CN107132346A - A kind of ferroso-ferric oxide immune nanometer magnetic bead and its preparation method and application - Google Patents
A kind of ferroso-ferric oxide immune nanometer magnetic bead and its preparation method and application Download PDFInfo
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- CN107132346A CN107132346A CN201710366724.1A CN201710366724A CN107132346A CN 107132346 A CN107132346 A CN 107132346A CN 201710366724 A CN201710366724 A CN 201710366724A CN 107132346 A CN107132346 A CN 107132346A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Abstract
The present invention is a kind of ferroso-ferric oxide immune nanometer magnetic bead and its preparation method and application.A kind of preparation method of ferroso-ferric oxide immune nanometer magnetic bead, comprises the following steps：(1) nanometer magnetic bead is prepared；(2) surface modification of nanometer magnetic bead；(3) preparation of immunomagnetic beads.The present invention also disclosed the application of the ferroso-ferric oxide immune nanometer magnetic bead.A kind of ferroso-ferric oxide immune nanometer magnetic bead of the present invention and its preparation method and application, the preparation method can not be limited by nitrogen, and improve Fe3O4Magnetic property；By to Fe3O4Nanometer magnetic bead surface is modified, and magnetic macromolecular microsphere is made, and is combined with nut allergies serum antibody and immunomagnetic beads is made, and adds the species and preparation method of immune nanometer magnetic bead；Immunomagnetic beads is combined with ELISA, without expensive devices such as hexavalent chrome bio-removal, mass spectrographs, cost is low, prediction can be improved with assessing the accuracy of Cross-reactivity with effective detection nut allergies albumen.
Description
Technical field
The invention belongs to the technical field of immune nanometer magnetic bead, and in particular to a kind of Fe3O4Immune nanometer magnetic bead and its preparation
Methods and applications.
Background technology
With the fast development of food service industry, increasing environmental pollution, allergenic foods are more and more, the incidence of disease of food irritability
Increasingly sharpen with popularity, food allergy disease has turned into emerging, public character, a global health problem.Its
In, the allergy of nut based article is even more to occupy very big proportion, because nut based food has high nutritive value and uniqueness
Local flavor, is well received by consumers as remedy diet and leisure food, but nut fruits food irritability is related to phagopyrism
90%.Therefore the presence of SD anaphylactogen is the most effective way for avoiding autopath from eating potential anaphylactogen on food labelling
Footpath, the detection of nut allergen is necessary.After existing detection method is essentially all separation and enrichment by early stage,
Again by hexavalent chrome bio-removal, mass spectrometric analysis method, and then confirm target proteinses, detection speed is slow, high to equipment requirement, cost
It is high.
Nanometer magnetic bead has wide because of its unique magnetism characteristic, such as superparamagnetism and high-coercive force in biomedical sector
Wealthy application prospect and receive much concern.Nanometer magnetic bead is in cell separation, immobilised enzymes, immunodiagnosis and neoplasm targeted therapy, DNA
Be widely used in terms of separation and nucleic acid hybridization, wherein nanometer magnetic bead and antibody binding are made into immunomagnetic beads, because its
Application in terms of immune detection, cell separation and biological macromolecule purifying, by the widely studied of people.Immunomagnetic beads
(Immunomagnetic Beads, IMB) is the magnetic microsphere that pan coating has antibody (or part).Immunomagnetic beads is main by carrying
Body microballoon and immunoligand two parts composition.Carrier microballoons include the core of small metal particles composition and are wrapped in core outer layer
The high polymer material of modification is (such as：Polystyrene, Aminosilylation reagent, glutaraldehyde etc.), outermost layer is that function basic unit is
Immunoligand, generally comprises antigen, antibody, biology enzyme, cell, agglutinin, metal ion and organic matter etc..The system of immunomagnetic beads
During standby, people have done substantial amounts of research.1979, John Ugelstad etc. were successfully prepared for a kind of uniformity and grain
The suitable polystyrene microsphere of degree, after being magnetized and being connected with antibody, the splendid immune magnetic of cell effect is separated as a kind of
Pearl -- Dynabeads.So far, China does not produce the ability of high-quality immunomagnetic beads still, and import magnetic bead is expensive, because
And limit application of the immunomagnetic beads in China's field of biomedical research.Secondly, immunomagnetic beads preparation difficulty is larger, to obtain
Obtain homogeneous, spherical, superparamagnetism and be easy to protein-bonded magnetic bead and be difficult, but with the reach of science, immunomagnetic beads is whole
The effect of individual medical domain especially clinically can be increasing.
In numerous nano materials, Fe3O4Nano particle is with its excellent property and widely applies and receives much concern.
Fe3O4The preparation method of nano particle mainly includes coprecipitation, thermal decomposition method etc..Wherein, coprecipitation is that having two kinds or many
Precipitating reagent is added in the solution for planting cation, the solution of this multicomponent system can obtain uniform component after precipitation reaction
Precipitation.The method most generally used at present is by equation：Fe2++2Fe3++8OH-=Fe3O4↓+4H2O is what principle was carried out,
According to certain mol proportion example configuration Fe2+, Fe3+The aqueous solution, excessive NaOH solution is added thereto as precipitating reagent, certain
Under the conditions of Fe is made3O4Nanometer magnetic bead.But can just be carried out under the protection of nitrogen gas in preparation process, with certain
Restricted, the nanometer magnetic bead saturation magnetization prepared is low.
In view of this, it is necessary to propose a kind of new Fe3O4Immune nanometer magnetic bead and its preparation method and application.
The content of the invention
It is an object of the invention to provide a kind of ferroso-ferric oxide immune nanometer magnetic bead and preparation method thereof, this method can be with
Do not limited by hydrogen peroxide or nitrogen, improve Fe3O4The magnetic property of nanometer magnetic bead, is repaiied by the surface to nanometer magnetic bead
Decorations, immunomagnetic beads can be made by being combined with nut allergies serum antibody.
To achieve these goals, the technical scheme used is：
A kind of preparation method of ferroso-ferric oxide immune nanometer magnetic bead, comprises the following steps：
(1) nanometer magnetic bead is prepared：
By Fe2+Molysite and Fe3+Iron salt dissolved in water, obtain iron salt solutions；The Fe2+Molysite in Fe2+And Fe3+
Molysite in Fe3+Mol ratio be 1:1-2；
At a temperature of 65-75 DEG C, first ultrasound iron salt solutions 4-6 minutes, add excessive sodium hydroxide solution and stir
Mix 25-35 minutes, then stand crystallization 2-3 hours, obtain ferroso-ferric oxide precipitation；
Ferroso-ferric oxide precipitation is separated by Magneto separate, distillation water washing is first used 2-3 times, then absolute ethyl alcohol washing
2-3 times, obtain ferriferrous oxide nano magnetic bead absolute ethyl alcohol mixed liquor；
(2) surface modification of nanometer magnetic bead：
Ferriferrous oxide nano magnetic bead absolute ethyl alcohol mixed liquor is diluted into 5-7 times, the nanometer after must diluting with absolute ethyl alcohol
Magnetic bead absolute ethyl alcohol mixed liquor；
Nanometer magnetic bead absolute ethyl alcohol mixed liquor after sonic oscillation dilution adds silylating reagent, room temperature after 25-35 minutes
Lower stirring 6-8 hours, Magneto separate obtains silanization ferriferrous oxide nano magnetic bead；The silylating reagent is APTES；
Silanization ferriferrous oxide nano magnetic bead is washed with ethanol solution 2-3 times, obtain silanization ferroso-ferric oxide
Nanometer magnetic bead absolute ethyl alcohol mixed liquor；
Silanization ferriferrous oxide nano magnetic bead absolute ethyl alcohol mixed liquor 2-3 is washed with PBS again
It is secondary, obtain magnetic bead mixed liquor；
Glutaraldehyde solution is added into magnetic bead mixed liquor, vibration crosslinking 1.5-2.5 hours, then Magneto separate at room temperature is discarded
Supernatant, obtains amination ferriferrous oxide nano magnetic bead mixed liquor mixed liquor；
(3) preparation of immunomagnetic beads：
Amination ferriferrous oxide nano magnetic bead mixed liquor mixed liquor is coated with antibody, fixed 1.5-2.5 is vibrated at room temperature
Hour, Magneto separate, the amination ferriferrous oxide nano magnetic bead mixed liquor after must being coated with is distinguished with distilled water and PBS
Washing 3-4 times, obtains ferroso-ferric oxide immune nanometer magnetic bead；The antibody is nut allergies mouse positive serum.
Further, in the step (1), Fe in iron salt solutions2+Molar concentration be 0.2-0.35mol/L, Fe3+'s
Molar concentration is 0.2-0.4mol/L；
The molar concentration of the sodium hydroxide solution is 0.5-1mol/L.
Further, the Fe2+Molysite be FeSO4·7H2O, Fe3+Molysite be FeCl3·6H2O。
Further, in the step (2), the APTES and the nanometer magnetic bead absolute ethyl alcohol mixed liquor after dilution body
Product is than being 1:350-400；
The consumption of the glutaraldehyde solution is 3-5 times of silanization ferriferrous oxide nano magnetic bead absolute ethyl alcohol mixed liquor.
Further, the volume fraction of the glutaraldehyde solution is 3-7%.
Further, in the step (3), the volume ratio of antibody and amination ferriferrous oxide nano magnetic bead mixed liquor
0.8-1.2:1。
Further, in the step (3), the dilution factor of antibody is 1:600-1000.
Further, the dilution factor of the antibody is 1:800.
A further object of the invention, is the application for providing above-mentioned ferroso-ferric oxide immune nanometer magnetic bead, and the application can
With effective detection nut allergies albumen.
To achieve these goals, the technical scheme used is：
A kind of application of ferroso-ferric oxide immune nanometer magnetic bead, it is characterised in that including：
Antigen is detected using enzyme linked immunological competition law；Wherein, the antigen is nut allergies albumen, and the first antibody is
Ferroso-ferric oxide immune nanometer magnetic bead.
Further, the secondary antibody is sheep anti mouse IgE-HRP antibody.
Compared with prior art, beneficial effect is：
1st, a kind of ferroso-ferric oxide immune nanometer magnetic bead of the present invention and preparation method thereof, this method can not be by nitrogen
The limitation of gas, obtained ferriferrous oxide nano magnetic bead has very high saturation magnetization, also improves ferroso-ferric oxide
Magnetic responsiveness, and show weak ferromagnetism feature, improve the magnetic property of ferroso-ferric oxide.
2nd, a kind of ferroso-ferric oxide immune nanometer magnetic bead of the present invention and preparation method thereof, this method is to four oxidations three
Iron nanometer magnetic bead surface is modified, so that magnetic macromolecular microsphere is made, is combined to be made with nut allergies serum antibody and exempted from
Epidemic disease magnetic bead, adds the species and preparation method of immune nanometer magnetic bead.
3rd, a kind of application of ferroso-ferric oxide immune nanometer magnetic bead of the present invention, makes immunomagnetic beads and ELISA
With reference to without expensive devices such as hexavalent chrome bio-removal, mass spectrographs, cost is low, can improve pre- with effective detection nut allergies albumen
The accuracy with assessing Cross-reactivity is surveyed, the implementation for China's food allergen tag identifier system provides detection technique
Method.
Brief description of the drawings
The Fe that Fig. 1 obtains for step (1) in embodiment 23O4The X ray diffracting spectrum of nanometer magnetic bead.
The Fe that Fig. 2 obtains for step (1) in embodiment 43O4The X ray diffracting spectrum of nanometer magnetic bead.
Embodiment
In order to which a kind of ferroso-ferric oxide immune nanometer magnetic bead of the invention and its preparation method and application is expanded on further, reach
It is expected that goal of the invention, below in conjunction with preferred embodiment, to according to a kind of ferroso-ferric oxide immune nanometer magnetic bead proposed by the present invention
And its preparation method and application, its embodiment, structure, feature and its effect are described in detail as after.In the description below
In, what different " embodiment " or " embodiment " referred to is not necessarily same embodiment.In addition, the spy in one or more embodiments
Determining feature, structure or feature can be combined by any suitable form.
Before a kind of ferroso-ferric oxide immune nanometer magnetic bead of the invention and its preparation method and application is elaborated, having must
Associated materials, method for being referred in the present invention etc. are described further, to reach more preferable effect.
The present invention prepares nanometer magnetic bead using chemical coprecipitation, and it is added in the solution for have two or more cations
Enter precipitating reagent, the solution of this multicomponent system can obtain the precipitation of uniform component after precipitation reaction, be by equation：
Fe2++2Fe3++8OH-=Fe3O4↓+4H2O is what principle was carried out.Under without the restrictive condition such as nitrogen gas shield or deoxygenated water, press
According to certain mol proportion example configuration Fe2+、Fe3+The aqueous solution, excessive NaOH solution is added thereto as precipitating reagent, in certain bar
Fe is made under part3O4Nanometer magnetic bead.
The present invention is using the coupling agent modification in organic molecule method of modifying, just in nano magnetic after being handled by silane agent
Bead surface introduces reactive group, and further chemo-selective is provided for its function.The amido modified magnetic bead glutaraldehyde of the present invention
Activation, two aldehyde radicals using glutaraldehyde are as bridge, and the aldehyde radical of one end is combined with the active group of magnetic bead surfaces, and another aldehyde
Base is then combined with the amino in positive nut allergies serum antibody molecule, so that antibody coupling is to magnetic bead surfaces, in detection,
Uncombined site is closed with bSA, so that immunomagnetic beads can be used for detecting nut allergen.
Green vitriol, molecular formula is FeSO4·7H2O, molecular weight 278.01, pale bluish green monoclinic crystal, alias
Green vitriol, soluble in water, insoluble in ethanol, fusing point is 64 DEG C, and boiling point is 330 DEG C, is heated to 70-73 DEG C and loses 3 molecular waters, extremely
80-123 DEG C loses 6 molecular waters, and ferric subsulfate is transformed into more than 156 DEG C.
Iron(III) chloride hexahydrate, molecular formula is FeCl3·6H2O, molecular weight is 270.2962, and Chinese nickname has ferric trichloride
(six water), high-purity ferric trichloride, ferric trichloride (III) hexahydrate, Iron trichloride hexahydrate, ferric chloride (FeCl36H2O), are crocus
Crystal, 37 DEG C of fusing point, 280-285 DEG C of boiling point is soluble in water, insoluble in glycerine, is soluble in methanol, ethanol, acetone, ether.
Ferroso-ferric oxide, chemical formula Fe3O4, iron oxide black, magnet, magnet, black iron are commonly called as, to have magnetic black
Crystal, therefore also known as magnetic iron oxide.This material is a kind of mixed valence oxide of iron, and fusing point is 1597 DEG C, and density is
5.17g/cm3, it is dissolved in acid solution, the organic solvent such as water insoluble, aqueous slkali and ethanol, ether.
Ethanol (Ethanol) is commonly called as alcohol, is a kind of organic matter, skeleton symbol CH3CH2OH or C2H5OH, molecular formula
C2H6O, is most common monohydric alcohol.Ethanol liquid density is 0.789g/cm3, alcohol gas density is 1.59kg/m3, relative point
Protonatomic mass is 46.07g/mol, and boiling point is 78.4 DEG C, and fusing point is -114.3 DEG C.Straight alcohol is the liquid of water white transparency, there is special
Fragrance, it is volatile.
Phosphate buffer (Phosphate Buffered Saline, abbreviation PBS) is conventional to be ground for biology
The cushioning liquid studied carefully, for molecular cloning and cell culture, main component is potassium dihydrogen phosphate, disodium hydrogen phosphate, chlorination
Sodium and potassium chloride, its compound method are different, and pH value is different, and the biological action of performance is also incomplete same.It is mainly used in one
Experiment is not influenceed to adjust pH value in the case of reacting in a little chemical experiments, to allow the chemical reaction of experiment to enter at optimum conditions
OK, partly processed for food or feedstuff industry, such as food or feed, the board-washing of Elisa methods during mycotoxin is examined
Or the dilution buffer in Food microbe testing.The preferential phosphate buffer from pH=7.4 in the present invention.
APTES is a kind of chemical substance, and 3- aminopropyl triethoxysilanes, molecular formula is H2NCH2CH2CH2Si
(OC2H5)3, 217 DEG C of boiling point, refractive index 1.420 is light yellow liquid, sucks poisonous, facile hydrolysis, releases ethanol, and generation is corresponding
Silanol condensation thing；C-NH in molecule2Amino can be carried out with acid, carboxylate, aldehyde, ketone, halogenated hydrocarbons, acid amides and nitrile etc. in key
Reaction.APTES can be for synthesizing organo-silicon intermediate and high-molecular compound, it is also possible to make silane coupler.
Glutaraldehyde, molecular formula is C5H8O2, molecular weight is 100.1158, fusing point：- 5 DEG C, boiling point：189 DEG C, flash-point：66 DEG C,
Density：0.947g/cm3, the colourless transparent oil liquid with penetrating odor is dissolved in hot water；It can be processed as food industry
Auxiliary agent, bacterium disinfectant, tanning extracts, timber preservative, medicine and Polymer Synthesizing raw material etc..
Magnetic separation technique is a kind of technology that material is carried out to magnetic field processing, is divided using the magnetic of particle in liquid
From the application of the technology has penetrated into every field.In the present invention, by applying externally-applied magnetic field, magnetic bead is just pooled to magnetic
On one side, magnetic bead can then be isolated by siphoning away supernatant for field.
Enzyme-linked immunosorbent assay method, abbreviation ELISA, or ELISA method.Its center be exactly allow antibody with
Multienzyme complex is combined, and is then detected by developing the color.Its general principle is 1. to make antigen or antibody binding to certain solid phase carrier
Surface, and keep its immunocompetence；2. antigen or antibody is made to connect into enzyme-labelled antigen or antibody, this enzyme-labelled antigen with certain enzyme
Or antibody had both retained its immunocompetence, the activity of enzyme is retained again.When determining, by inspection sample, (measure antibody therein is anti-
It is former) and enzyme-labelled antigen or antibody reacted by the antigen or antibody of different step and surface of solid phase carriers.With the method for washing
The antigen antibody complex formed on solid phase carrier is set to be separated with other materials, finally with reference to the enzyme amount and mark on solid phase carrier
The amount of tested substance is into certain ratio in this.After the substrate for adding enzyme reaction, substrate is changed into color products, product by enzymatic
Amount it is directly related with the amount of tested substance in sample, therefore qualitative or quantitative analysis can be carried out according to the depth of color reaction.
Because the catalysis frequency of enzyme is very high, thus can greatly iodine effect so that assay method reaches very high susceptibility.It
Type there is double antibody sandwich method, double site one-step method, indirect method to survey antibody, competition law, prize law etc., of the present invention one
Plant the Fe for being applied to detection nut allergies albumen3O4Immune nanometer magnetic bead, uses competition law.
Competition law can be used for determining antigen, it can also be used to determine antibody.It is used to detect antigen in the present invention, operating procedure is such as
Under:(1) specific antibody is connected with solid phase carrier, forms insolubilized antibody；Washing；(2) treat in test tube plus by inspection sample and a certain amount of
The mixed solution of enzyme-labelled antigen, is allowed to react with insolubilized antibody；Such as by inspection sample in it is nonantigenic, then enzyme-labelled antigen can successfully with
Insolubilized antibody is combined；Such as contained antigen by inspection sample, then combined with enzyme-labelled antigen with same chance with insolubilized antibody, competed
The chance that enzyme-labelled antigen is combined with solid phase carrier has been accounted for property, the binding capacity of enzyme-labelled antigen and solid phase carrier is reduced；With reference to
Only add enzyme-labelled antigen in pipe, after insulation, the combination of enzyme-labelled antigen and insolubilized antibody is up to most sufficient amount；Washing；Plus substrate (3)
Colour developing:It is most with reference to the enzyme-labelled antigen in pipe due to combination, therefore color is most deep；With reference to pipe color depth with treating test tube color depth
Difference, represent by inspection sample antigen amount；Treat that test tube color is lighter, represent that antigenic content is more in sample.The present invention is coating
Antigen, the indirect competitive that ELIAS secondary antibody is carried out, this method is advantageous in that first antibody is not present operation influence (does not recognize
For be marked or other processing), a resistant activity is good, and the coating of antigen is typically not in that excessive coating causes IC50
It is worth higher, the situation of sensitivity decrease.
Coating, refers to the process by antigen or antibody fixation, phosphate buffer, pH that conventional coating buffer is pH=7.4
=9.6 carbonate buffer solution, pH=7-8 Tris-HCL buffer solutions.Only with one kind therein in the embodiment of the present invention
Or it is several, can it be served the same role using with use.
In board-like ELISA, the PBS that conventional cleaning solution is the pH=7.4 containing 0.05% Tween-20 is (referred to as
PBST)。
It is to allow a large amount of not phases with the irrelevant protein solution of high concentration coated process again that closing, which is after coating,
The protein of pass fills these spaces, so as to repel adsorbing again for interfering material in the step after ELISA.Conventional sealer
Have：0.05%-0.5%BSA (bovine serum albumin(BSA)), 10% calf serum, 1% gelatin, skimmed milk power etc., the present invention are preferential
From BSA；BSA containing 0.5g and 100ml cleaning solution in confining liquid per 100ml.
Terminate liquid, conventional terminate liquid is sulfuric acid, the general sulfuric acid solution using 2mol/L in board-like ELISA.
Substrate solution in the present invention contains pH=7.4 PBS, o-phenylenediamine and hydrogen peroxide, it is necessary to existing with existing
Match somebody with somebody, and be kept in dark place.The volume ratio of pH=7.4PBS buffer solutions and hydrogen peroxide is 1 in substrate solution：0.0015, per 10ml
There is o-phenylenediamine 0.004g in pH=7.4 PBS.
OD is the abbreviation of optical density (optical density), represents the optical density that detected material is sponged, is detection side
Proper noun in method, detection unit represents that OD=lg (1/trans), wherein trans is the printing opacity value of detectable substance with OD values.
OD450It is certain solution in the light measurement how much of 450nm wavelength absorptions.
Secondary antibody is the antibody of energy and antibody binding, i.e. antibody.Mainly for detection of the presence of antibody.Secondary antibody
It should select preferential from sheep anti mouse IgE-HRP antibody in the first antibody identical source of species with using, the present invention.
Heretofore described washing sensing is treated to add cleaning solution in washings, is well mixed, by Magneto separate, magnetic bead collects
To magnetic field on one side, siphon away supernatant then, isolate magnetic bead.But magnetic bead can not remove cleaning solution completely, may eventually form and contain
There is the mixed liquor of cleaning solution and magnetic bead.
In perfect solution, the volume of solution has an additivity, cumulative volume be equal to the volume before all compositions are mixed and.Now
Volume fraction be alternatively referred to as volumetric concentration, refer to certain composition percentage shared in cumulative volume.
It is excessive, refer in chemical reaction and add a certain reactant, another reactant is all reacted, itself also have surplus
It is remaining.
After associated materials, method referred in having understood the present invention etc., below in conjunction with specific embodiments, to this
A kind of ferroso-ferric oxide immune nanometer magnetic bead and its preparation method and application is invented to be further described in detail：
Embodiment 1.
A kind of ferroso-ferric oxide immune nanometer magnetic bead and its preparation method and application, comprises the following steps：
(1) nanometer magnetic bead is prepared：
1. 2.78g FeSO is weighed4·7H2O and 2.703g FeCl3·6H2O, is dissolved in 50ml pure steaming water, obtains
Iron salt solutions；(FeSO4·7H2The amount of O materials is 2.78 ÷ 278.01 ≈ 0.01mol, Fe2+Molar concentration be 0.2mol/L,
FeCl3·6H2The amount of O materials is 2.703 ÷ 270.2962 ≈ 0.01mol, Fe3+Molar concentration be 0.2mol/L)
2. at a temperature of 65 DEG C, first ultrasound iron salt solutions 6 minutes, place into rotor and are placed in high-speed stirring on magnetic stirring apparatus
Mix 25 minutes, while 160ml 0.5mol/L sodium hydroxide solutions are added dropwise, with NaOH addition, crystallization occurs in reaction solution
Black level value peony is precipitated, then stands crystallization 2 hours, obtains Fe3O4Precipitation；
3. Magneto separate frame is used by Fe3O4Precipitation is separated, and first uses distillation water washing 2 times, then absolute ethyl alcohol is washed 2 times, is obtained
Fe3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor；
(2) surface modification of nanometer magnetic bead：
1. 25ml Fe is measured3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor, is diluted to 150ml with absolute ethyl alcohol, obtains after dilution
Nanometer magnetic bead absolute ethyl alcohol mixed liquor；
2. the nanometer magnetic bead absolute ethyl alcohol mixed liquor after sonic oscillation dilution adds 0.4ml APTES, room after 30 minutes
The lower stirring of temperature 7 hours, is separated with Magneto separate frame, obtains silanization Fe3O4Nanometer magnetic bead (APTES with dilution after nanometer magnetic bead it is anhydrous
The volume ratio of alcohol mixeding liquid is 0.4:150=1:375)；
3. silanization Fe is washed with ethanol solution3O4Nanometer magnetic bead 2 times, obtains silanization Fe3O4Nanometer magnetic bead is anhydrous
Alcohol mixeding liquid；
4. 1ml silanization Fe is drawn3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor, is washed with pH=7.4 PBS
Silanization Fe3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor 3 times, obtains magnetic bead mixed liquor；
5. the volume fraction that 4ml is added into magnetic bead mixed liquor is 5% glutaraldehyde solution, and vibration crosslinking 2 is small at room temperature
When, then separated with Magneto separate frame, abandoning supernatant obtains amination Fe3O4Nanometer magnetic bead mixed liquor；(glutaraldehyde solution and silanization
Fe3O4The volume ratio of nanometer magnetic bead absolute ethyl alcohol mixed liquor is 4:1)
(3) preparation of immunomagnetic beads：
It is 1 with 0.8ml dilution factor：600 nut allergies mouse positive serum is coated with 1ml amination Fe3O4Nano magnetic
Pearl mixed liquor, vibrates fix 1.5-2.5 hours at room temperature, then is separated by magnetic separator, supernatant is discarded after must being coated with
Amination Fe3O4Nanometer magnetic bead mixed liquor, is washed 4 times, obtains Fe respectively with distilled water and pH=7.4 PBS3O4Exempt from
Epidemic disease nanometer magnetic bead；
(4)Fe3O4Immune nanometer magnetic bead detects nut allergies albumen by ELISA
The nut proteins of different dilution factors, by taking walnut allergen protein as an example：
1 times of dilution：For walnut protein extract solution stoste.
2 times of dilutions：Walnut protein liquid 2ml is drawn, pH=7.4 PBS 2ml is added and mixes.
4 times of dilutions：2 times of dilution 2ml are drawn, adds after pH=7.4 PBS 2ml and mixes.
8 times of dilutions：4 times of dilution 2ml are drawn, adds after pH=7.4 PBS 2ml and mixes.
16 times of dilutions：8 times of dilution 2ml are drawn, adds after pH=7.4 PBS 2ml and mixes.
1. it is separately added into walnut protein extract solution stoste and is diluted to 2 times, 4 times, 8 times, 16 times of protein liquid, sets simultaneously
PBS is negative control, 100 μ L/ holes；The PBS for adding pH=7.4 is coated with, 100 μ L/ holes, at 37 DEG C
It is incubated in insulating box one hour, 4 DEG C overnight.
2. close：With cleaning solution board-washing 3 times, confining liquid is added, 300 μ L/ holes, 37 DEG C of concussions are incubated 2 hours on shaking table,
Board-washing.(cleaning solution is：The PBS of pH=7.4 containing 0.05% Tween-20；Contain 0.5g in confining liquid per 100ml
BSA and 100ml cleaning solution)
3. compete：50 μ L walnut protein extract solution stostes are added per hole and 2 times, 4 times, 8 times, 16 times of protein liquid is diluted to
With 50 μ L Fe3O4Immune nanometer magnetic bead, 37 DEG C of concussions are incubated 2 hours, use cleaning solution board-washing；Then 100 μ L sheep is added per hole
Anti- mouse IgE-HRP antibody, 37 DEG C of concussions are incubated 1 hour, use cleaning solution board-washing.(cleaning solution is：PH containing 0.05% Tween-20
=7.4 PBS)
4. develop the color：The substrate solution of 50 μ L Extemporaneous is added per hole and is sufficiently mixed；After 37 DEG C of lucifuges develop the color 20 minutes,
50 μ L terminate liquids are added per hole and are sufficiently mixed, then OD is determined with ELIASA450.(the preparation of substrate solution：By 10ml pH=
The hydrogen peroxide mixing of 7.4 PBS, 0.004g o-phenylenediamine and 15 μ L, matching while using is kept in dark place；Terminate liquid：
2mol/l sulfuric acid solution)
A kind of ferroso-ferric oxide immune nanometer magnetic bead described in the present embodiment and its preparation method and application, the preparation method
It can not be limited by nitrogen, and improve Fe3O4Magnetic property；By to Fe3O4Nanometer magnetic bead surface is modified,
Magnetic macromolecular microsphere is made, is combined with nut allergies serum antibody and immunomagnetic beads is made, add immune nanometer magnetic bead
Species and preparation method；Immunomagnetic beads is set to be combined with ELISA, without expensive devices such as hexavalent chrome bio-removal, mass spectrographs,
Cost is low, can be improved prediction with effective detection nut allergies albumen with assessing the accuracy of Cross-reactivity, be China's food
The implementation of anaphylactogen tag identifier system provides detection technique method.
Embodiment 2.
A kind of ferroso-ferric oxide immune nanometer magnetic bead and its preparation method and application, comprises the following steps：
(1) nanometer magnetic bead is prepared：
1. 2.78g FeSO is weighed4·7H2O and 5.406g FeCl3·6H2O, is dissolved in 50ml pure steaming water, obtains
Iron salt solutions；(FeSO4·7H2The amount of O materials is 2.78 ÷ 278.01 ≈ 0.01mol, Fe2+Molar concentration be 0.2mol/L,
FeCl3·6H2The amount of O materials is 5.406 ÷ 270.2962 ≈ 0.02mol, Fe3+Molar concentration be 0.4mol/L)
2. at a temperature of 75 DEG C, first ultrasound iron salt solutions 4 minutes, are placed into S21-I constant temperature blender with magnetic force at a high speed
Stirring 35 minutes, while 180ml 0.75mol/L sodium hydroxide solutions are added dropwise, with NaOH addition, crystallization goes out in reaction solution
Existing black level value peony precipitation, then stand crystallization 3 hours, obtain Fe3O4Precipitation；
3. Magneto separate frame is used by Fe3O4Precipitation is separated, and first uses distillation water washing 3 times, then absolute ethyl alcohol is washed 3 times, is obtained
Fe3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor；
Take out 10 1mlFe3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor is put into centrifuge tube, is whetheing there is externally-applied magnetic field situation
Lower its magnetic of observation.By obtained Fe3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor is divided in plate, is freezed, is frozen in refrigerator
12 hours are dried in freeze drier afterwards, nanometer magnetic bead powder is obtained, observation is taken out, spreads out in microscope and x-ray powder
Penetrate and its form is observed under instrument, obtain collection of illustrative plates as shown in Figure 1.
(2) surface modification of nanometer magnetic bead：
1. 50ml Fe is measured3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor, is diluted to 250ml with absolute ethyl alcohol, obtains after dilution
Nanometer magnetic bead absolute ethyl alcohol mixed liquor；
2. the nanometer magnetic bead absolute ethyl alcohol mixed liquor after sonic oscillation dilution adds 0.7ml APTES, room after 25 minutes
The lower stirring of temperature 6 hours, is separated with Magneto separate frame, obtains silanization Fe3O4Nanometer magnetic bead；(APTES with dilution after nanometer magnetic bead without
The volume ratio of water-ethanol mixed liquor is 0.7:250=1:357.143)；
3. silanization Fe is washed with ethanol solution3O4Nanometer magnetic bead 2 times, obtains silanization Fe3O4Nanometer magnetic bead is anhydrous
Alcohol mixeding liquid；
4. 4ml silanization Fe is drawn3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor, is washed with pH=7.4 PBS
Silanization Fe3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor 2 times, obtains magnetic bead mixed liquor；
5. the volume fraction that 12ml is added into magnetic bead mixed liquor is 7% glutaraldehyde solution, at room temperature vibration crosslinking 2.5
Hour, then separated with Magneto separate frame, abandoning supernatant obtains amination Fe3O4Nanometer magnetic bead mixed liquor；(glutaraldehyde solution and silane
Change Fe3O4The volume ratio of nanometer magnetic bead absolute ethyl alcohol mixed liquor is 3:1)
(3) preparation of immunomagnetic beads：
It is 1 with 1.2ml dilution factor：1000 nut allergies mouse positive serum is coated with 1ml amination Fe3O4Receive
Rice magnetic bead mixed liquor, is vibrated fix 1.5-2.5 hours at room temperature, then is separated by magnetic separator, and supernatant, which is discarded, to be wrapped
Amination Fe by after3O4Nanometer magnetic bead mixed liquor, is washed 4 times, obtained respectively with distilled water and pH=7.4 PBS
Fe3O4Immune nanometer magnetic bead；
(4)Fe3O4Immune nanometer magnetic bead detects nut allergies albumen by ELISA
The nut proteins of different dilution factors, by taking Cashew nut allergy albumen as an example：
1 times of dilution：For cashew nut protein extract stoste.
2 times of dilutions：Draw after cashew nut protein liquid 2ml adds pH=7.4 PBS 2ml and mix.
4 times of dilutions：Draw after 2 times of dilution 2ml add pH=7.4 PBS 2ml and mix.
8 times of dilutions：Draw after 4 times of dilution 2ml add pH=7.4 PBS 2ml and mix.
16 times of dilutions：Draw after 8 times of dilution 2ml add pH=7.4 PBS 2ml and mix.
1. it is separately added into cashew nut protein extract stoste and is diluted to 2 times, 4 times, 8 times, 16 times of protein liquid, sets simultaneously
PBS is negative control, 100 μ L/ holes；The PBS for adding pH=7.4 is coated with, 100 μ L/ holes, at 37 DEG C
It is incubated 1 hour in insulating box, then 4 DEG C are stayed overnight.
2. close：With cleaning solution board-washing 3 times, confining liquid is added, 300 μ L/ holes, 37 DEG C of concussions are incubated 2 hours on shaking table,
Board-washing.(cleaning solution is：The PBS of pH=7.4 containing 0.05% Tween-20；Contain 0.5g in confining liquid per 100ml
BSA and 100ml cleaning solution)
3. compete：50 μ L cashew nut protein extract stostes are added per hole and 2 times, 4 times, 8 times, 16 times of protein liquid is diluted to
With 50 μ L Fe3O4Immune nanometer magnetic bead, 37 DEG C of concussions are incubated 2 hours, use cleaning solution board-washing；Then 100 μ L sheep is added per hole
Anti- mouse IgE-HRP antibody, 37 DEG C of concussions are incubated 1h, use cleaning solution board-washing.(cleaning solution is：PH=containing 0.05% Tween-20
7.4 PBS)
4. develop the color：The substrate solution of 50 μ L Extemporaneous is added per hole and is sufficiently mixed；After 37 DEG C of lucifuges develop the color 20 minutes,
50 μ L terminate liquids are added per hole and are sufficiently mixed, then OD is determined with ELIASA450.(the preparation of substrate solution：By 10ml pH=
The hydrogen peroxide mixing of 7.4 PBS, 0.004g o-phenylenediamine and 15 μ L, matching while using is kept in dark place；Terminate liquid：
2mol/l sulfuric acid solution)
A kind of ferroso-ferric oxide immune nanometer magnetic bead described in the present embodiment and its preparation method and application, the preparation method
It can not be limited by nitrogen, and improve Fe3O4Magnetic property；By to Fe3O4Nanometer magnetic bead surface is modified,
Magnetic macromolecular microsphere is made, is combined with nut allergies serum antibody and immunomagnetic beads is made, add immune nanometer magnetic bead
Species and preparation method；Immunomagnetic beads is set to be combined with ELISA, without expensive devices such as hexavalent chrome bio-removal, mass spectrographs,
Cost is low, can be improved prediction with effective detection nut allergies albumen with assessing the accuracy of Cross-reactivity, be China's food
The implementation of anaphylactogen tag identifier system provides detection technique method.
Embodiment 3.
A kind of ferroso-ferric oxide immune nanometer magnetic bead and its preparation method and application, comprises the following steps：
(1) nanometer magnetic bead is prepared：
1. 4.17g FeSO is weighed4·7H2O and 5.406g FeCl3·6H2O, is dissolved in 50ml pure steaming water, obtains
Iron salt solutions；(FeSO4·7H2The amount of O materials is 4.17 ÷ 278.01 ≈ 0.015mol, Fe2+Molar concentration be 0.3mol/
L, FeCl3·6H2The amount of O materials is 5.406 ÷ 270.2962 ≈ 0.02mol, Fe3+Molar concentration be 0.4mol/L)
2. at a temperature of 70 DEG C, first ultrasound iron salt solutions 5 minutes, are placed into S21-I constant temperature blender with magnetic force at a high speed
Stirring 30 minutes, while 150ml 1mol/L sodium hydroxide solutions are added dropwise, with NaOH addition, crystallization occurs in reaction solution
Black level value peony is precipitated, then stands crystallization 2.5 hours, obtains Fe3O4Precipitation；
3. Magneto separate frame is used by Fe3O4Precipitation is separated, and first uses distillation water washing 3 times, then absolute ethyl alcohol is washed 2 times, is obtained
Fe3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor；
(2) surface modification of nanometer magnetic bead：
1. 25ml Fe is measured3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor, is diluted to 175ml with absolute ethyl alcohol, obtains after dilution
Nanometer magnetic bead absolute ethyl alcohol mixed liquor；
2. the nanometer magnetic bead absolute ethyl alcohol mixed liquor after sonic oscillation dilution adds 0.5ml APTES, room after 35 minutes
The lower stirring of temperature 8 hours, is separated with Magneto separate frame, obtains silanization Fe3O4Nanometer magnetic bead；(APTES with dilution after nanometer magnetic bead without
The volume ratio of water-ethanol mixed liquor is 0.5:175=1:350)；
3. silanization Fe is washed with ethanol solution3O4Nanometer magnetic bead 3 times, obtains silanization Fe3O4Nanometer magnetic bead is anhydrous
Alcohol mixeding liquid；
4. 1ml silanization Fe is drawn3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor, is washed with pH=7.4 PBS
Silanization Fe3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor 3 times, obtains magnetic bead mixed liquor；
5. the volume fraction that 5ml is added into magnetic bead mixed liquor is 3% glutaraldehyde solution, at room temperature vibration crosslinking 1.5
Hour, then separated with Magneto separate frame, abandoning supernatant obtains amination Fe3O4Nanometer magnetic bead mixed liquor；(glutaraldehyde solution and silane
Change Fe3O4The volume ratio of nanometer magnetic bead absolute ethyl alcohol mixed liquor is 5:1)
(3) preparation of immunomagnetic beads：
It is 1 with 1ml dilution factor：800 nut allergies mouse positive serum is coated with 1ml amination Fe3O4Nanometer magnetic bead
Mixed liquor, vibration fixes 1.5 hours at room temperature, then is separated by magnetic separator, and supernatant is discarded into the amino after must being coated with
Change Fe3O4Nanometer magnetic bead mixed liquor, is washed 4 times, obtains Fe respectively with distilled water and pH=7.4 PBS3O4Immune nano
Magnetic bead；
(4)Fe3O4Immune nanometer magnetic bead detects nut allergies albumen by ELISA
The nut proteins of different dilution factors, by taking pine nut allergen protein as an example：
1 times of dilution：For pine nut protein extract stoste.
2 times of dilutions：Draw after pine nut protein liquid 2ml adds pH=7.4 PBS 2ml and mix.
4 times of dilutions：Draw after 2 times of dilution 2ml add pH=7.4 PBS 2ml and mix.
8 times of dilutions：Draw after 4 times of dilution 2ml add pH=7.4 PBS 2ml and mix.
16 times of dilutions：Draw after 8 times of dilution 2ml add pH=7.4 PBS 2ml and mix.
1. it is separately added into pine nut protein extract stoste and is diluted to 2 times, 4 times, 8 times, 16 times of protein liquid, sets simultaneously
PBS is negative control, 100 μ L/ holes；The PBS for adding pH=7.4 is coated with, 100 μ L/ holes, at 37 DEG C
After being incubated one hour in insulating box, 4 DEG C overnight.
2. close：With cleaning solution board-washing 3 times, confining liquid is added, 300 μ L/ holes, 37 DEG C of concussions are incubated 2 hours on shaking table,
Board-washing.(cleaning solution is：The PBS of pH=7.4 containing 0.05% Tween-20；Contain 0.5g in confining liquid per 100ml
BSA and 100ml cleaning solution)
3. compete：50 μ L pine nut protein extract stostes are added per hole and 2 times, 4 times, 8 times, 16 times of protein liquid is diluted to
With 50 μ L Fe3O4Immune nanometer magnetic bead, 37 DEG C of concussions are incubated 2 hours, use cleaning solution board-washing；Then 100 μ L sheep is added per hole
Anti- mouse IgE-HRP antibody, 37 DEG C of concussions are incubated 1 hour, use cleaning solution board-washing.(cleaning solution is：PH containing 0.05% Tween-20
=7.4 PBS)
4. develop the color：The substrate solution of 50 μ L Extemporaneous is added per hole and is sufficiently mixed；After 37 DEG C of lucifuges develop the color 20 minutes,
50 μ L terminate liquids are added per hole and are sufficiently mixed, then OD is determined with ELIASA450.(the preparation of substrate solution：By 10ml pH=
The hydrogen peroxide mixing of 7.4 PBS, 0.004g o-phenylenediamine and 15 μ L, matching while using is kept in dark place；Terminate liquid：
2mol/l sulfuric acid solution)
A kind of ferroso-ferric oxide immune nanometer magnetic bead described in the present embodiment and its preparation method and application, the preparation method
It can not be limited by nitrogen, and improve Fe3O4Magnetic property；By to Fe3O4Nanometer magnetic bead surface is modified,
Magnetic macromolecular microsphere is made, is combined with nut allergies serum antibody and immunomagnetic beads is made, add immune nanometer magnetic bead
Species and preparation method；Immunomagnetic beads is set to be combined with ELISA, without expensive devices such as hexavalent chrome bio-removal, mass spectrographs,
Cost is low, can be improved prediction with effective detection nut allergies albumen with assessing the accuracy of Cross-reactivity, be China's food
The implementation of anaphylactogen tag identifier system provides detection technique method.
Embodiment 4.
A kind of ferroso-ferric oxide immune nanometer magnetic bead and its preparation method and application, comprises the following steps：
(1) nanometer magnetic bead is prepared：
1. 9.73g FeSO is weighed4·7H2O and 10.812g FeCl3·6H2O, is dissolved in 100ml pure steaming water,
Obtain iron salt solutions；(FeSO4·7H2The amount of O materials is 9.73 ÷ 278.01 ≈ 0.035mol, Fe2+Molar concentration be
0.35mol/L, FeCl3·6H2The amount of O materials is 10.812 ÷ 270.2962 ≈ 0.04mol, Fe3+Molar concentration be
0.4mol/L)
2. at a temperature of 68 DEG C, first ultrasound iron salt solutions 5 minutes place into that (rotor is placed on magnetic stirring apparatus at a high speed
Stirring 28 minutes, while 340ml 0.9mol/L sodium hydroxide solutions are added dropwise, with NaOH addition, crystallization goes out in reaction solution
Existing black level value peony precipitation, then stand crystallization 3 hours, obtain Fe3O4Precipitation；
3. Magneto separate frame is used by Fe3O4Precipitation is separated, and first uses distillation water washing 2 times, then absolute ethyl alcohol is washed 3 times, is obtained
Fe3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor；
Take out 10 1mlFe3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor is put into centrifuge tube, is whetheing there is externally-applied magnetic field situation
Lower its magnetic of observation.By obtained Fe3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor is divided in plate, is freezed, is frozen in refrigerator
12 hours are dried in freeze drier afterwards, nanometer magnetic bead powder is obtained, observation is taken out, spreads out in microscope and x-ray powder
Penetrate and its form is observed under instrument, obtain the collection of illustrative plates shown in Fig. 2.
(2) surface modification of nanometer magnetic bead：
1. 25ml Fe is measured3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor, is diluted to 140ml with absolute ethyl alcohol, obtains after dilution
Nanometer magnetic bead absolute ethyl alcohol mixed liquor；
2. the nanometer magnetic bead absolute ethyl alcohol mixed liquor after sonic oscillation dilution adds 0.4ml APTES, room after 28 minutes
The lower stirring of temperature 6.5 hours, is separated with Magneto separate frame, obtains silanization Fe3O4Nanometer magnetic bead；(APTES and the nanometer magnetic bead after dilution
The volume ratio of absolute ethyl alcohol mixed liquor is 0.4:140=1:350)；
3. silanization Fe is washed with ethanol solution3O4Nanometer magnetic bead 3 times, obtains silanization Fe3O4Nanometer magnetic bead is anhydrous
Alcohol mixeding liquid；
4. 1ml silanization Fe is drawn3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor, is washed with pH=7.4 PBS
Silanization Fe3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor 2 times, the consumption of each pH=7.4 PBS is 2ml, obtains magnetic bead
Mixed liquor；
5. the volume fraction that 3ml is added into magnetic bead mixed liquor is 6% glutaraldehyde solution, and vibration crosslinking 2 is small at room temperature
When, then separated with Magneto separate frame, abandoning supernatant obtains amination Fe3O4Nanometer magnetic bead mixed liquor；(glutaraldehyde solution and silanization
Fe3O4The volume ratio of nanometer magnetic bead absolute ethyl alcohol mixed liquor is 3:1)
(3) preparation of immunomagnetic beads：
It is 1 with 0.9ml dilution factor：700 nut allergies mouse positive serum is coated with 1ml amination Fe3O4Nano magnetic
Pearl mixed liquor, vibration fixes 2.5 hours at room temperature, then is separated by magnetic separator, and supernatant is discarded into the ammonia after must being coated with
Base Fe3O4Nanometer magnetic bead mixed liquor, is washed 3 times, obtains Fe respectively with distilled water and pH=7.4 PBS3O4It is immune to receive
Rice magnetic bead；
(4)Fe3O4Immune nanometer magnetic bead detects nut allergies albumen by ELISA
The nut proteins of different dilution factors, by taking fibert allergen protein as an example：
1 times of dilution：For fibert protein extract stoste.
2 times of dilutions：Draw after fibert protein liquid 2ml adds pH=7.4 PBS 2ml and mix.
4 times of dilutions：Draw after 2 times of dilution 2ml add pH=7.4 PBS 2ml and mix.
8 times of dilutions：Draw after 4 times of dilution 2ml add pH=7.4 PBS 2ml and mix.
16 times of dilutions：Draw after 8 times of dilution 2ml add pH=7.4 PBS 2ml and mix.
1. it is separately added into fibert protein extract stoste and is diluted to 2 times, 4 times, 8 times, 16 times of protein liquid, sets simultaneously
PBS is negative control, 100 μ L/ holes；The PBS for adding pH=7.4 is coated with, 100 μ L/ holes, at 37 DEG C
After being incubated one hour in insulating box, 4 DEG C overnight.
2. close：With cleaning solution board-washing 3 times, confining liquid is added, 300 μ L/ holes, 37 DEG C of concussions are incubated 2 hours on shaking table,
Board-washing.(cleaning solution is：The PBS of pH=7.4 containing 0.05% Tween-20；Contain 0.5g in confining liquid per 100ml
BSA and 100ml cleaning solution)
3. compete：50 μ L fibert protein extract stostes are added per hole and 2 times, 4 times, 8 times, 16 times of protein liquid is diluted to
With 50 μ L Fe3O4Immune nanometer magnetic bead, 37 DEG C of concussions are incubated 2 hours, use cleaning solution board-washing；Then 100 μ L sheep is added per hole
Anti- mouse IgE-HRP antibody, 37 DEG C of concussions are incubated 1 hour, use cleaning solution board-washing.(cleaning solution is：PH containing 0.05% Tween-20
=7.4 PBS)
4. develop the color：The substrate solution of 50 μ L Extemporaneous is added per hole and is sufficiently mixed；After 37 DEG C of lucifuges develop the color 20 minutes,
50 μ L terminate liquids are added per hole and are sufficiently mixed, then OD is determined with ELIASA450.(the preparation of substrate solution：By 10ml pH=
The hydrogen peroxide mixing of 7.4 PBS, 0.004g o-phenylenediamine and 15 μ L, matching while using is kept in dark place；Terminate liquid：
2mol/l sulfuric acid solution)
Table 1n (Fe2+)∶n(Fe3+) it is 1.75:Sample X-ray powder diffraction tables of data when 2
From table 1 and Fig. 1 and Fig. 2 contrast, n (Fe are used2+)∶n(Fe3+) it is 1.75:Receiving prepared by 2 ratios
Rice magnetic microsphere purity is higher.Due to preparing Fe3O4Magnetic microsphere n (Fe in theory2+)∶n(Fe3+) it is 1:2, so reacting
Should be to have partition losses in journey, comprehensive analysis is drawn, such a loss is due to Fe2+Easily it is oxidized in atmosphere.
A kind of ferroso-ferric oxide immune nanometer magnetic bead described in the present embodiment and its preparation method and application, the preparation method
It can not be limited by nitrogen, and improve Fe3O4Magnetic property；By to Fe3O4Nanometer magnetic bead surface is modified,
Magnetic macromolecular microsphere is made, is combined with nut allergies serum antibody and immunomagnetic beads is made, add immune nanometer magnetic bead
Species and preparation method；Immunomagnetic beads is set to be combined with ELISA, without expensive devices such as hexavalent chrome bio-removal, mass spectrographs,
Cost is low, can be improved prediction with effective detection nut allergies albumen with assessing the accuracy of Cross-reactivity, be China's food
The implementation of anaphylactogen tag identifier system provides detection technique method.
Embodiment 5.
A kind of ferroso-ferric oxide immune nanometer magnetic bead and its preparation method and application, comprises the following steps：
(1) nanometer magnetic bead is prepared：
1. 2.78g FeSO is weighed4·7H2O and 4.055g FeCl3·6H2O, is dissolved in 50ml pure steaming water, obtains
Iron salt solutions；(FeSO4·7H2The amount of O materials is 2.78 ÷ 278.01 ≈ 0.01mol, Fe2+Molar concentration be 0.2mol/L,
FeCl3·6H2The amount of O materials is 4.055 ÷ 270.2962 ≈ 0.015mol, Fe3+Molar concentration be 0.3mol/L)
2. at a temperature of 72 DEG C, first ultrasound iron salt solutions 5 minutes, are placed into S21-I constant temperature blender with magnetic force at a high speed
Stirring 33 minutes, while 140ml 0.8mol/L sodium hydroxide solutions are added dropwise, with NaOH addition, crystallization goes out in reaction solution
Existing black level value peony precipitation, then stand crystallization 3 hours, obtain Fe3O4Precipitation；
3. Magneto separate frame is used by Fe3O4Precipitation is separated, and first uses distillation water washing 2 times, then absolute ethyl alcohol is washed 2 times, is obtained
Fe3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor；
(2) surface modification of nanometer magnetic bead：
1. 25ml Fe is measured3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor, is diluted to 160ml with absolute ethyl alcohol, obtains after dilution
Nanometer magnetic bead absolute ethyl alcohol mixed liquor；
2. the nanometer magnetic bead absolute ethyl alcohol mixed liquor after sonic oscillation dilution adds 0.4ml APTES, room after 32 minutes
The lower stirring of temperature 7.5 hours, is separated with Magneto separate frame, obtains silanization Fe3O4Nanometer magnetic bead；(APTES and the nanometer magnetic bead after dilution
The volume ratio of absolute ethyl alcohol mixed liquor is 0.4:160=1:400)；
3. silanization Fe is washed with ethanol solution3O4Nanometer magnetic bead 3 times, obtains silanization Fe3O4Nanometer magnetic bead is anhydrous
Alcohol mixeding liquid；
4. 1ml silanization Fe is drawn3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor, is washed with pH=7.4 PBS
Silanization Fe3O4Nanometer magnetic bead absolute ethyl alcohol mixed liquor 2 times, obtains magnetic bead mixed liquor；
5. the volume fraction that 5ml is added into magnetic bead mixed liquor is 4% glutaraldehyde solution, at room temperature vibration crosslinking 2.5
Hour, then separated with Magneto separate frame, abandoning supernatant obtains amination Fe3O4Nanometer magnetic bead mixed liquor；(glutaraldehyde solution and silane
Change Fe3O4The volume ratio of nanometer magnetic bead absolute ethyl alcohol mixed liquor is 5:1)
(3) preparation of immunomagnetic beads：
It is 1 with 1.1ml dilution factor：700 nut allergies mouse positive serum is coated with 1ml amination Fe3O4Nano magnetic
Pearl mixed liquor, vibrates fix 1.5-2.5 hours at room temperature, then is separated by magnetic separator, supernatant is discarded after must being coated with
Amination Fe3O4Nanometer magnetic bead mixed liquor, is washed 3 times, obtains Fe respectively with distilled water and pH=7.4 PBS3O4Exempt from
Epidemic disease nanometer magnetic bead；
(4)Fe3O4Immune nanometer magnetic bead detects nut allergies albumen by ELISA
The nut proteins of different dilution factors, by taking almond allergen protein as an example：
1 times of dilution：For almond protein extract solution stoste.
2 times of dilutions：Draw after almond protein liquid 2ml adds pH=7.4 PBS 2ml and mix.
4 times of dilutions：Draw after 2 times of dilution 2ml add pH=7.4 PBS 2ml and mix.
8 times of dilutions：Draw after 4 times of dilution 2ml add pH=7.4 PBS 2ml and mix.
16 times of dilutions：Draw after 8 times of dilution 2ml add pH=7.4 PBS 2ml and mix.
1. it is separately added into almond protein extract solution stoste and is diluted to 2 times, 4 times, 8 times, 16 times of protein liquid, sets simultaneously
PBS is negative control, 100 μ L/ holes；The PBS for adding pH=7.4 is coated with, 100 μ L/ holes, at 37 DEG C
It is incubated in insulating box 1 hour, 4 DEG C overnight.
2. close：With cleaning solution board-washing 3 times, confining liquid is added, 300 μ L/ holes, 37 DEG C of concussions are incubated 2 hours on shaking table,
Board-washing.(cleaning solution is：The PBS of pH=7.4 containing 0.05% Tween-20；Contain 0.5g in confining liquid per 100ml
BSA and 100ml cleaning solution)
3. compete：50 μ L almond protein extract solution stostes are added per hole and 2 times, 4 times, 8 times, 16 times of protein liquid is diluted to
With 50 μ L Fe3O4Immune nanometer magnetic bead, 37 DEG C of concussions are incubated 2 hours, use cleaning solution board-washing；Then 100 μ L sheep is added per hole
Anti- mouse IgE-HRP antibody, 37 DEG C of concussions are incubated 1 hour, use cleaning solution board-washing.(cleaning solution is：PH containing 0.05% Tween-20
=7.4 PBS)
4. develop the color：The substrate solution of 50 μ L Extemporaneous is added per hole and is sufficiently mixed；After 37 DEG C of lucifuges develop the color 20 minutes,
50 μ L terminate liquids are added per hole and are sufficiently mixed, then OD is determined with ELIASA450.(the preparation of substrate solution：By 10ml pH=
The hydrogen peroxide mixing of 7.4 PBS, 0.004g o-phenylenediamine and 15 μ L, matching while using is kept in dark place；Terminate liquid：
2mol/l sulfuric acid solution)
A kind of ferroso-ferric oxide immune nanometer magnetic bead described in the present embodiment and its preparation method and application, the preparation method
It can not be limited by nitrogen, and improve Fe3O4Magnetic property；By to Fe3O4Nanometer magnetic bead surface is modified,
Magnetic macromolecular microsphere is made, is combined with nut allergies serum antibody and immunomagnetic beads is made, add immune nanometer magnetic bead
Species and preparation method；Immunomagnetic beads is set to be combined with ELISA, without expensive devices such as hexavalent chrome bio-removal, mass spectrographs,
Cost is low, can be improved prediction with effective detection nut allergies albumen with assessing the accuracy of Cross-reactivity, be China's food
The implementation of anaphylactogen tag identifier system provides detection technique method.
It is described above, only it is the preferred embodiment of the embodiment of the present invention, not makees any shape to the embodiment of the present invention
Limitation in formula, according to the embodiment of the present invention technical spirit above example is made any simple modification, equivalent variations
With modification, in the range of still falling within technical scheme of the embodiment of the present invention.
Claims (10)
1. a kind of preparation method of ferroso-ferric oxide immune nanometer magnetic bead, it is characterised in that comprise the following steps：
(1) nanometer magnetic bead is prepared：
By Fe2+Molysite and Fe3+Iron salt dissolved in water, obtain iron salt solutions；The Fe2+Molysite in Fe2+And Fe3+Iron
Fe in salt3+Mol ratio be 1:1-2；
At a temperature of 65-75 DEG C, first ultrasound iron salt solutions 4-6 minutes, add excessive sodium hydroxide solution and stir 25-
35 minutes, then stand crystallization 2-3 hours, obtain ferroso-ferric oxide precipitation；
Ferroso-ferric oxide precipitation is separated by Magneto separate, distillation water washing is first used 2-3 times, then absolute ethyl alcohol washing 2-3
It is secondary, obtain ferriferrous oxide nano magnetic bead absolute ethyl alcohol mixed liquor；
(2) surface modification of nanometer magnetic bead：
Ferriferrous oxide nano magnetic bead absolute ethyl alcohol mixed liquor is diluted into 5-7 times, the nanometer magnetic bead after must diluting with absolute ethyl alcohol
Absolute ethyl alcohol mixed liquor；
Nanometer magnetic bead absolute ethyl alcohol mixed liquor after sonic oscillation dilution adds silylating reagent, stirred at room temperature after 25-35 minutes
Mix 6-8 hours, Magneto separate, obtain silanization ferriferrous oxide nano magnetic bead；The silylating reagent is APTES；
Silanization ferriferrous oxide nano magnetic bead is washed with ethanol solution 2-3 times, obtain silanization ferriferrous oxide nano
Magnetic bead absolute ethyl alcohol mixed liquor；
Silanization ferriferrous oxide nano magnetic bead absolute ethyl alcohol mixed liquor is washed with PBS again 2-3 times, obtain magnetic bead mixing
Liquid；
Glutaraldehyde solution is added into magnetic bead mixed liquor, vibration crosslinking 1.5-2.5 hours, then Magneto separate at room temperature, supernatant discarding
Liquid, obtains amination ferriferrous oxide nano magnetic bead mixed liquor mixed liquor；
(3) preparation of immunomagnetic beads：
Amination ferriferrous oxide nano magnetic bead mixed liquor mixed liquor is coated with antibody, vibrates fix 1.5-2.5 hours at room temperature,
Magneto separate, the amination ferriferrous oxide nano magnetic bead mixed liquor after must being coated with, 3- is washed with distilled water and PBS respectively
4 times, obtain ferroso-ferric oxide immune nanometer magnetic bead；The antibody is nut allergies mouse positive serum.
2. preparation method according to claim 1, it is characterised in that wherein,
In the step (1), Fe in iron salt solutions2+Molar concentration be 0.2-0.35mol/L, Fe3+Molar concentration be 0.2-
0.4mol/L；
The molar concentration of the sodium hydroxide solution is 0.5-1mol/L.
3. preparation method according to claim 2, it is characterised in that wherein,
The Fe2+Molysite be FeSO4·7H2O, Fe3+Molysite be FeCl3·6H2O。
4. preparation method according to claim 1, it is characterised in that wherein,
In the step (2), the volume ratio of the APTES and the nanometer magnetic bead absolute ethyl alcohol mixed liquor after dilution is 1:350-
400；
The consumption of the glutaraldehyde solution is 3-5 times of silanization ferriferrous oxide nano magnetic bead absolute ethyl alcohol mixed liquor.
5. preparation method according to claim 4, it is characterised in that wherein,
The volume fraction of the glutaraldehyde solution is 3-7%.
6. preparation method according to claim 1, it is characterised in that wherein,
In the step (3), the volume ratio 0.8-1.2 of antibody and amination ferriferrous oxide nano magnetic bead mixed liquor:1.
7. preparation method according to claim 6, it is characterised in that wherein,
In the step (3), the dilution factor of antibody is 1:600-1000.
8. preparation method according to claim 7, it is characterised in that wherein,
The dilution factor of the antibody is 1:800.
9. a kind of application of ferroso-ferric oxide immune nanometer magnetic bead, it is characterised in that including：
Antigen is detected using enzyme linked immunological competition law；Wherein, the antigen is nut allergies albumen, and the first antibody is four oxygen
Change three-iron immune nanometer magnetic bead.
10. application according to claim 9, it is characterised in that wherein, the secondary antibody is anti-for sheep anti mouse IgE-HRP
Body.
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