CN108931637A - A kind of preparation method of biological immune magnetic bead - Google Patents
A kind of preparation method of biological immune magnetic bead Download PDFInfo
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- CN108931637A CN108931637A CN201810567906.XA CN201810567906A CN108931637A CN 108931637 A CN108931637 A CN 108931637A CN 201810567906 A CN201810567906 A CN 201810567906A CN 108931637 A CN108931637 A CN 108931637A
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
The present invention relates to a kind of preparation methods of biological immune magnetic bead, belong to detection technique field.Present invention hydro-thermal reaction method prepares of uniform size and tiny, ferromagnetism nanoparticle Fe3O4, and it is modified with ethyl orthosilicate, obtain surface silanol group, dispersibility improves and keeps ferromagnetic modified Nano magnetic bead Fe3O4@SiO2, then it is further modified to it with activator, more preferably, still it is easy to modify variety classes antibody, SiO in ferromagnetism and surface to obtain dispersibility2It is wrapped good, since surface is rich in active silicone hydroxyl or amino, its dispersion stabilization can be not only effectively improved, and it is convenient for and at ester or amide in conjunction with several functional molecules, it is expected synchronous realization spectral response and magnetic responsiveness, the quick detection and separated in synchronization technology that food-safe field is badly in need of have significant potential using value.
Description
Technical field
The present invention relates to a kind of preparation methods of biological immune magnetic bead, belong to detection technique field.
Background technique
Immunomagnetic beads are combined by carrier microballoons and immunoligand.The core of carrier microballoons is small metal particles,
It is a kind of magnetic material that magnetism is high and more stable, wraps up one layer of high molecular material outside core（Such as polyvinyl chloride, polystyrene,
Polyethyleneimine）, outermost layer is function base, such as hydroxyl, amino, aldehyde radical, Suo Ji.Not due to carrier microballoons performance physical property
It together, can the different immunoligand of covalent bond（Such as enzyme, cell, antibody, antigen, DNA, RNA）Equal bioactive substances.
Ideal immunomagnetic beads are that general partial size is smaller, uniform spherical, the particle with protectiveness shell and superparamagnetism,
Its structural core is magnetic material, and core outer layer wraps up high molecular material, and outermost layer is immunoligand.
The key for forming immunomagnetic beads is magnetic carrier, can be divided into three kinds according to the difference of its structure：（1）Shell-core structure,
I.e. using high molecular material as core, outside coated magnetic material.（2）Shell-nucleocapsid structure, centre are magnetic material, internal layer and outer
Layer is high molecular material.（3）Nucleocapsid structure, magnetic material are that high molecular material is wrapped up outside core.As immunomagnetic beads carrier
Magnetic microsphere is mainly that core shell structure is most.Magnetic material is mostly Fe, the oxidation of the transition metal specific crystal formation such as Ni, Co
Object.Most widely used at present is iron and its oxi.Magnetic microsphere is that inside contains nano magnetic particle, is external for macromolecule
Composite material of the shell as carrier is widely used in biomolecule mobilization and Organic Solid-Phase synthesis, in bioengineering and
In biomedical research and practice, the biomolecule of immobilization is usually used as the aglucon of affinity analyzing, also can be used as biological anti-
The catalyst or drug answered.
Existing immunomagnetic beads are all based on Fe3O4Magnetic particle, and Fe3O4Magnetic particle chemical stability is not high, easily
It is oxidized, and the active group on surface is less, is inclined to, is unfavorable in conjunction with immunoligand with strong aggregation, limitation expansion
Using.
Summary of the invention
The technical problems to be solved by the invention：Fe is all based on for existing immunomagnetic beads3O4Magnetic particle, and
Fe3O4Magnetic particle chemical stability is not high, is easily oxidized, and the active group on surface is less, and there is strong aggregation to incline
To providing a kind of preparation method of biological immune magnetic bead the problem of being unfavorable in conjunction with immunoligand.
In order to solve the above technical problems, the technical solution adopted by the present invention is that：
A kind of preparation method of biological immune magnetic bead, the specific steps are：
（1）Iron trichloride hexahydrate and ferrous gluconate are dissolved in ethylene glycol, sodium acetate, Macrogol 4000 stirring are added
It is fitted into reaction kettle and reacts after uniformly, separated under magnetic fields after cooling, and alternately washed with deionized water and dehydrated alcohol
After dry, obtain magnetic Fe3O4Nanoparticle；
（2）By magnetic Fe3O4Nanoparticle and mass fraction be 25% tetramethyl ammonium hydroxide solution be added after mixing go from
10~15min is stirred in sub- water, obtains magnetic Fe3O4Dispersion liquid；
（3）By magnetic Fe3O4Dispersion liquid is added in borate buffer solution and is uniformly mixed, then ethyl orthosilicate is added dropwise, and drips
PH to 8~9 is adjusted for 25% ammonium hydroxide with mass fraction after finishing, separates, is washed with deionized water under magnetic fields after standing 3~5h
It is transparent to cleaning solution to wash sediment, obtains modified magnetic Fe3O4Nanoparticle；
（4）Take modified magnetic Fe3O4Nanoparticle is scattered in dry toluene after washing 2~3 times with toluene, adds activator,
With 300~400r/min stirring 20~for 24 hours, separated under magnetic fields, obtain biological immune magnetic bead.
Step（1）The molar ratio of the Iron trichloride hexahydrate and ferrous gluconate is 1：1~1：4.
Step（1）Described weight of material part is the mixture of 0.15~0.20 part of Iron trichloride hexahydrate and ferrous gluconate,
40~50 parts of ethylene glycol, 1.8~3.6 parts of sodium acetates, 0.5~1.0 part of Macrogol 4000.
Step（1）The reaction process is 6~8h of insulation reaction at 200~220 DEG C.
Step（2）The magnetic Fe3O4Nanoparticle, tetramethyl ammonium hydroxide solution mass ratio be 1：1~1：3, it is described
Deionized water dosage is magnetic Fe3O450~200 times of nanoparticle quality.
Step（3）The magnetic Fe3O4Dispersion liquid, borate buffer solution, ethyl orthosilicate mass ratio be 1：1：1.
Step（3）The borate buffer solution is the borate buffer solution of pH7~9.
Step（4）The activator is 3- glycidylpropyl trimethoxy silane, γ-(2,3- glycidoxy) propyl
One or more of trimethoxy silane, aminopropyl triethoxysilane, aminopropyl trimethoxysilane.
Step（4）The activator level is modified magnetic Fe3O410~40 times of nanoparticle quality.
The present invention is compared with other methods, and advantageous effects are：
（1）Present invention hydro-thermal reaction method prepares of uniform size and tiny, ferromagnetism nanoparticle Fe3O4, and with positive silicic acid second
Ester is modified it, obtains surface silanol group, dispersibility improves and keeps ferromagnetic modified Nano magnetic bead Fe3O4@
SiO2, then it is further modified to it with activator, with obtain it is dispersed it is more preferable, still in ferromagnetism and surface be easy to modify it is not of the same race
Class antibody, SiO2It is wrapped good, since surface is rich in active silicone hydroxyl or amino, it can not only be effectively improved its stably dispersing
Property, and be convenient for and at ester or amide in conjunction with several functional molecules, it is expected to it is synchronous to realize spectral response and magnetic responsiveness,
The quick detection and separated in synchronization technology that food-safe field is badly in need of have significant potential using value；
（2）Small size, magnetic nano-particle of uniform size, magnetic nano-particle can be obtained by shirtsleeve operation in the present invention
It is easy to modify variety classes antibody, the selectable wide variety of silane coupling agent, according to the amino of connection, epoxy group, carboxyl base
The difference of group can preferably can realize the coupling with antibody, coupling method is quick, simple, institute in conjunction with different immunoligands
It is low with cost of material, it is suitable for a wide range of promote.
Specific embodiment
The mixture of 0.15~0.20g Iron trichloride hexahydrate and ferrous gluconate is dissolved in 40~50g ethylene glycol, then
1.8~3.6g sodium acetate is added, 0.5~1.0g Macrogol 4000 is transferred to after stirring 30~40min with 400~500r/min
In reaction kettle, 6~8h of insulation reaction at 200~220 DEG C separates to obtain precipitating, respectively under magnetic fields after being cooled to room temperature
With deionized water and dehydrated alcohol alternately washing precipitating 2~3 times, then it is placed in drying box, dry 6~8h at 40~50 DEG C,
Obtain magnetic Fe3O4Nanoparticle, by 0.1~0.2g magnetic Fe3O4Nanoparticle and 0.2~0.3g mass fraction are 25% tetramethyl
Ammonia is added after mixing in 10~20mL deionized water, is stirred 10~15min with 300~400r/min, is obtained
Magnetic Fe3O4Dispersion liquid, by 10~20g magnetic Fe3O4Dispersion liquid is added in 10~20g borate buffer solution and is uniformly mixed, then
With 0.1~0.2g/min be added dropwise 10~20g ethyl orthosilicate, after being added dropwise with mass fraction be 25% ammonium hydroxide adjust pH to 8~
9, it is separated under magnetic fields after standing 3~5h, removes supernatant, it is transparent to cleaning solution to be washed with deionized sediment, obtains
Modified magnetic Fe3O4Nanoparticle takes 50~100mg modified magnetic Fe3O4Nanoparticle is scattered in after washing 2~3 times with toluene
In 10~20g dry toluene, 1~2g activator is added, with 300~400r/min stirring 20~for 24 hours, under magnetic fields point
From obtaining biological immune magnetic bead.
The mixture of 0.15g Iron trichloride hexahydrate and ferrous gluconate is dissolved in 40g ethylene glycol, 1.8g vinegar is added
Sour sodium, 0.5g Macrogol 4000 are transferred in reaction kettle, the insulation reaction 6h at 200 DEG C after stirring 30min with 400r/min,
Precipitating is separated to obtain after being cooled to room temperature under magnetic fields, respectively with deionized water and dehydrated alcohol alternately washing precipitating 2 times, then
It is placed in drying box, dry 6h, obtains magnetic Fe at 40 DEG C3O4Nanoparticle, by 0.1g magnetic Fe3O4Nanoparticle and 0.2g
Mass fraction is that 25% tetramethyl ammonium hydroxide solution is added after mixing in 10mL deionized water, is stirred with 300r/min
10min obtains magnetic Fe3O4Dispersion liquid, by 10g magnetic Fe3O4Dispersion liquid is added in 10g borate buffer solution and is uniformly mixed, then
With 0.1g/min be added dropwise 10g ethyl orthosilicate, after being added dropwise with mass fraction be 25% ammonium hydroxide adjust pH to 8, stand 3h after
It is separated under magnetic fields, removes supernatant, it is transparent to cleaning solution to be washed with deionized sediment, obtains modified magnetic Fe3O4Nanometer
Particle takes 50mg modified magnetic Fe3O4Nanoparticle is washed after 2 times with toluene and is scattered in 10g dry toluene, and 1g activation is added
Agent stirs 20h with 300r/min, separates under magnetic fields, obtain biological immune magnetic bead.
The mixture of 0.18g Iron trichloride hexahydrate and ferrous gluconate is dissolved in 45g ethylene glycol, 2.0g vinegar is added
Sour sodium, 0.8g Macrogol 4000 are transferred in reaction kettle, the insulation reaction 7h at 210 DEG C after stirring 35min with 450r/min,
Precipitating is separated to obtain after being cooled to room temperature under magnetic fields, respectively with deionized water and dehydrated alcohol alternately washing precipitating 2 times, then
It is placed in drying box, dry 7h, obtains magnetic Fe at 45 DEG C3O4Nanoparticle, by 0.1g magnetic Fe3O4Nanoparticle and 0.2g
Mass fraction is that 25% tetramethyl ammonium hydroxide solution is added after mixing in 15mL deionized water, is stirred with 350r/min
12min obtains magnetic Fe3O4Dispersion liquid, by 15g magnetic Fe3O4Dispersion liquid is added in 15g borate buffer solution and is uniformly mixed, then
With 0.2g/min be added dropwise 15g ethyl orthosilicate, after being added dropwise with mass fraction be 25% ammonium hydroxide adjust pH to 9, stand 4h after
It is separated under magnetic fields, removes supernatant, it is transparent to cleaning solution to be washed with deionized sediment, obtains modified magnetic Fe3O4Nanometer
Particle takes 80mg modified magnetic Fe3O4Nanoparticle is washed after 3 times with toluene and is scattered in 15g dry toluene, and 1g activation is added
Agent stirs 21h with 350r/min, separates under magnetic fields, obtain biological immune magnetic bead.
The mixture of 0.20g Iron trichloride hexahydrate and ferrous gluconate is dissolved in 50g ethylene glycol, 3.6g vinegar is added
Sour sodium, 1.0g Macrogol 4000 are transferred in reaction kettle, the insulation reaction 8h at 220 DEG C after stirring 40min with 500r/min,
Precipitating is separated to obtain after being cooled to room temperature under magnetic fields, respectively with deionized water and dehydrated alcohol alternately washing precipitating 3 times, then
It is placed in drying box, dry 8h, obtains magnetic Fe at 50 DEG C3O4Nanoparticle, by 0.2g magnetic Fe3O4Nanoparticle and 0.3g
Mass fraction is that 25% tetramethyl ammonium hydroxide solution is added after mixing in 20mL deionized water, is stirred with 400r/min
15min obtains magnetic Fe3O4Dispersion liquid, by 20g magnetic Fe3O4Dispersion liquid is added in 20g borate buffer solution and is uniformly mixed, then
With 0.2g/min be added dropwise 20g ethyl orthosilicate, after being added dropwise with mass fraction be 25% ammonium hydroxide adjust pH to 9, stand 5h after
It is separated under magnetic fields, removes supernatant, it is transparent to cleaning solution to be washed with deionized sediment, obtains modified magnetic Fe3O4Nanometer
Particle takes 100mg modified magnetic Fe3O4Nanoparticle is washed after 3 times with toluene and is scattered in 20g dry toluene, and it is living to add 2g
Agent for 24 hours with 400r/min stirring separates under magnetic fields, obtains biological immune magnetic bead.
Reference examples：The immunomagnetic beads of Hunan company production.
The immunomagnetic beads of example and reference examples are detected, specific testing result such as table 1.
1 performance characterization contrast table of table
| Detection project | Example 1 | Example 2 | Example 3 | Reference examples |
| Magnetic eum/g | 75.9 | 76.2 | 76.8 | 65.5 |
| Investigation of stabilized dispersion of nano d in ethyl alcohol | 12 | 13 | 14 | 8 |
As shown in Table 1, biological immune magnetic bead magnetism with higher prepared by the present invention, and stability shows well, be compared with
For ideal immunomagnetic beads.
Claims (9)
1. a kind of preparation method of biological immune magnetic bead, which is characterized in that the specific steps are：
（1）Iron trichloride hexahydrate and ferrous gluconate are dissolved in ethylene glycol, sodium acetate, Macrogol 4000 stirring are added
It is fitted into reaction kettle and reacts after uniformly, separated under magnetic fields after cooling, and alternately washed with deionized water and dehydrated alcohol
After dry, obtain magnetic Fe3O4Nanoparticle；
（2）By magnetic Fe3O4Nanoparticle and mass fraction be 25% tetramethyl ammonium hydroxide solution be added after mixing go from
10~15min is stirred in sub- water, obtains magnetic Fe3O4Dispersion liquid；
（3）By magnetic Fe3O4Dispersion liquid is added in borate buffer solution and is uniformly mixed, then ethyl orthosilicate is added dropwise, and is added dropwise
It is afterwards that 25% ammonium hydroxide adjusts pH to 8~9 with mass fraction, separates, be washed with deionized under magnetic fields after standing 3~5h
Sediment is transparent to cleaning solution, obtains modified magnetic Fe3O4Nanoparticle；
（4）Take modified magnetic Fe3O4Nanoparticle is scattered in dry toluene after washing 2~3 times with toluene, adds activator,
With 300~400r/min stirring 20~for 24 hours, separated under magnetic fields, obtain biological immune magnetic bead.
2. a kind of preparation method of biological immune magnetic bead as described in claim 1, which is characterized in that step（1）Six water
The molar ratio of iron chloride and ferrous gluconate is 1：1~1：4.
3. a kind of preparation method of biological immune magnetic bead as described in claim 1, which is characterized in that step（1）The material
Mixture of the parts by weight for 0.15~0.20 part of Iron trichloride hexahydrate and ferrous gluconate, 40~50 parts of ethylene glycol, 1.8~3.6
Part sodium acetate, 0.5~1.0 part of Macrogol 4000.
4. a kind of preparation method of biological immune magnetic bead as described in claim 1, which is characterized in that step（1）The reaction
Process is 6~8h of insulation reaction at 200~220 DEG C.
5. a kind of preparation method of biological immune magnetic bead as described in claim 1, which is characterized in that step（2）The magnetism
Fe3O4Nanoparticle, tetramethyl ammonium hydroxide solution mass ratio be 1：1~1：3, the deionized water dosage is magnetic Fe3O4
50~200 times of nanoparticle quality.
6. a kind of preparation method of biological immune magnetic bead as described in claim 1, which is characterized in that step（3）The magnetism
Fe3O4Dispersion liquid, borate buffer solution, ethyl orthosilicate mass ratio be 1：1：1.
7. a kind of preparation method of biological immune magnetic bead as described in claim 1, which is characterized in that step（3）The boric acid
Salt buffer solution is the borate buffer solution of pH7~9.
8. a kind of preparation method of biological immune magnetic bead as described in claim 1, which is characterized in that step（4）The activation
Agent is 3- glycidylpropyl trimethoxy silane, γ-(2,3- glycidoxy) propyl trimethoxy silicane, three second of aminopropyl
One or more of oxysilane, aminopropyl trimethoxysilane.
9. a kind of preparation method of biological immune magnetic bead as described in claim 1, which is characterized in that step（4）The activation
Agent dosage is modified magnetic Fe3O410~40 times of nanoparticle quality.
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Application publication date: 20181204 |