 0；When detecting negative sample, as a result it is all negative, it is known that negative match-rate 100%, false positive rate
It is 0.Illustrate that the remaining fluorescent micro-ball immune chromatography test paper strip of detection pendimethalin of the invention can be to pendimethalin in tobacco leaf
Residual is used for quickly detecting.
3, specific test
The dinitroanilines such as flumetralim, butralin, trefanocide, the benfluralin of 5 mg/L are detected with pendimethalin test strips to remove
Careless agent, is negative as the result is shown.Illustrate this test strips to the pendimethalin of 5 mg/L, butralin, trefanocide, benfluralin etc. two
Nitroaniline herbicide no cross reaction, specificity are good.
Claims (7)
1. a kind of remaining fluorescent micro-ball immune chromatography test paper strip of detection pendimethalin, it is characterised in that: including bottom plate and the bottom of at
Sample bonding pad, nitrocellulose filter and the water absorption pad of stickup are successively overlapped on plate, are embedded with fluorescence on the sample bonding pad
The pendimethalin monoclonal antibody of microballoon label is fixed with detection zone and quality control region, detection zone spray on the nitrocellulose filter
It is coated with pendimethalin hapten-carrier protein conjugate, quality control region is coated with sheep anti mouse antiantibody, the pendimethalin monoclonal
Antibody is prepared using pendimethalin hapten-carrier protein conjugate as immunogene, the pendimethalin haptens-load
Body protein conjugate is to be obtained by pendimethalin haptens with carrier protein couplet, and the pendimethalin haptens is by 3- (1-
The third ammonia of ethyl) -6- methyl -2,4- dinitrobenzal-dehyde reacts to obtain with 3- hydrazinepropionic acid, molecular structural formula are as follows:
。
2. the detection remaining fluorescent micro-ball immune chromatography test paper strip of pendimethalin as described in claim 1, it is characterised in that: institute
The preparation reaction process for stating pendimethalin haptens is as follows:
。
3. a kind of remaining fluorescent micro-ball immune chromatography test paper strip of detection pendimethalin as described in claim 1, feature exist
It is the microballoon that fluorescent material is wrapped up with polystyrene that diameter is 100 ~ 300nm in: the fluorescent microsphere, surface is connected with-
COOH group, the fluorescent material are fluorescein isothiocynate.
4. a kind of remaining fluorescent micro-ball immune chromatography test paper strip of detection pendimethalin as described in claim 1, feature exist
In: the carrier protein is bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
5. a kind of remaining fluorescent micro-ball immune chromatography test paper strip of detection pendimethalin as described in claim 1, feature exist
In: the sheep anti mouse antiantibody is to obtain source of mouse antibody mediated immunity sheep.
6. a kind of system of the described in any item detection remaining fluorescent micro-ball immune chromatography test paper strips of pendimethalin of claim 1-5
Preparation Method, it is characterised in that the following steps are included:
1) preparation of sample bonding pad: pendimethalin monoclonal antibody is marked with commercially available fluorescent microsphere, and by it with specific slow
After rushing system dilution, sample bonding pad is soaked in dilution buffer, is prepared after vacuum freeze drying；
2) preparation of nitrocellulose filter: pendimethalin hapten-carrier protein conjugate is sprayed on nitrocellulose filter
Detection zone range, detection zone is made；Sheep anti mouse antiantibody is sprayed into the quality control region range on nitrocellulose filter, matter is made
Control area；
3) it assembles and shears: pasting with successively being overlapped on bottom plate and be embedded with fluorescent microsphere label pendimethalin monoclonal antibody
Sample bonding pad, the nitrocellulose filter and water absorption pad for being fixed with detection zone and quality control region, and cut into required width and be
Fluorescent micro-ball immune chromatography test paper strip.
7. a kind of described in any item remaining fluorescent micro-ball immune chromatography test paper strips of detection pendimethalin of claim 1-5 are being examined
Survey the application in pendimethalin, it is characterised in that comprising steps of
1) sample pre-treatments；
2) it is detected with the test strips；
3) fluorescence detector analysis detection result is used.
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