CN106732437A - A kind of affine magnetic microsphere and its preparation and application for purifying and detecting carbamate and organophosphorus pesticide - Google Patents
A kind of affine magnetic microsphere and its preparation and application for purifying and detecting carbamate and organophosphorus pesticide Download PDFInfo
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- CN106732437A CN106732437A CN201710142671.5A CN201710142671A CN106732437A CN 106732437 A CN106732437 A CN 106732437A CN 201710142671 A CN201710142671 A CN 201710142671A CN 106732437 A CN106732437 A CN 106732437A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28009—Magnetic properties
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28016—Particle form
- B01J20/28021—Hollow particles, e.g. hollow spheres, microspheres or cenospheres
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/405—Concentrating samples by adsorption or absorption
Abstract
The invention discloses a kind of affine magnetic microsphere and its preparation and application for purifying and detecting carbamate and organophosphorus pesticide, this is affine magnetic microsphere is magnetic ion in porous or hollow microsphere interior polymeric, has acetylcholine esterase or anti-carbamate and organophosphorus insecticide antibody in porous or hollow microsphere Surface coating；Its preparation method include acetylcholinesterase magnetic microsphere surface fix and magnetic microsphere surface-closed.The present invention can effectively extract the carbamate and organophosphorus insecticide in sample, to solve the result of false positive/false negative that complex matrices of the prior art bring, improve the accuracy and reliability of actual sample Pesticides scene residue detection.
Description
Technical field
The present invention relates to field of pesticide detection, and in particular to one kind is for purifying and detecting carbamate and organophosphor agriculture
The affine magnetic microsphere and its preparation and application of medicine.
Background technology
For many years, Pesticide Residues in Vegetables excessive problem is more prominent always, and one of reason is the agricultural chemicals that present standard is implemented
Residues detecton needs by Solvent Extract methods, purification and is carried out with large-sized analytic instrument, it is impossible to cheap vegetables are carried out with
When quick detection everywhere.
The method for being applied to the residual quick detection of agriculture at present mainly has inhibiting AChE and enzyme linked immunosorbent assay.Inhibiting AChE is
Can specifically suppress acetylcholine ester in insect maincenter and peripheral nervous system using organophosphor and carbamate chemicals for agriculture
The activity of enzyme (AChE), neurolytic normal conduction makes insect lethal this toxicology principle of poisoning, and AChE is anti-with sample
Should, according to the situation that AChE activity is suppressed, can determine whether out whether contain organophosphor and carbamate chemicals for agriculture in sample.
Though inhibiting AChE detection technique have easily and fast, low cost and other advantages, some are there is also in use urgently to be resolved hurrily
Problem：Such as selection in enzyme source, the esterase in animal body is sufficiently complex, such as acetylcholinesterase (AChE), butyrylcholine esterase
(BuChE) etc., reaction of the separate sources difference esterase to agricultural chemicals differs widely, therefore the selection and extraction in sensitive enzyme source are detections
The key of accuracy, same batch sample must select same batch enzyme source；Enzymatic condition, enzyme is a species specific albumen
Matter, enzymatic reaction requirement is very strict, and temperature, acid-base value, oxidation-reduction potential are the essential conditions of left and right enzymatic reaction；Some
Agricultural product species can not detect or have an impact that such as celery, leek sample contain excessive chlorophyll, anthocyanidin, Plant Secondary Materials
Material, has interference to testing result, causes testing result in false positive etc..
The Fast Detection Technique to organophosphor and carbamate chemicals for agriculture is concentrated on based on enzyme level and paper disk method at present,
It is, using acetylcholinesterase (AChE) and the specific binding of agricultural chemicals, to cause the drop of enzyme activity based on common Cleaning Principle
Low or disappearance, the detection of residues of pesticides is carried out by indirect determination enzyme activity.Such as the profits of Chinese patent ZL 201310116604.8
Test paper is made with temperature sensitive microcapsules technology, in specified temp critical zone, the cholinesterase of parcel is discharged, indophenols second is catalyzed
Acid esters hydrolysis colour developing, and then judge residues of pesticides situation.The present invention is single use, cheap, it is not necessary to particular formulation
Reagent, saves the maintenance work of analytical instrument；Using simpler compared with manual compression, result judgement is very clear, without training；Examination
Agent consumption is less, and detection time is shorter, is completed in 1min；Cholinesterase is protected more stable by microcapsules, and the holding time is long.
Chinese patent ZL discloses a kind of coloration method based on acetylcholinesterase, the party for 201010523463.8 times
Method employs special developer so that yin and yang attribute colour developing result clearly, and without spectrophotometer, visually can directly be sentenced
It is disconnected.
Chinese patent application CN201510056439.0 discloses a kind of Organophosphorus and carbamate pesticides pesticide residue speed
Survey card, including lower cover and with the upper lid that lower cover is interlocked, be above covered with well and peep hole, test agricultural chemicals is fixed with lower cover
The speed of residual surveys paper, speed survey paper include base plate and in the soleplate portion paste it is fixed climb sample film, the one end for climbing sample film is pasted successively
Colour developing pad and adsorptive pads, the other end for climbing sample film paste barrier film and enzyme pad successively, the position correspondence observation colour developing pad of peep hole
Position, the position of the position correspondence enzyme pad of well, detection time is short, and simple to operate, lateral chromatography causes strong antijamming capability,
Color developing effect is good, can be widely applied to the quick detection of organophosphor and carbamate chemicals for agriculture.
Above-mentioned Patents, all fail to solve influence of the actual sample matrix to detecting, such as celery, leek sample contains
Excessive chlorophyll, anthocyanidin, plant secondary substance, has interference to testing result, causes testing result in false positive etc..
The content of the invention
It is an object of the invention to provide micro- for purifying and detecting the affine magnetic of carbamate and organophosphorus pesticide
Ball and its preparation and application, are applicable in Site Detection.With solve prior art Pesticides Site Detection exist compared with
Big false positive issue.After acetylcholinesterase and magnetic microsphere are reacted a period of time by the present invention jointly, prepare with spy
The affine magnetic microsphere of the opposite sex.Thick by actual sample is carried after solution reacts a period of time jointly with the magnetic microsphere, you can real
Existing carbamate and organophosphorus insecticide are adsorbed onto magnetic microsphere surface.After Magnetic Isolation, you can realize carbamic acid
Ester and organophosphorus insecticide sample clean, eliminate the influence of pigment matrix, obtain relatively pure sample to be checked, are further used for glimmering
Light detection, immune detection, enzyme level, enzyme colorimetric etc., illustrate design of the invention in detail below.
First, the present invention provides a kind of affine magnetic microsphere for purifying and detecting carbamate and organophosphorus pesticide
Preparation method, concrete technical scheme is as follows：
A kind of preparation method for purifying and detecting the affine magnetic microsphere of carbamate and organophosphorus pesticide, including
Following steps：
A, acetylcholinesterase are fixed on magnetic microsphere surface
Take magnetic particle suspension to be added in centrifuge tube, jog is resuspended；It is placed on magnetic separator, Magnetic Isolation is abandoned
Clearly；Coupling buffer is added, jog is resuspended；Centrifuge tube is placed on magnetic separator, Magnetic Isolation, abandons standby after supernatant；
Take acetylcholinesterase preparation and obtain stock solution, take in appropriate addition magnetic particle, make magnetic particle and acetyl
Cholinesterase or anti-carbamate and organophosphorus insecticide antibody response concentration cause that it reaches maximum for 0.1~10mg/ml
Conjugate ratio；Reaction is finished, Magnetic Isolation, abandons supernatant；Add cleaning buffer solution, the resuspended magnetic grain of jog；Magnetic Isolation, abandons supernatant,
Non-specific adsorption is removed in the enzyme on magnetic particle surface, affine magnetic microsphere crude product is obtained；
The surface-closed of B, magnetic microsphere
Add sealer in the affine magnetic particle crude product that step A is obtained, on closing magnetic particle crude product not with enzyme knot
The site of conjunction；It is cleaned multiple times with cleaning buffer solution, is finally suspended from preservation buffer solution, 2~8 DEG C of preservations is obtained affine magnetic
Microballoon.
Preferably, the coupling buffer is the PBS of 0.05~0.5M, pH7.0~8.0, or 0.05~0.5M,
The tris-HCI buffer of pH7.0~7.6, or 0.05~0.5M, the MES cushioning liquid of pH4~6.
Cleaning buffer solution is the coupling buffer containing 0.05% tween；
Sealer is the bovine serum albumin(BSA) BSA containing 5~10% defatted milk 1~10%, or 1~5% calf serum, or 1~
The coupling buffer of 5% polyethylene glycol；
It is to contain the 0.05~0.5M of 0.1~1%BSA, the trishydroxymethylaminomethane-hydrochloric acid of pH7.4 to delay to preserve buffer solution
Fliud flushing.
Secondly, the present invention also provides the application method of above-mentioned affine magnetic microsphere, comprises the following steps：
(1) affine magnetic microsphere, is obtained according to above-mentioned preparation method；
(2), affine magnetic microsphere capture carbamate and organophosphorus insecticide
A. in sample carbamate and organophosphorus insecticide extraction
Sampling is originally dissolved in methanol-water or acetonitrile-water, is fully extracted, and extract solution is diluted with PBS after filtering；
B. affine magnetic microsphere obtained in step (1) is taken, Magnetic Isolation abandons supernatant, adds capture buffer solution, met most
Carbamate and organophosphorus insecticide in limits capture sample；Jog is resuspended, Magnetic Isolation, abandons supernatant；Add dilution
Actual sample afterwards, 37 DEG C in shaking table, 180r/min reacts 10~15min；Affine magnetic microsphere capture carbamate and
After organophosphorus insecticide, then through Magnetic Isolation, so as to realize the purification of carbamate and organophosphorus insecticide sample；Removal magnetic
Property separate after supernatant, precipitation is and captures the affine magnetic microsphere of carbamate and organophosphorus insecticide；
The capture buffer solution used in this step is 1 × PBS of pH6.5~7.6；
(3) wash-out of magnetic particle
To adding eluent to be eluted in the affine magnetic microsphere obtained in step (2), room temperature is shaken, Magnetic Isolation,
Supernatant is carbamate and organophosphorus insecticide sample to be checked after purification；
(4) agricultural chemicals quick detection
The sample to be checked that will be obtained in step (3), is added in fluoroscopic examination, enzyme level, scraps of paper detection, realizes amino first
The quick detection of acid esters and organophosphorus insecticide.
Preferably, in step (2) b, the carbamate and organophosphorus insecticide sample after dilution are added, is entered in 37 DEG C
Row is incubated, and reacts 10-15min；Sample after being extracted through wash-out can be directly used for follow-up rapid detection system.
Preferably, step 3) described in eluent use 20%~40% acetonitrile, affine magnetic microsphere is in eluent
Concentration is 0.05~50mg/ml；0.5~5min of shaken at room temperature.
Finally, it is yet another object of the invention to provide a kind of magnetic microsphere, concrete technical scheme is as follows：
A kind of affine magnetic microsphere for purifying and detecting carbamate and organophosphorus insecticide, porous or hollow
Microballoon interior polymeric is magnetic ion, has acetylcholine esterase or anti-carbamate in porous or hollow microsphere Surface coating
With organophosphorus insecticide antibody.
Preferably, the magnetic microsphere particle diameter is in 20-10000nm, and with superparamagnetism.
The present invention can effectively extract the carbamate and organophosphorus insecticide in sample, to solve in the prior art
The result of false positive/false negative brought of complex matrices, improve actual sample Pesticides scene residue detection accuracy and
Reliability, specifically with advantages below：
1st, carbamate and organophosphorus insecticide sample purification efficiency high.Magnetic microsphere has larger specific surface area,
Have the advantages that immobilization capacity is high, the immobilization capacity to biomolecule is very high so that secure purpose biology using this kind of
The magnetic microsphere of macromolecular antibody/enzyme is very high to the separative efficiency of carbamate and organophosphorus insecticide.
2nd, carbamate and organophosphorus insecticide purification separation process are simple and convenient.The inventive method need not be to used
Carrier matrix be magnetic microsphere carry out use preceding activation process and centrifugally operated, can be used directly, it is easy to operate, contracting
The time required to short removal, without using expensive laboratory apparatus.Also can be needed to adjust reaction volume according to experiment, it is whole net
Change process is completed in 15min.
3rd, the carbamate and organophosphorus insecticide for being purified ensure that the correctness and reliability of rapid field detection,
Especially to intractable body no specimen, such as containing the residues of pesticides sample in pigment spinach higher.
4th, residues of pesticides purification run process safety, the present invention is simple to sample treatment, reduces poisonous extract to behaviour
The injury of author and pollution environment.Carbamate and the treatment of organophosphorus insecticide sample clean are also the reaction vessel in closing
In carry out, the contact of agricultural chemicals and people and operator is avoided to greatest extent.
Specific embodiment
Below in conjunction with specific embodiment, invention is described in detail.
With superparamagnetism microballoon as carrier, enzyme/antibody is affinity ligand to the present invention, prepares a kind of affine magnetic microsphere,
For carbamate and the purified treatment of organophosphorus insecticide sample, for follow-up fluophotometer, ELISA, enzyme suppression
System, enzyme colorimetric etc. are detected.Magnetic microsphere refers to particle diameter in 20nm~10000nm, answering with superparamagnetism and high-specific surface area
Close microballoon.
Acetylcholinesterase/anti-carbamate and organophosphorus insecticide are reacted one section by the present invention jointly with magnetic particle
After time, prepare with specific affine magnetic microsphere, wherein anti-carbamate and organophosphorus insecticide can be a class
Or it is a kind of for carbamate and the antibody of organophosphorus insecticide.The sample of coarse extraction is reacted jointly with affine magnetic microsphere
After a period of time, you can realize carbamate and organophosphorus insecticide is affine is adsorbed onto magnetic microsphere surface.Through Magnetic Isolation
Afterwards, you can carbamate and organophosphorus insecticide sample clean are obtained relatively pure carbamate and organic phosphates by realization
Agricultural chemicals sample, is further used for the detection such as fluophotometer, ELISA, enzyme level, enzyme colorimetric.
Following examples are to the solution of the present invention example, experiment condition therein and setting in the form of specific experiment operation
Parameter is not construed as the limitation to basic technical scheme of the present invention.
Embodiment 1
Acid esters and organophosphorus insecticide and magnetic microsphere preparation method is purified and detected in sample, and its in sample
The purification of carbamate and organophosphorus insecticide and detection detailed process are as follows：
1) preparation of affine magnetic microsphere：
A. acetylcholinesterase is fixed in isosulfocyanate radical magnetic microsphere surface：
Take 1mg magnetic microsphere suspensions to be added in a centrifuge tube for 2ml capacity, jog is resuspended.It is placed in magnetic separator
On, Magnetic Isolation abandons supernatant.1ml coupling buffers (PBS of 0.01M, PH 7.4) is added, jog is resuspended.It is placed in magnetic
On property separator, Magnetic Isolation abandons supernatant.Repeat once.By the μ g of acetylcholinesterase 500, it is certain to be configured to concentration
The solution (0.05~10mg/ml) of concentration, takes in suitable amount addition magnetic particle, and 37 DEG C in shaking table, 200r/min reacts
60min。
Reaction is finished, Magnetic Isolation, abandons supernatant.Add the 1ml cleaning buffer solutions (PBS of the pH 7.4 containing 0.05% tween
Buffer solution), the resuspended magnetic grain of jog.Magnetic Isolation, abandons supernatant, removes non-specific adsorption in the enzyme on magnetic particle surface, obtains
Affine magnetic microsphere crude product.
B. the closing of magnetic particle：
The sealer (coupling buffer of 5% defatted milk) of 1ml is added in the magnetic grain of previous step, 37 DEG C in shaking table,
200r/min reacts 2h.Cleaned 3 times with the above-mentioned cleaning buffer solution of 2ml, be finally suspended from 1ml and preserve buffer solution (containing 0.1%NaN3
With the PBS of 0.1%BSA) in, 2~8 DEG C save backup.
2) affine magnetic microsphere capture agricultural chemicals
A. the extraction of sample Pesticides (carbamate and organophosphorus insecticide)：Sample this 5g, be dissolved in 25mL methyl alcohol-
Water, or acetonitrile-water is extracted, and extract solution is diluted with PBS after filtering.
B. by the magnetic microsphere Magnetic Isolation in 1) step, supernatant is abandoned.Add 1ml capture buffer solution (the 1 of pH6.5 ×
PBS), the resuspended magnetic microsphere of jog, Magnetic Isolation abandons supernatant.The sample in a steps is added, 37 DEG C in shaking table,
200r/min reacts 15min.After the completion of affine magnetic microsphere capture carbamate and organophosphorus insecticide, then through magnetic point
From so as to realize the purification of carbamate and organophosphorus insecticide sample.Supernatant after removal Magnetic Isolation, precipitation is and catches
The magnetic microsphere of carbamate and organophosphorus insecticide is obtained.
3) wash-out of magnetic particle：
To step 2) in add 100 μ l eluents (20% acetonitrile) in the immune magnetic particulate that obtains, room temperature concussion 1min,
Magnetic Isolation, supernatant is yellow carbamate and organophosphorus insecticide sample after purification.By the Sample preservation after purification in-
20 DEG C, in case fluophotometer, ELISA or enzyme level, enzyme colorimetric etc. are further detected.
4) step 3) obtain purification after spinach Chlorpyrifos and 'Miduowei ' agricultural chemical sample, be directly used in enzyme colorimetric detection
In system：It is added dropwise 2 and drips extract solution to white tablet, react 10min, red tablet and white medicine is then carried out into contact
Reaction 3min, is negative findings if white tablet is sky blue or identical with blank control group color；If white medicine is shallow
It is blue or it is constant it is blue be then positive findings, show containing residues of pesticides can exceed that safe defining standard, gas phase color need to be used
The analysis methods such as spectrum are further confirmed that.
5) step 3) obtain purification after leek Chlorpyrifos and Methomyl sample, also can be used for enzyme colorimetric detection system
In, improve the accuracy and reliability of detection.The above-mentioned prepare liquids of 2.5mL are added in clean cuvette, then is separately added into 100ul
Enzyme and 100ul developers, mix, and 37 DEG C of water-bath 10min add 100ul substrates, and instrument is put into after mixing carries out sample
Test, instrument shows the absorbance change △ A of 3min automatically, if sample inhibiting rate >=50%, contains in the sample of surface
Some residues of pesticides can exceed that safe defining standard, need to be further confirmed that with analysis methods such as gas-chromatographies.
The inhibition assay result of table 1 (chlopyrifos)
| Sample | Addition concentration level | This method inhibiting rate | Non-purified method |
| 1 | 0.05mg/kg | 48% | 20% |
| 2 | 0.1mg/kg | 60% | 35% |
| 3 | 0.2mg/kg | 76% | 46% |
The inhibition assay result of table 2 (Methomyl)
| Sample | Addition concentration level | This method inhibiting rate | Non-purified method |
| 1 | 0.05mg/kg | 50% | 10% |
| 2 | 0.1mg/kg | 66% | 46% |
| 3 | 0.2mg/kg | 87% | 63% |
If magnetic particle of the invention has the amino or carboxyl of reactivity or the magnetic microsphere of sulfydryl to make with end
It is carrier, because its characteristic is similar, through experiment, the methods for making and using same of this several affine magnetic microsphere can also reference
The method of above example (affine isosulfocyanate radical magnetic magnetic microsphere), only accommodation coupling buffer, envelope
Close agent.Specific optimum choice is as follows：
Coupling buffer is with 0.05~0.5M, and the tris-HCI buffer of pH7.0~8.0 is relatively good,
Sealer is relatively good with the coupling buffer of 1~10% calf serum and 1~5% polyethylene glycol.
The inhibiting rate that the purification method of present invention offer is obtained can be seen that by the inhibition assay result contrast of table 1 and table 2
All there is greatly lifting in chlopyrifos and Methomyl sample, illustrate that the present invention has splendid inhibition.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any one skilled in the art in the technical scope of present disclosure, technology according to the present invention scheme and its
Inventive concept is subject to equivalent or change, should all be included within the scope of the present invention.
Claims (7)
1. a kind of preparation method for purifying and detecting the affine magnetic microsphere of carbamate and organophosphorus insecticide, it is special
Levy and be, comprise the following steps：
A, acetylcholinesterase are fixed on magnetic microsphere surface
Take magnetic particle suspension to be added in centrifuge tube, jog is resuspended；It is placed on magnetic separator, Magnetic Isolation abandons supernatant；Plus
Enter coupling buffer, jog is resuspended；Centrifuge tube is placed on magnetic separator, Magnetic Isolation, abandons standby after supernatant；
Take acetylcholinesterase preparation and obtain stock solution, take in appropriate addition magnetic particle, make magnetic particle and acetylcholine
Esterase or anti-carbamate and organophosphorus insecticide antibody response concentration cause that it reaches maximum coupling for 0.1~10mg/ml
Rate；Reaction is finished, Magnetic Isolation, abandons supernatant；Add cleaning buffer solution, the resuspended magnetic grain of jog；Magnetic Isolation, abandons supernatant, removes
Non-specific adsorption obtains affine magnetic microsphere crude product in the enzyme on magnetic particle surface；
The surface-closed of B, magnetic microsphere
Sealer is added in the affine magnetic particle crude product that step A is obtained, is not combined with enzyme on closing magnetic particle crude product
Site；It is cleaned multiple times with cleaning buffer solution, is finally suspended from preservation buffer solution, 2~8 DEG C of preservations is obtained affine magnetic microsphere.
2. preparation method according to claim 1, it is characterised in that：
The coupling buffer is the PBS of 0.05~0.5M, pH7.0~8.0, or 0.05~0.5M, pH7.0~7.6
Tris-HCI buffer, or 0.05~0.5M, the MES cushioning liquid of pH4~6.
Cleaning buffer solution is the coupling buffer containing 0.05% tween；
Sealer is the bovine serum albumin(BSA) BSA containing 5~10% defatted milk 1~10%, or 1~5% calf serum, or 1~5%
The coupling buffer of polyethylene glycol；
It is 0.05~0.5M, the tris-HCI buffer of pH7.4 containing 0.1~1%BSA to preserve buffer solution.
3. a kind of application method for purifying and detecting the affine magnetic microsphere of carbamate and organophosphorus insecticide, it is special
Levy and be, comprise the following steps：
(1) affine magnetic microsphere, is obtained according to the preparation method described in claim 1 or 2；
(2), affine magnetic microsphere capture carbamate and organophosphorus insecticide
A. in sample carbamate and organophosphorus insecticide extraction
Sampling is originally dissolved in methanol-water or acetonitrile-water, is fully extracted, and extract solution is diluted with PBS after filtering；
B. affine magnetic microsphere obtained in step (1) is taken, Magnetic Isolation abandons supernatant, adds capture buffer solution, meets maximum limit
Carbamate and organophosphorus insecticide in degree capture sample；Jog is resuspended, Magnetic Isolation, abandons supernatant；After adding dilution
Actual sample, 37 DEG C in shaking table, 180r/min reacts 10~15min；Affine magnetic microsphere capture carbamate and organic
After Phosphorus agricultural chemicals, then through Magnetic Isolation, so as to realize the purification of carbamate and organophosphorus insecticide sample；Removal magnetic point
Supernatant after, precipitation is the affine magnetic microsphere for capturing carbamate and organophosphorus insecticide；
The capture buffer solution used in this step is 1 × PBS of pH6.5~7.6；
(3) wash-out of magnetic particle
To adding eluent to be eluted in the affine magnetic microsphere obtained in step (2), room temperature is shaken, Magnetic Isolation, supernatant
Carbamate and organophosphorus insecticide sample to be checked after as purifying；
(4) agricultural chemicals quick detection
The sample to be checked that will be obtained in step (3), is added in fluoroscopic examination, enzyme level, scraps of paper detection, realizes carbamate
With the quick detection of organophosphorus insecticide.
4. application method according to claim 3, it is characterised in that in step (2) b, adds the carbamic acid after dilution
Ester and organophosphorus insecticide sample, are incubated in 37 DEG C, react 10-15min；Sample after being extracted through wash-out can be direct
For follow-up rapid detection system.
5. application method according to claim 3, it is characterised in that step 3) described in eluent use 20%~40%
Acetonitrile, concentration of the affine magnetic microsphere in eluent is 0.05~50mg/ml；0.5~5min of shaken at room temperature.
6. a kind of affine magnetic microsphere for purifying and detecting carbamate and organophosphorus insecticide, it is characterised in that
Porous or hollow microsphere interior polymeric is magnetic ion, has acetylcholine esterase or anti-in porous or hollow microsphere Surface coating
Carbamate and organophosphorus insecticide antibody.
7. magnetic microsphere according to claim 6, it is characterised in that the magnetic microsphere particle diameter has in 20-10000nm
There is superparamagnetism.
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