CN109490293A - A kind of colorimetric reagent box of quick detection chicken yolk antibody - Google Patents
A kind of colorimetric reagent box of quick detection chicken yolk antibody Download PDFInfo
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- CN109490293A CN109490293A CN201811560333.4A CN201811560333A CN109490293A CN 109490293 A CN109490293 A CN 109490293A CN 201811560333 A CN201811560333 A CN 201811560333A CN 109490293 A CN109490293 A CN 109490293A
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- 210000002969 egg yolk Anatomy 0.000 title claims abstract description 48
- 238000001514 detection method Methods 0.000 title claims abstract description 41
- 241000287828 Gallus gallus Species 0.000 title claims abstract description 24
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 15
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims abstract description 41
- 229920000344 molecularly imprinted polymer Polymers 0.000 claims abstract description 32
- 239000002122 magnetic nanoparticle Substances 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 24
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000002245 particle Substances 0.000 claims abstract description 12
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000005406 washing Methods 0.000 claims abstract description 10
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims abstract description 7
- 239000007983 Tris buffer Substances 0.000 claims abstract description 7
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims abstract description 7
- 238000010907 mechanical stirring Methods 0.000 claims abstract description 7
- 238000003756 stirring Methods 0.000 claims abstract description 7
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000001291 vacuum drying Methods 0.000 claims abstract description 7
- 229960003638 dopamine Drugs 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims description 29
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- 235000015165 citric acid Nutrition 0.000 claims description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid group Chemical class C(CC(O)(C(=O)O)CC(=O)O)(=O)O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 3
- 239000013049 sediment Substances 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 2
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims 1
- 235000019800 disodium phosphate Nutrition 0.000 claims 1
- 229920000642 polymer Polymers 0.000 claims 1
- 238000007789 sealing Methods 0.000 abstract 1
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- 239000001257 hydrogen Substances 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
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- 238000012360 testing method Methods 0.000 description 6
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- 230000003197 catalytic effect Effects 0.000 description 4
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- 241000607272 Vibrio parahaemolyticus Species 0.000 description 3
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- 238000004737 colorimetric analysis Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
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- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
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- 241001465754 Metazoa Species 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
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- 230000003248 secreting effect Effects 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- General Health & Medical Sciences (AREA)
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- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention discloses a kind of colorimetric reagent boxes of quickly detection chicken yolk antibody, including a kind of magnetic molecularly imprinted polymer.The magnetic molecularly imprinted polymer, it is prepared by the following method: 1) preparing the Fe of size uniformity3O4 NPs；2) it is added in Tris buffer, ultrasonic disperse adds 1 ~ 5mg Yolk antibody and 2 ~ 5mg dopamine, at room temperature 6 ~ 12h of mechanical stirring；3) it washs, vacuum drying obtains magnetic molecularly imprinted polymer；The magnetic nanoparticle Fe3O4NPs, it is prepared by the following method: with vigorous stirring, by 2.7g FeCl3·6H2O and 7.2 g NaAc are dissolved in 100 mL ethylene glycol；Sealing, in pyroreaction, is cooled to room temperature, Magneto separate, obtains black magnetite particle；Washing, vacuum drying, obtains magnetic nanoparticle Fe3O4 NPs。
Description
Technical field
The invention belongs to Biological Detection fields, and in particular to a kind of colorimetric reagent box of quickly detection chicken yolk antibody.
Background technique
Yolk antibody (immunoglobulin of egg yolk, IgY), also known as Yolk immunoglobulin refer to special
Determine antigenic stimulus birds bone-marrow-derived lymphocyte, plasmablast broken up by B cell and thus secreting specificity antibody enters blood circulation,
It when blood flows through ovary, is gradually accumulated in egg cell, forms specific immunoglobulin, i.e. Yolk antibody.Yolk antibody
IgY is initially considered similar with mammalian immunoglobulin IgG structure, with the further investigation to IgY, it has been found that the two
There is part variation in structure and properties.IgY is similar to IgG basic structure, all by two heavy chains (H chain) and two light chains
(L chain) composition.The difference is that: 1. IgY heavy chain is made of 1 variable region and 4 constant regions, and IgG heavy chain is then
It is made of 1 variable region and 3 constant regions；2. IgY molecular weight is 180 kDa or so, heavy chain molecule amount is 67 ~ 70
KDa, and IgG molecular weight is 150 kDa or so, heavy chain molecule amount is 50 kDa；3. IgG has a hinge area, and
IgY does not have hinge arrangement, therefore its structure is more stable；4. IgY hydrophobicity is strong compared with IgG；5. IgY isoelectric point is in pH value
Between 5.7 ~ 7.6, and IgG isoelectric point, between 6.1 ~ 8.5, IgY IgY in pH value 3.5 ~ 11 is able to maintain surely
Fixed, immunocompetence is almost unchanged.Under room temperature, IgY can be reserved for 6 months its activity and be barely affected, and IgY also has resistance to height
It seeps, the characteristic of high pressure resistant, resistance to multigelation.Chicken yolk antibody makees common one of Yolk antibody.Chicken yolk antibody IgY is chicken
Most important immunoglobulin present in yolk.The antibody has economic easily acquisition, yield height, property stabilization, noresidue, no
The advantages that being also easy to produce drug resistance, having no adverse reaction.Chicken yolk antibody IgY is because having some special constructions and biological property,
It is widely used in the fields such as agricultural, animal husbandry, medical treatment.Currently, the development of IgY product and exploitation have been increasingly becoming countries in the world
The hot spot of research, many commercialization IgY products that some countries develop list in succession, and achieve good result.However, such as
This uses IgY on a large scale, and in secure context, there are many hidden danger.Such as China there is presently no unified quality standard with
Testing process；There may be some diseases propagated through egg in IgY, cause the quality and safety of product irregular；
Immature due to process, most of product is all semifinished product, containing a large amount of foreign protein and lipochondrion, is being infused
It is likely to result in and does not absorb or allergic reaction etc. when penetrating treatment.Therefore, a kind of quick and easy, high sensitivity, high specificity are developed
Chicken yolk antibody IgY detection kit be to promote one of widely used necessary condition of chicken yolk antibody IgY.
Currently, the detection method of antibody mainly includes traditional detection method, label immunoassay, Western blot etc..
Traditional detection method includes precipitation reaction, agglutination test, complement fixation test (CFT) etc., that there are detection times is long for these methods, by
The deficiencies such as antibody interference effect is big, Antibody positive rate is low；Label immunoassay such as enzyme-linked immunosorbent assay (ELISA), the party
Time-consuming for method, the disadvantages of false negative, cross reaction easily occurs.Immunoblotting rule is due to the harm of complex for operation step and reagent is big
Limit its application in quick detection.Therefore a kind of efficient and sensible, fast and convenient, specific high and economic is established
IgY detection kit be production and operation enterprise, Quality Control personnel and government administration section there is an urgent need to food, Environmental security
Powerful guarantee.
It is by surface molecule print technology, magnetic separation technique and to receive that chicken yolk antibody of the present invention, which quickly detects colorimetric reagent box,
Rice enzymatic Colorimetric techniques combine and a kind of Novel colorimetric test agent box for constructing.Nano enzyme is the only of a kind of existing nano material
Characteristic energy, and have the analogue enztme of catalysis.Nano enzyme has the characteristics that high catalytic efficiency, stabilization, economy and prepare with scale,
It has been widely used in the fields such as medicine, chemical industry, food, agricultural and environment.Research shows that Fe3O4Magnetic nano-particle is in peroxide
Change hydrogen (H2O2) in the presence of there is similar oxidase active, when pH=5,3,3', 5,5'- tetramethyl benzidines (TMB) can be catalyzed
Oxidation reaction occurs, generates blue product.Magnetic molecularly imprinted polymer makes cavity portion since object trace forms hole
Fe3O4Magnetic nano-particle is exposed, thus in H2O2With still have in the presence of TMB catalysis oxidation chromogenic substrate generate blue product
Ability.When detecting object, the content of object and the depth of blue form inversely prroportional relationship, so that with the colorimetric sensing
Device Visual retrieval object is possibly realized.Magnetic molecularly imprinted polymer has higher concentration and separation ability and spy as a kind of
The material of anisotropic recognition capability, the present invention are applied in chicken yolk antibody IgY detection, on the one hand substantially increase detection speed
Degree, the accuracy of another aspect testing result also improve a lot, can effectively improve in conjunction with nano enzyme efficient catalytic colorimetric
The sensitivity of detection architecture.
Summary of the invention
Object of the present invention is to be the traditional antibody detection method detection time length of solution, the big, antibody by antibody interference effect
The problems such as recall rate is low, and a kind of efficient and sensible, fast and convenient, specific high and economic detection chicken yolk antibody ratio are provided
Color reagent box.
A kind of magnetic molecularly imprinted polymer, it is prepared by the following method:
1) magnetic nanoparticle Fe3O4NPs is dispersed in ultrapure water, 3 ~ 15min of centrifugation at 2000 ~ 10000rpm, in abandoning
Clearly, it is resuspended with ultrapure water, Magneto separate, obtains sediment, it is dry, obtain the Fe of size uniformity3O4 NPs；
2) Fe3O4 NPs obtained by step 1), be added 8 ~ 15mL, pH=8.5 Tris buffer in, 3 ~ 15min of ultrasonic disperse,
1 ~ 5mg Yolk antibody and 2 ~ 5mg dopamine are added, at room temperature 6 ~ 12h of mechanical stirring；
3) it washs 6 ~ 8 times, vacuum drying obtains magnetic molecularly imprinted polymer；
5min is centrifuged at 7000rpm described in step 1)；
2mg Yolk antibody and 2 mg dopamines are added, at room temperature mechanical stirring 8 in 5 min of ultrasonic disperse described in step 2
h；
Washing described in step 3) is alternately washed with lauryl sodium sulfate and ultrapure water；
The magnetic nanoparticle Fe3O4NPs, it is prepared by the following method:
A. with vigorous stirring, by 2.7g FeCl3·6H2O and 7.2 g NaAc are dissolved in 100 mL ethylene glycol, are obtained molten
Liquid；
B. the obtained solution of step a is transferred in reaction kettle, is sealed, 8 ~ 18 h are reacted at 180 ~ 200 DEG C, it is cold to reaction kettle
But to room temperature, with permanent magnet from reaction solution isolated black magnetite particle；
C. dehydrated alcohol and ultrapure water are used, by alternately washing 6 ~ 7 times of black magnetite particle, is dried in vacuo 12 at 50 ~ 60 DEG C
~ 18 h obtain magnetic nanoparticle Fe3O4NPs；
8h is reacted at 200 DEG C described in step b；
12 h are dried in vacuo described in step c at 60 DEG C.
A kind of colorimetric reagent box of quick detection chicken yolk antibody, it includes: a kind of magnetic molecularly imprinted polymerization
Object, A liquid, B liquid, 1% BSA, TMB, H2O2；
The A liquid is that 21.0 g citric acids are dissolved in 1 L ultrapure water, and B liquid is 71.6 g Na2HPO4It is dissolved in 1 L ultrapure water
In；
Magnetic molecularly imprinted polymer 0.5mg, A liquid 206.77mL, B liquid 242.73mL, 1% BSA 500mL, 50 TMB
µL、H2O2 0.5 µL。
It is by surface molecule print technology, magnetic separation technique and to receive that chicken yolk antibody of the present invention, which quickly detects colorimetric reagent box,
Rice enzymatic Colorimetric techniques combine and a kind of Novel colorimetric test agent box for constructing.Nano enzyme is the only of a kind of existing nano material
Characteristic energy, and have the analogue enztme of catalysis.Nano enzyme has the characteristics that high catalytic efficiency, stabilization, economy and prepare with scale,
It has been widely used in the fields such as medicine, chemical industry, food, agricultural and environment.Research shows that Fe3O4Magnetic nano-particle is in peroxide
Change hydrogen (H2O2) in the presence of there is similar oxidase active, when pH=5,3,3', 5,5'- tetramethyl benzidines (TMB) can be catalyzed
Oxidation reaction occurs, generates blue product.Magnetic molecularly imprinted polymer makes cavity portion since object trace forms hole
Fe3O4Magnetic nano-particle is exposed, thus in H2O2With still have in the presence of TMB catalysis oxidation chromogenic substrate generate blue product
Ability.When detecting object, the content of object and the depth of blue form inversely prroportional relationship, so that with the colorimetric sensing
Device Visual retrieval object is possibly realized.Magnetic molecularly imprinted polymer has higher concentration and separation ability and spy as a kind of
The material of anisotropic recognition capability, the present invention are applied in chicken yolk antibody IgY detection, on the one hand substantially increase detection speed
Degree, the accuracy of another aspect testing result also improve a lot, can effectively improve in conjunction with nano enzyme efficient catalytic colorimetric
The sensitivity of detection architecture.
The present invention provides a kind of magnetic molecularly imprinted polymers, it is prepared by the following method: 1) magnetic Nano
Grain Fe3O4NPs is dispersed in ultrapure water, and supernatant is abandoned in centrifugation, is resuspended, and Magneto separate obtains sediment, dry, and it is more equal to obtain size
One Fe3O4 NPs；2) Fe3O4 NPs obtained by step 1) is added in Tris buffer, and ultrasonic disperse adds 1 ~ 5mg yolk
Antibody and 2 ~ 5mg dopamine, at room temperature 6 ~ 12h of mechanical stirring；3) it washs, vacuum drying obtains magnetic molecularly imprinted polymer；Institute
The magnetic nanoparticle Fe stated3O4NPs, it is prepared by the following method: 1) with vigorous stirring, by 2.7g FeCl3·
6H2O and 7.2 g NaAc are dissolved in 100 mL ethylene glycol；Be transferred in reaction kettle, seal, at 180 ~ 200 DEG C react 8 ~
18 h, are cooled to room temperature, and are separated with permanent magnet, obtain black magnetite particle；3) it washs, vacuum drying obtains magnetic Nano
Particle Fe3O4NPs；A kind of colorimetric reagent box of quick detection chicken yolk antibody, it includes: that described one kind is magnetic molecularly imprinted
Polymer, A liquid, B liquid, 1% BSA, TMB, H2O2；Magnetic molecularly imprinted polymer prepared by the present invention has superparamagnetism, magnetic
Saturation intensity is 68.407 emu/g, is easily isolated, and can specificity capture Yolk antibody；With traditional molecularly imprinted polymer
It compares, novel magnetic molecularly imprinted polymer of the invention not only has molecule by introducing dopamine and magnetic Nano material
The characteristics of engram technology specific recognition object, and have the advantages that quick separating under the action of externally-applied magnetic field, and magnetic
Property nano material in the presence of chromogenic substrate can catalysis substrate colour developing, be very suitable for construct colorimetric reagent box；Of the invention is fast
Speed detection Yolk antibody colorimetric reagent box, for detecting chicken yolk antibody IgY, with easy to operate, instrument and equipment is simple, detection
The advantages that signal is sensitive, and the strength of Magnetic Isolation is big, high-efficient, is particularly suitable for execute-in-place.Colorimetric reagent of the invention
Box can be applied to the quick specific detection of chicken yolk antibody, to achieve the purpose that monitor egg yolk antibody product quality.
Detailed description of the invention
The DLS of the isolated magnetic nanoparticle of Fig. 1 different rotating speeds schemes；(A) 2000rpm and 4000rpm；(B)
5000rpm and 6000rpm；(C) 7000rpm and 10000rpm；
Fig. 2 magnetic nano-particle is saturated magnetic intensity；
The detection process schematic diagram of Fig. 3 chicken yolk antibody.
Specific embodiment
1 magnetic nano-particle Fe of embodiment3O4The preparation of NPs
With vigorous stirring by 2.7g FeCl3·6H2O and 7.2 g NaAc are dissolved in 100 mL ethylene glycol, are obtained homogeneous
Brown yellow solution；Homogeneous brown yellow solution is transferred in Teflon liner reaction kettle and is sealed, and is reacted in 200 DEG C of baking ovens
8 h, are cooled to room temperature to reaction kettle, by permanent magnet from reaction solution isolated black magnetite particle；With anhydrous second
Alternately washing 6 ~ 7 times of black magnetite particle are dried in vacuo 12 h by pure and mild ultrapure water at 60 DEG C, obtain magnetic Nano
Particle Fe3O4NPs；
To obtain the magnetic nanoparticle of size uniformity, this research separates magnetic nanoparticle using centrifugal, using dynamic
State light scattering (DLS) characterizes gained magnetic nanoparticle, as a result as shown in Figure 1, centrifugal rotational speed is from 2000 ~ 10000rpm
Between when converting, obtained magnetic nanoparticle partial size gradually tends to uniform, and when revolving speed is 7000rpm, obtained magnetism is received
Rice grain is the most uniform, and therefore, subsequent experimental separates the size that magnetic nanoparticle is carried out with 7000rpm.
2 magnetic nano-particle Fe of embodiment3O4The preparation of NPs
With vigorous stirring by 2.7g FeCl3·6H2O and 7.2 g NaAc are dissolved in 100 mL ethylene glycol, are obtained homogeneous
Brown yellow solution；Homogeneous brown yellow solution is transferred in Teflon liner reaction kettle and is sealed, and is reacted in 180 DEG C of baking ovens
10 h, are cooled to room temperature to reaction kettle, by permanent magnet from reaction solution isolated black magnetite particle；With anhydrous second
Alternately washing 6 ~ 7 times of black magnetite particle are dried in vacuo 18 h by pure and mild ultrapure water at 60 DEG C, obtain magnetic Nano
Particle Fe3O4 NPs。
3 magnetic nano-particle Fe of embodiment3O4The preparation of NPs
With vigorous stirring by 2.7g FeCl3·6H2O and 7.2 g NaAc are dissolved in 100 mL ethylene glycol, are obtained homogeneous
Brown yellow solution；Homogeneous brown yellow solution is transferred in Teflon liner reaction kettle and is sealed, and is reacted in 200 DEG C of baking ovens
18h is cooled to room temperature to reaction kettle, by permanent magnet from reaction solution isolated black magnetite particle；With anhydrous second
Pure and mild ultrapure water alternately washs black magnetite particle for several times, and is dried in vacuo 15h at 55 DEG C, obtains magnetic nanoparticle
Fe3O4 NPs。
The preparation of 4 magnetic molecularly imprinted polymer of embodiment
Fe prepared by embodiment 13O4 NPs is dispersed in ultrapure water, and packing 7000rpm into 10mL centrifuge tube is centrifuged 5min,
Supernatant is abandoned, is resuspended with ultrapure water, Magneto separate, precipitated product is dried in vacuo, obtains the more uniform Fe of size3O4 NPs；It weighs
10.0mgFe3O4NPs, using ultrasonic disperse in 10.0mL Tris buffer (pH=8.5) 5 min, add 2 mg gold
Staphylococcus aureus Yolk antibody and 2 mg dopamines (DA) use resulting mixed solution 8 h of mechanical stirring at room temperature
SDS(lauryl sodium sulfate) and the washing of ultrapure water alternating is for several times, to remove the template protein of deentrainment, meanwhile, take washing supernatant
Albumen absorbance at ultraviolet 280 is surveyed, is done finally, the magnetic molecularly imprinted polymer after washing is placed in a vacuum drying oven vacuum
It is dry；Prepared magnetic molecularly imprinted polymer has superparamagnetism, and table 1 is shown, at magnetic molecularly imprinted washing the 6th time, magnetic
Property molecularly imprinted polymer supernatant exist substantially without albumen, as shown in Fig. 2, the magnetic molecularly imprinted polymer has superparamagnetic
Property, magnetic saturation intensity is 68.407 emu/g, is easily isolated, and can specificity capture staphylococcus aureus Yolk antibody；
Tris buffer (pH=8.5) configuration method is as follows: 12.141 g Tris are dissolved in the ultrapure water of 90 mL,
PH to 8.5 is adjusted with 5 M NaOH.
The detection of 5 Staphylococcus aureus antibody of embodiment
It takes 0.5 mg with cuniculate magnetic molecularly imprinted polymer, is resuspended in the PBS buffer solution of 500mL, not same amount is added
Staphylococcus aureus Yolk antibody, be incubated for 1 h at room temperature, Magneto separate abandons supernatant, and PBS buffer solution is washed 3 times, is added 500
1% BSA of mL closes 1 h, and 449.5 mL A+ B liquid (pH=5.0), 50 μ L TMB and 0.5 μ L H is added2O2Colour developing 5
Min surveys absorption spectrum in ultraviolet specrophotometer；
The PBS buffer solution configuration method is as follows:
8 g NaCl, 0.2g KCl, 3.63g Na2HPO4.12H2O, 0.24 g KH2PO4It is dissolved in 1 L ultrapure water, with 5 M
NaOH adjusts pH to 7.4；
Confining liquid (the 1 % BSA) configuration method is as follows:
5 g bovine serum albumin(BSA)s (BSA) are taken to be added in 500 mL PBS buffers, matching while using；
The A liquid, the configuration method of B liquid are as follows:
A liquid: 21.0 g citric acids are dissolved in 1 L ultrapure water；B liquid: 71.6 g Na2HPO4It is dissolved in 1 L ultrapure water；A liquid and
B liquid first presses 2.5mL and 2.5mL volume mixture, tests pH, then according to pH value, increases and decreases the volume of A liquid and B liquid accordingly, most
Afterwards when A liquid product be 2.3 mL, B liquid product be 2.7 mL when, pH 5.0, then A liquid and B liquid proportional needed for subsequent experimental be
2.3:2.7；
According to staphylococcus aureus Yolk antibody linear regression curves, the detection range content of chicken yolk antibody is determined；Standard
Curve is y=- 0.5958x+0.9235, and standard concentration has good linear correlation, line in 0.001 ~ 1 μ g range
Property coefficient R2= 0.9911；The method of the present invention detection is stablized, and detection limit can be down to 0.066 μ g, and detection time is short, is stablized
Property is good.0.066 μ g is limited to the lowest detection of staphylococcus aureus Yolk antibody；Table 2 the result shows that, kit of the present invention
Cross reaction not occurring with other ions and protein, specificity is good, and it is easily operated, it is anti-to can be applied to field quick detection yolk
The content of body.
The detection of 6 vibrio parahemolyticus antibody of embodiment
It takes 0.5 mg with cuniculate magnetic molecularly imprinted polymer, is resuspended in the PBS buffer solution of 500mL, not same amount is added
Vibrio parahemolyticus Yolk antibody (VP-IgY), be incubated for 1 h at room temperature, Magneto separate abandons supernatant, and PBS buffer solution is washed 3 times,
500 mL, 1% BSA is added, closes 1 h, 449.5 mL A+ B liquid (pH=5.0), 50 μ L TMB and 0.5 μ L H is added2O2
Develop the color 5 min, surveys absorption spectrum in ultraviolet specrophotometer；
According to vibrio parahemolyticus Yolk antibody linear regression curves, the detection range content of chicken yolk antibody is determined；Standard is bent
Line is y=- 0.5736x+0.9217, and standard concentration has good linear correlation in 0.001 ~ 1 μ g range, linearly
Coefficient R2= 0.9909；The method of the present invention detection is stablized, and detection limit can be down to 0.048 μ g, and detection time is short, stability
It is good.Table 3 shows that with other ions and protein cross reaction does not occur for the invention kit, and specificity is good, easily operated, can
Content applied to field quick detection Yolk antibody.
。
Claims (9)
1. a kind of magnetic molecularly imprinted polymer, it is prepared by the following method:
1) magnetic nanoparticle Fe3O4NPs is dispersed in ultrapure water, and 3 ~ 15min is centrifuged at 2000 ~ 10000rpm, abandons supernatant,
It is resuspended with ultrapure water, Magneto separate, obtains sediment, it is dry, obtain the Fe of size uniformity3O4 NPs；
2) Fe3O4 NPs obtained by step 1), be added 8 ~ 15mL, pH=8.5 Tris buffer in, 3 ~ 15min of ultrasonic disperse,
1 ~ 5mg Yolk antibody and 2 ~ 5mg dopamine are added, at room temperature 6 ~ 12h of mechanical stirring；
3) it washs 6 ~ 8 times, vacuum drying obtains magnetic molecularly imprinted polymer.
2. a kind of magnetic molecularly imprinted polymer according to claim 1, it is characterised in that: described in step 1)
5min is centrifuged under 7000rpm.
3. a kind of magnetic molecularly imprinted polymer according to claim 2, it is characterised in that: surpass described in step 2
Sound disperses 5 min, 2mg Yolk antibody and 2 mg dopamines is added, at room temperature 8 h of mechanical stirring.
4. a kind of magnetic molecularly imprinted polymer according to claim 3, it is characterised in that: washed described in step 3)
It washs, is alternately washed with lauryl sodium sulfate and ultrapure water.
5. a kind of magnetic molecularly imprinted polymer according to claim 4, it is characterised in that: the magnetic nanoparticle
Fe3O4NPs, it is prepared by the following method:
A. with vigorous stirring, by 2.7g FeCl3·6H2O and 7.2 g NaAc are dissolved in 100 mL ethylene glycol, are obtained molten
Liquid；
B. the obtained solution of step a is transferred in reaction kettle, is sealed, 8 ~ 18 h are reacted at 180 ~ 200 DEG C, it is cold to reaction kettle
But to room temperature, with permanent magnet from reaction solution isolated black magnetite particle；
C. dehydrated alcohol and ultrapure water are used, by alternately washing 6 ~ 7 times of black magnetite particle, is dried in vacuo 12 at 50 ~ 60 DEG C
~ 18 h obtain magnetic nanoparticle Fe3O4 NPs。
6. a kind of magnetic molecularly imprinted polymer according to claim 5, it is characterised in that: at 200 DEG C described in step b
Lower reaction 8h.
7. a kind of magnetic molecularly imprinted polymer according to claim 6, it is characterised in that: 60 described in step c
12 h are dried in vacuo at DEG C.
8. a kind of colorimetric reagent box of quickly detection chicken yolk antibody, it includes: a kind of magnetic molecule described in claim 1
Imprinted polymer, A liquid, B liquid, 1% BSA, TMB, H2O2；
The A liquid is that 21.0 g citric acids are dissolved in 1 L ultrapure water, and B liquid is that 71.6 g Na2HPO4 are dissolved in 1 L ultrapure water
In.
9. a kind of colorimetric reagent box of quickly detection chicken yolk antibody according to claim 8, it is characterised in that: described
Magnetic molecularly imprinted polymer 0.5mg, A liquid 206.77mL, B liquid 242.73mL, 1% BSA 500mL, 50 TMB μ L, H2O2
0.5 µL。
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| CN111323596A (en) * | 2020-03-11 | 2020-06-23 | 赵薇 | Staphylococcus aureus detection kit and preparation method thereof |
| CN112649602A (en) * | 2020-06-17 | 2021-04-13 | 吉林大学 | Visual kit for detecting staphylococcus aureus based on immunomagnetic beads |
| CN114113055A (en) * | 2021-11-03 | 2022-03-01 | 湖南农业大学 | Method for detecting norfloxacin based on colorimetric chemical sensing technology |
| CN116328742A (en) * | 2023-04-27 | 2023-06-27 | 大连理工大学 | Core-shell magnetic molecularly imprinted polymer material, preparation method and application |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111323596A (en) * | 2020-03-11 | 2020-06-23 | 赵薇 | Staphylococcus aureus detection kit and preparation method thereof |
| CN111323596B (en) * | 2020-03-11 | 2023-07-04 | 赵薇 | Staphylococcus aureus detection kit and preparation method thereof |
| CN112649602A (en) * | 2020-06-17 | 2021-04-13 | 吉林大学 | Visual kit for detecting staphylococcus aureus based on immunomagnetic beads |
| CN114113055A (en) * | 2021-11-03 | 2022-03-01 | 湖南农业大学 | Method for detecting norfloxacin based on colorimetric chemical sensing technology |
| CN114113055B (en) * | 2021-11-03 | 2024-01-30 | 湖南农业大学 | Method for detecting norfloxacin based on colorimetric chemical sensing technology |
| CN116328742A (en) * | 2023-04-27 | 2023-06-27 | 大连理工大学 | Core-shell magnetic molecularly imprinted polymer material, preparation method and application |
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