CN104927010B - Core-shell magnetic composite microsphere containing polyelectrolyte and its preparation method and application - Google Patents
Core-shell magnetic composite microsphere containing polyelectrolyte and its preparation method and application Download PDFInfo
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Abstract
The invention belongs to function nano field of material technology, specially a kind of core-shell magnetic composite microsphere rich in polyelectrolyte and its preparation method and application.The core of magnetic composite microsphere of the invention is magnetic ferroferric oxide nano-particles cluster, and inner casing is the polymer network rich in hydroxyl of crosslinking, and shell is one layer of PAA of Surface RAFT polymerization.The preparation method of the complex microsphere includes：Magnetic nano-particle cluster is prepared, in magnetic cluster surface modification vinyl-functional, the cross-linked network of hydroxyl polymer-containing is coated, RAFT reagents are modified, is triggered on RAFT reagents surface and is connect one layer of PAA shell etc..The inventive method process is simple.Obtained magnetic composite microsphere can be used for the super amount albumen of fast separating concentration, the high-abundance proteins and enrichment method low-abundance protein being such as used in high flux removal blood.
Description
Technical field
The invention belongs to function nano field of material technology, and in particular to a kind of core-shell type magnetic rich in polyelectrolyte is answered
Close microballoon and its preparation method and application.
Background technology
Protein is the regulation and control person of the various physiological functions of organism, is also the most direct agent of biological phenomena.To albumen
The structure of matter, function carry out systematic research analysis for we have appreciated that living things system and announcement life entity are in pathological conditions
Under change mechanism have great importance, for the diseases such as cancer ahead of time find and diagnosis and treatment also have vital work
With.The albumen that important vital movement is undertaken in organism is all often low-abundance protein, after the extremely low content of these albumen is given
Continuous analysis and detection brings very big difficulty.Therefore by these low-abundance proteins from complex biological system selective enrichment
Out, then it is analyzed again and identifies and become current study hotspot.
Magnetic composite microsphere to complicated living things system under externally-applied magnetic field due to that with unique magnetic responsiveness, can enter
Row microoperation is so as to be particularly well-suited to the research of proteomics.Richness can be reached by carrying out various modifications to magnetic microsphere surface
Collect the purpose of various specific protein.The method of traditional use magnetic composite microsphere isolating biologically active material is by anti-
Antibody, i.e., be fixed on magnetic composite microsphere surface by the interaction of body-antigen, then the corresponding antigen of specific isolation.Though
The specificity of right antibody-antigene effect is fine, but the price of antibody is sufficiently expensive, and can only a certain egg of specific recognition
In vain.And for protein science, what is generally required is the albumen or polypeptide for being enriched with all or a certain class.Therefore antibody-anti-
Original work are with being not to be well suited for.Enrichment target protein or polypeptide can be roughly divided into two classes by purpose at present：One kind is selection
Property a certain class of enrichment have the albumen or polypeptide of special modification, the recombinant protein of such as selective enrichment His-tag marks, selection
Property enrichment acid albumen or phosphoeptide, selective enrichment glycoprotein or glycopeptide etc.；Another kind is that do not have selective enrichment system
In all albumen or polypeptide, its main purpose is the salinity and other compositions that analysis and detection are disturbed in removal system.The
The enrichment that one species specific enrichment is particularly well-suited to known biomarker is detected and caused widely to be paid close attention to, but in recent years
, for the raising that unknown flag thing finds to require, do not knowing marker protein type with for disease detections such as cancers
In the case of, all possible marker protein is particularly important in unspecific enrichment whole blood.
Typically utilize is all hydrophobic interaction not to have all albumen or polypeptide in selective enrichment system.So
Magnetic composite microsphere surface for such application should be relatively hydrophobic material.Report has C8, C60, PMMA etc. at present.
With the help of this kind of material strong-hydrophobicity, complex microsphere is enriched with preferable effect for protein.But due to hydrophobic-thin
Water interacts still certain selectivity for protein classes, is such as more suitable for hydrophobin, so being enriched with low abundance
Some hydrophilic proteins can be missed when mark unavoidably.And the albumen major part that obtains of such material enrichment will be with organic molten
Agent is eluted, and condition is relatively harsh and elution efficiency and the rate of recovery be not high.For actual mechanical process, such hydrophobic material is often
Dispersed and bad in enrichment solution and easily stick in tube wall, so it operates cumbersome and stock utilization also not high.
Accordingly, it would be desirable to seek the albumen that new method is used in the enriched biological system of non-selectivity, then the present invention is carried
A kind of method of counter ion evaporation has been supplied to go enrichment.Mainly in two steps, complicated actual sample is removed with such magnetic material first
Such as the high-abundance proteins in serum, then proceed to utilize material enrichment to concentrate remaining low-abundance protein in the system.Such
Material is mainly magnetic material outer cladding polyelectrolyte（Such as PAA（Polyacrylic acid））, can form substantial amounts of anti-in aqueous
Ion（Such as cation）, protein no matter its isoelectric point how to be generally made up of positive charge and negative electrical charge two parts, it is therein just
Electric charge section can just go to replace the counter ion of positively charged in polyelectrolyte so that protein is adsorbed, and this is that an entropy increases
Process, so be a kind of very general method, it is adaptable to the richness of nearly all protein of different molecular weight difference isoelectric point
Collection.
The content of the invention
It is an object of the invention to propose that a kind of preparation process is simple, surface functional group density is high, can largely be enriched with various
Core-shell magnetic composite microsphere of various kinds albumen and its preparation method and application.
The present invention is for the problem in the presence of background technology, it is proposed that being capable of efficiently concentrating and wash-out either high abundance
Or the material and method of low-abundance protein.The nanocrystalline cluster of inorganic magnetic is prepared first, then poly hydroxy ethyl acrylate
Magnetic cluster is coated to as inner casing up, then in its one layer of RAFT reagent of outer modification（S-1- dodecyls-S'- (α, α '-diformazan
Base-α ' '-acetate) three thioesters）, then trigger RAFT polymerizations to prepare nucleocapsid shell-type magnetic coupling by surface micro-
Ball, is designated as Fe3O4/ PHEMA-RAFT-PAA, due to the microballoon is very hydrophilic and can be formed in the solution substantial amounts of positive charge it is anti-from
Son, albumen all has positive charge and a negative electrical charge part, thus positive charge part can displace the positive charge that refers to before it is anti-from
Son, so as to realize the absorption for all proteins.
The preparation method of the core-shell magnetic composite microsphere rich in polyelectrolyte proposed by the present invention, concretely comprises the following steps：
（1）First, the magnetic of citrate-stable is prepared as raw material with Iron(III) chloride hexahydrate, acetate and citrate
Nano-particle cluster（Abbreviation magnetic cluster）；
（2）Then, magnetic cluster surface is modified using sol-gal process, its surface is taken the vinyl functional of activity
Group；
（3）Then, the magnetic cluster of vinyl is contained as seed with surface, by the method for the precipitation polymerization that flows back on magnetic cluster surface
One layer of cross-linked network of the hydroxyl polymer-containing of densification of cladding, obtains with magnetic cluster as core, magnetic of the hydroxyl polymer-containing network as shell
Property polymer composite microsphere；
（4）Then, using the hydroxyl polymer-containing network in esterification modification previous step, the RAFT on its surface grafting
Reagent；
（5）Finally, substantial amounts of PAA in RAFT polymerization modifications is triggered on the magnetic composite microsphere surface（Polyacrylic acid）Chain,
Obtain final product required magnetic composite microsphere.
With magnetic composite microsphere of the surface rich in PAA chains, high-abundance proteins and the low abundance egg of enrichment method are removed
White experiment.
The specific operation process of the inventive method each step is as follows：
Step (1)：1 ~ 30g Iron(III) chloride hexahydrates, 1 ~ 60g acetate and 0.1 ~ 20g citrates are dissolved in 20 ~
In 500mL ethylene glycol, 0.5 ~ 5h of mechanical agitation at 100 ~ 200 DEG C is subsequently placed in the stainless steel containing polytetrafluoroethyllining lining
In autoclave, reactor is positioned over 10 ~ 50h in 100 ~ 300 DEG C of baking oven, taken out, room is allowed to cool to running water
Temperature；Product magnetic cluster is isolated with magnet, and unreacted reactant is removed with absolute ethanol washing, finally disperse product magnetic cluster
It is standby in absolute ethyl alcohol；
Step (2)：0.1g ~ 3g magnetic cluster that step (1) is obtained, 20 ~ 400mL absolute ethyl alcohols, 5 ~ 100mL deionized waters,
The silane coupler of 0.5 ~ 10mL ammoniacal liquor and 0.2 ~ 10g with double bond is added in there-necked flask, is 50 ~ 100 DEG C in reaction temperature
The lower h of mechanical agitation 10 ~ 60, makes the vinyl-functional of activity in magnetic cluster surface modification；After reaction terminates, obtained with Magneto separate
Surface modification has the magnetic cluster of vinyl, and removes excessive silane coupler with absolute ethyl alcohol；Being then placed in vacuum drying oven is carried out
Dry；
Step (3)：25 ~ 500mg surface modifications that step (2) is obtained are had magnetic cluster, 0.1 ~ 5mL side chain bands of vinyl
The vinyl monomer of hydroxyl, 2mg ~ 2gN, N’- methylene-bisacrylamide, 1 ~ 80mg 2,2- azodiisobutyronitriles and solvent
20 ~ 400mL acetonitriles are added in 50 ~ 1000mL single-necked flasks, and ultrasound is well mixed it；Flask is connected to equipped with rectifying column
On reflux；From room temperature to fluidized state, then control reaction keeps 1 ~ 5h at 90 ~ 150 DEG C；Reaction uses magnetic after terminating
Separate, and washed with absolute ethyl alcohol, obtain the hydroxyl magnetic composite microsphere in surface；
Step (4)：Hydroxyl 0.1 ~ the 5g of magnetic composite microsphere in surface that step (3) is obtained adds 20 ~ 500mL acetonitriles
In, it is subsequently adding 0.1 ~ 3g DCC（N, N'- dicyclohexylcarbodiimide）, 0.01~0.5g DMAP（DMAP）,
0.1 ~ 3g RAFT reagents, ultrasonic disperse reacts 10 ~ 30 h in 20 ~ 120 DEG C.Reaction uses Magneto separate after terminating, and uses deionization
Water is washed, and obtaining surface modification has the magnetic composite microsphere of RAFT reagents.
Step (5)：By the surface modification that step (4) is obtained have RAFT reagents 0.1 ~ 5g of magnetic composite microsphere add 10 ~
In the dioxane of 500mL, vinyl monomer of 1 ~ 10mL side chains with carboxyl is subsequently adding, 1 ~ 50mg 2,2- azodiisobutyronitriles,
Lead to nitrogen 20-100 min after ultrasonic disperse, then heat to 60 ~ 100 DEG C, reaction carries out 5 ~ 50h.Reaction uses Magneto separate after terminating,
And washed with deionized water, obtaining surface modification has the magnetic composite microsphere of a large amount of PAA chains, obtains final product required product.
In the present invention, the acetate described in step (1) can be sodium acetate, ammonium acetate, potassium acetate, lithium acetate or magnesium acetate
In one kind, described citrate can be the one kind in citric acid or sodium citrate.
In the present invention, the silane coupler with double bond described in step (2) be KH570, VTES or
One kind in vinyltrimethoxy silane, or it is therein several.
In the present invention, the hydroxyl vinyl monomer of side chain described in step (3) is hydroxyethyl methacrylate, methyl-prop
One kind in olefin(e) acid hydroxypropyl acrylate or N hydroxymethyl acrylamide monomer, or it is therein several.
In the present invention, the hydroxyl vinyl monomer of side chain described in step (3) andN, N’- methylene-bisacrylamide
Concentration sum is the wt% of 0.001 wt% to 10.
In the present invention, described in step (3)N, N’The consumption of-methylene-bisacrylamide, withN, N’- di-2-ethylhexylphosphine oxide
The percent value of acrylamide consumption and the hydroxyl vinyl monomer consumption summation of side chain is 10 wt of wt % to 50 %.
In the present invention, vinyl monomer of the side chain with carboxyl described in step (4) is in acrylic or methacrylic acid
It is a kind of.
Nucleocapsid shell-type magnetic composite microsphere rich in polyelectrolyte prepared by the inventive method, its core is the oxidation of magnetic four three
Fe nanometer particles cluster, inner casing is the polymer network rich in hydroxyl of crosslinking, and shell is the strata electricity of Surface RAFT polymerization
Solution matter PAA chains.By the positive charge counter ion produced with the positive charge section displacement PAA chains of protein surface, it is possible to achieve Ji Husuo
There is the enrichment of albumen.
The magnetic composite microsphere that preparation method of the present invention is obtained, particle diameter distribution is homogeneous, compound with regular structure, and surface functional group
Density is very high, and separation high-abundance proteins are very competent, can be used for the super amount albumen of fast separating concentration, such as gone for high flux
Except the high-abundance proteins in blood and enrichment method low-abundance protein etc., excellent effect.The mg albumen of enrichment capacity ＞ 1100/g materials
Material, specially 1498 mg BSA（Bovine serum albumin(BSA)）/ g materials, 1178 mg HRP（Horseradish peroxidase）/ g materials,
1555 mg MYO（Myoglobins）/ g materials, 1233 mg LYS（Lysozyme）/ g materials.Therefore, the magnetic core shell-type is combined
Microballoon is a kind of bio-separation material for having very much an application prospect.
The problems such as magnetic composite microsphere is primarily present particle diameter distribution heterogeneity, surface functional group density is not high at present.This hair
It is bright be polymerized by RAFT prepare surface there are functional group densities high, the core-shell structure magnetic complex microsphere of uniform particle diameter, tool
There are following characteristics：(1) particle diameter distribution is homogeneous, compound with regular structure；(2) surface of core-shell magnetic composite microsphere is rich in a large amount of linear
Carboxyl functional group；(3) preparation process of core-shell magnetic composite microsphere is efficient；(4) the microballoon hydrophily is fine and can be used for
Separate various protein and separating effect is fabulous, principle is unique.
Brief description of the drawings
Fig. 1 be embodiment 1 in shell thickness be 100 nm core-shell type Fe3O4The transmission electricity of/PHEMA-RAFT-PAA microballoons
Mirror photo.
Fig. 2 is the magnetic material Fe of embodiment 23O4The electrophoretogram run out of before and after/PHEMA-RAFT-PAA enrichment standard proteins.
Wherein, swimming lane 1 is the band of standard molecular weight, and 2 is mixed protein before enrichment（BSA+HRP+MYO+LYS）, 3 is upper after being enriched with
Clear liquid, 4 is eluent.
Fig. 3 is the magnetic material Fe of embodiment 33O4The electrophoretogram run out of before and after/PHEMA-RAFT-PAA enriched standard albumen.
Wherein, swimming lane 5 is the band of standard molecular weight, and 6 is the mixed protein before dilution, and 7 is the mixed protein being diluted to during 6ng/ μ L,
8-12 is the eluent after enrichment when albumen is diluted to 6,3,2,1.2,0.6 ng/ μ L respectively.
Fig. 4 is the magnetic material Fe of embodiment 43O4/ PHEMA-RAFT-PAA or Fe3O4/ PHEMA-RAFT removals are complicated actual
Sample（a：Human plasma, b：Rabbit blood）The electrophoretogram run out of before and after middle high-abundance proteins.Wherein, swimming lane 13 or 19 is standard molecular weight
Band, 14 or 20 is human plasma or rabbit blood before enrichment, and 15 or 21 is to use Fe3O4Human blood after/PHEMA-RAFT-PAA enrichments
Slurry or rabbit blood supernatant, 16 or 22 is the eluent after 0.5%TFA wash-outs, and 17 or 23 is to use Fe3O4After/PHEMA-RAFT enrichments
Human plasma or rabbit blood supernatant, 18 or 24 be 0.5%TFA wash-out after eluent.
Fig. 5 is the magnetic material Fe of embodiment 53O4/ PHEMA-RAFT-PAA is to (a) BSA, richnesses of (b) MYO in different time
Collection capacity.
Fig. 6 is the magnetic material Fe of embodiment 63O4/ PHEMA-RAFT-PAA feeds intake to (a) BSA, (b) MYO in different albumen
Corresponding enrichment capacity during amount.
Fig. 7 is the magnetic material Fe of embodiment 73O4/ PHEMA-RAFT-PAA is to (a) BSA, and (b) MYO is corresponding in different pH
Enrichment capacity.
Fig. 8 is the Fe of the embodiment 8-11 difference degrees of cross linking3O4The transmission electron microscope picture of/PAA, wherein (a) 2%, (b) 5%, (c)
10%, (d) 20%.
Fig. 9 is the magnetic material Fe of embodiment 93O4/ PHEMA-RAFT-PAA and different degree of cross linking Fe3O4/ PAA to (a) BSA,
The enrichment Capacity Ratio of (b) MYO compared with（Calculated by whole ball or calculated by shell）, wherein (i-iv) is respectively Fe3O4/ PAA (is handed over
Connection degree is followed successively by 2%, 5%, 10% and 20%), and v is Fe3O4/PHEMA-RAFT-PAA。
Specific embodiment
Embodiment 1：Shell thickness is the core-shell type Fe of 100 nm3O4The preparation of/PHEMA-RAFT-PAA microballoons
1st, the preparation of the magnetic cluster of sodium citrate stabilization
By 1.3g Iron(III) chloride hexahydrates（FeCl3•6H2O）, 3.8g ammonium acetates（NH4Ac）, 0.4g sodium citrates are dissolved in
After in 70mL ethylene glycol, add in 150mL there-necked flasks, 170 DEG C, after stirring reaction 1h are then warmed up to, by liquid in flask
It is transferred in the autoclave containing polytetrafluoroethyllining lining that capacity is 100mL, then the baking oven that reactor is put into 200 DEG C is anti-
Taken out after answering 16h, room temperature is allowed to cool to running water.Product is isolated with Magneto separate, and is removed not with absolute ethanol washing
, finally be dispersed in for product standby in absolute ethyl alcohol by the reactant of reaction.
2nd, active ethylene group modification is carried out to magnetic cluster surface
By magnetic cluster derived above, 40 ml absolute ethyl alcohols, 10 ml deionized waters, 1.5 ml ammoniacal liquor and 0.6 g silane
Coupling agent KH 570 is added in 150ml there-necked flasks, is warmed up to 70 DEG C, and after 24 h of reaction, Magneto separate obtains product and with anhydrous
Ethanol washing removes excessive silane coupler.Vacuum drying oven is then placed in be dried.
3、Fe3O4The preparation of/PHEMA
Will more than dry after the product that obtains take about 100 mg and 80 ml acetonitriles are added in 200 ml single-necked flasks point together
Dissipate, add 400 μ L hydroxyethyl methacrylates（HEMA）、100 mgN, N’- methylene-bisacrylamide（MBA）、10
Mg 2,2- azodiisobutyronitriles（AIBN）, in dissolving it in reaction system.Then flask is connected to returning equipped with rectifying column
On stream device.From room temperature to fluidized state, control reacts 1 h at 110 DEG C.Reaction terminates rear Magneto separate and obtains product, and
Washed with absolute ethyl alcohol, finally given the Fe that shell thickness is 10 nm or so3O4/ PHEMA microballoons.
4、Fe3O4The preparation of/PHEMA-RAFT
The hydroxyl g of magnetic composite microsphere 0.2 in surface that step (3) is obtained is added in 100mL acetonitriles, is subsequently adding 0.1
G DCC, 0.01 g DMAP, 0.1 g RAFT reagents, ultrasonic disperse reacts 10 h in 25 DEG C.Reaction is divided after terminating with magnetic
From, and washed with deionized water, obtaining surface modification has the magnetic composite microsphere of RAFT reagents.
5、Fe3O4The preparation of/PHEMA-RAFT-PAA
The g of magnetic composite microsphere 0.1 that the surface modification that step (4) is obtained there are RAFT reagents is added the dioxy six of 30 mL
In ring, 3 mL acrylic acid are subsequently adding, 2 mg 2,2- azodiisobutyronitriles lead to the min of nitrogen 30, then heat to after ultrasonic disperse
70 DEG C, reaction carries out 20 h.Reaction uses Magneto separate after terminating, and is washed with deionized water, and obtaining surface modification has largely
The magnetic composite microsphere of PAA chains, as required product（See Fig. 1）.
Embodiment 2：Using the Fe that above-mentioned shell thickness is 100 nm3O4/ PHEMA-RAFT-PAA carries out separating different albumen
1st, 0.1 mg Fe are weighed first3O4/ PHEMA-RAFT-PAA magnetic particles, two are washed with 100 μ L deionized waters
Secondary, Magneto separate goes water removal.
2nd, BSA, HRP, MYO and each 5 μ g of lysozyme are subsequently added into, plus deionized water is incubated at room temperature to 100 μ L
Educate 5 minutes.
3 then Magneto separate collect supernatant, add 100 μ L deionized waters wash twice.
4th, finally eluted with 50 μ L solution (0.5 % TFA), all of stoste of vacuum drying, supernatant and washed
De- liquid, is separately added into 10 μ L bromophenol blue loading buffer denaturation, runs electrophoresis（See Fig. 2）.
Embodiment 3：Using the Fe that above-mentioned shell thickness is 100 nm3O4It is different that/PHEMA-RAFT-PAA carries out enrichment method
Concentration difference albumen
1st, 0.1 mg Fe are weighed first3O4/ PHEMA-RAFT-PAA magnetic particles, two are washed with 100 μ L deionized waters
Secondary, Magneto separate goes water removal.
2nd, then by five groups of BSA, the mixed protein of HRP, MYO and each 6 μ g of lysozyme（Totally 24 μ g）It is diluted to respectively
Each protein concentration is five groups of samples of the ng/ μ L of 6,3,2,1.2 and 0.6, and corresponding volume is respectively 1 mL, 2 mL, 3
ML, 5 mL and 10 mL, by 0.1 mg Fe3O4/ PHEMA-RAFT-PAA magnetic particles are separately added into above-mentioned five groups of samples,
It is incubated 5 minutes at room temperature.
3 then Magneto separate collect supernatant, it is each to add 100 μ L deionized waters to wash twice.
4th, finally eluted, vacuum drying stoste with 50 μ L solution (0.5 % TFA), 50 μ L of supernatant liquid and richness
Eluent during collection various concentrations albumen, is separately added into 10 μ L bromophenol blue loading buffer denaturation, runs electrophoresis（See Fig. 3）.
Embodiment 4：Using the Fe that above-mentioned shell thickness is 100 nm3O4The complicated actual sample of/PHEMA-RAFT-PAA removals
（Rabbit blood and human plasma）In high-abundance proteins
1st, 0.1 mg Fe are weighed first3O4/ PHEMA-RAFT-PAA or 0.1 mg Fe3O4/ PHEMA-RAFT magnetic grain
Son, is washed twice with 100 μ L deionized waters, and Magneto separate goes water removal.
2nd, 1 μ L human plasmas or 1 μ L rabbit blood are subsequently added into, plus deionized water is incubated 5 minutes at room temperature to 100 μ L.
3 then Magneto separate collect supernatant, add 100 μ L deionized waters wash twice.
4th, finally eluted with 50 μ L solution (0.5 % TFA).By stoste, divide after supernatant and eluent drying
Do not add 10 μ L bromophenol blue loading buffer to be denatured, run electrophoresis（See Fig. 4）.
Embodiment 5：Magnetic particle is to protein enrichment dynamics research
1st, 1 mg Fe are weighed first3O4/ PHEMA-RAFT-PAA magnetic particles, are washed twice with 100 μ L deionized waters,
Magneto separate goes water removal.
2nd, 1.8 mg MYO or 1.8 mg BSA are then separately added into（Each 900 μ L aqueous solution, i.e. 2 mg/mL）, in room temperature
Lower incubation different time, then surveys ultraviolet with ELIASA respectively.
3rd, compare the front and rear protein concentration difference of reaction by ultraviolet, can be calculated not with reference to the standard curve for determining before
With time corresponding enriching quantity（It is curve such as Fig. 5）.
Embodiment 6：Magnetic particle is enriched with capacity with the change of albumen input amount
1st, 1mg Fe are weighed first3O4/ PHEMA-RAFT-PAA magnetic particles, are washed twice, magnetic with 100 μ L deionized waters
Water removal is gone in separation.
2nd, different amounts of MYO or BSA is then separately added into, 5 min is incubated at room temperature, then survey purple with ELIASA respectively
Outward.
3rd, compare the front and rear protein concentration difference of reaction by ultraviolet, can be calculated not with reference to the standard curve for determining before
With the relation of albumen inventory and final enriching quantity（It is curve such as Fig. 6）.
Embodiment 7：Magnetic particle is enriched with capacity with the change of pH
1st, 1mg Fe are weighed first3O4/ PHEMA-RAFT-PAA magnetic particles, are washed twice, magnetic with 100 μ L deionized waters
Water removal is gone in separation.
2nd, 1.8 mg MYO or 1.8 mg BSA are then separately added into, different pH are adjusted to respectively with HCl or NaOH, in room temperature
5 min of lower incubation, then survey ultraviolet with ELIASA respectively.
3rd, compare the front and rear protein concentration difference of reaction by ultraviolet, can be calculated not with reference to the standard curve for determining before
With the relation of pH and final enriching quantity（It is curve such as Fig. 7）.
Embodiment 8：The degree of cross linking is the Fe of 2 %3O4The preparation of/PAA
1st, the preparation of the magnetic cluster of sodium citrate stabilization is with described in embodiment 1-1.
2nd, active ethylene group modification is carried out to magnetic cluster surface with described in embodiment 1-2.
3rd, core-shell type Fe3O4/PAA（The degree of cross linking is 2 %）Preparation
Will more than dry after the product that obtains take about 100 mg and 80 ml acetonitriles are added in 150 ml single-necked flasks point together
Dissipate, add 490 μ L acrylic acid, 10 mgN, N’- methylene-bisacrylamide, 10 mg 2,2- azodiisobutyronitriles make
It is dissolved in reaction system.Then flask is connected on the reflux equipped with rectifying column.From room temperature to boiling-like
State, control reacts 1 h at 110 DEG C.Reaction terminates rear Magneto separate and obtains product, and is washed with absolute ethyl alcohol, finally gives
Shell thickness is the Fe of 10 nm or so3O4/ PAA microballoons（Such as Fig. 8 a）.
Embodiment 9：The degree of cross linking is 5% Fe3O4The preparation of/PAA
1st, the preparation of the magnetic cluster of sodium citrate stabilization is with described in embodiment 1-1.
2nd, active ethylene group modification is carried out to magnetic cluster surface with described in embodiment 1-2.
3rd, core-shell type Fe3O4/PAA（The degree of cross linking is 5 %）Preparation with described in embodiment 8-3.Except that propylene
Acid,N, N’- methylene-bisacrylamide, the consumption of 2,2- azodiisobutyronitriles are respectively 475 μ L, 25 mg, 10 mg（As schemed
8b）.
Embodiment 10：The degree of cross linking is 10% Fe3O4The preparation of/PAA
1st, the preparation of the magnetic cluster of sodium citrate stabilization is with described in embodiment 1-1.
2nd, active ethylene group modification is carried out to magnetic cluster surface with described in embodiment 1-2.
3rd, core-shell type Fe3O4/PAA（The degree of cross linking is 10 %）Preparation with described in embodiment 8-3.Except that propylene
Acid,N, N’- methylene-bisacrylamide, the consumption of 2,2- azodiisobutyronitriles are respectively 450 μ L, 50 mg, 10 mg（As schemed
8c）.
Embodiment 11：The degree of cross linking is 20% Fe3O4The preparation of/PAA
1st, the preparation of the magnetic cluster of sodium citrate stabilization is with described in embodiment 1-1.
2nd, active ethylene group modification is carried out to magnetic cluster surface with described in embodiment 1-2.
3rd, core-shell type Fe3O4/PAA（The degree of cross linking is 20 %）Preparation with described in embodiment 8-3.Except that propylene
Acid,N, N’- methylene-bisacrylamide, the consumption of 2,2- azodiisobutyronitriles are respectively 400 μ L, 100 mg, 10 mg（Such as
Fig. 8 d）.
Embodiment 12：Compare Fe3O4The Fe of/PHEMA-RAFT-PAA and the different degrees of cross linking3O4Enrichments of/the PAA to albumen is held
Amount
1st, 1mg Fe are weighed first3O4The Fe of/the PHEMA-RAFT-PAA or 1 mg difference degrees of cross linking3O4/ PAA magnetic particles,
Washed twice with 100 μ L deionized waters, Magneto separate goes water removal.
2nd, then distinguish each addition 1.8 mg MYO or 1.8 mg BSA, 5 min are incubated at room temperature, enzyme is then used respectively
Mark instrument surveys ultraviolet.
3rd, compare the front and rear protein concentration difference of reaction by ultraviolet, richness can be calculated with reference to the standard curve for determining before
Collection capacity, compares enrichment capacity of the different materials to different albumen（Such as Fig. 9）.Fe3O4/ PHEMA-RAFT-PAA for BSA or
The Fe of the enrichment capacity than the different degrees of cross linking of MYO3O4/ PAA is much higher.
Claims (10)
1. a kind of preparation method of the core-shell magnetic composite microsphere containing polyelectrolyte, it is characterised in that concretely comprise the following steps：
（1）First, the magnetic Nano of citrate-stable is prepared as raw material with Iron(III) chloride hexahydrate, acetate and citrate
Particle cluster, abbreviation magnetic cluster；
（2）Then, magnetic cluster surface is modified using sol-gal process, its surface is taken the vinyl-functional of activity；
（3）Then, the magnetic cluster of vinyl is contained as seed with surface, by the method for the precipitation polymerization that flows back in magnetic cluster Surface coating
One layer of cross-linked network of the hydroxyl polymer-containing of densification, obtain with magnetic cluster as core, magnetic of the hydroxyl polymer-containing network as shell gather
Compound complex microsphere；
（4）Then, using the hydroxyl polymer-containing network in esterification modification previous step, the RAFT examinations on its surface grafting
Agent；
（5）Finally, trigger PAA chains in RAFT polymerization modifications on the magnetic composite microsphere surface, obtain final product required magnetic composite microsphere.
2. preparation method according to claim 1, it is characterised in that the specific operation process of each step is：
Step (1) operating process：By the dissolving of 1 ~ 30g Iron(III) chloride hexahydrates, 1 ~ 60g acetate and 0.1 ~ 20g citrates
In 20 ~ 500mL ethylene glycol, 0.5 ~ 5h of mechanical agitation, is subsequently placed in containing polytetrafluoroethyllining lining not at 100 ~ 200 DEG C
In rust steel autoclave, reactor is positioned over 10 ~ 50h in 100 ~ 300 DEG C of baking oven, taken out, be allowed to cool with running water
To room temperature；Product magnetic cluster is isolated with magnet, and unreacted reactant is removed with absolute ethanol washing, finally by product magnetic cluster
It is dispersed in absolute ethyl alcohol, it is standby；
Step (2) operating process：0.1g ~ 3g magnetic cluster that step (1) is obtained, 20 ~ 400mL absolute ethyl alcohols, 5 ~ 100mL go from
The silane coupler of sub- water, 0.5 ~ 10mL ammoniacal liquor and 0.2 ~ 10g with double bond is added in there-necked flask, at a temperature of 50 ~ 100 DEG C
The h of mechanical agitation 10 ~ 60, makes the vinyl-functional of activity in magnetic cluster surface modification；After reaction terminates, table is obtained with Magneto separate
Face is modified with the magnetic cluster of vinyl, and removes excessive silane coupler with absolute ethyl alcohol；Vacuum drying oven is then placed in be done
It is dry；
Step (3) operating process：25 ~ 500mg surface modifications that step (2) is obtained are had magnetic cluster, 0.1 ~ 5mL sides of vinyl
The hydroxyl vinyl monomer of chain, 2mg ~ 2gN, N’- methylene-bisacrylamide, 1 ~ 80mg 2,2- azodiisobutyronitriles and
20 ~ 400mL of solvent acetonitriles are added in single-necked flask, and ultrasound is well mixed it；Flask is connected to the backflow equipped with rectifying column
On device；From room temperature to fluidized state, then control reaction keeps 1 ~ 5h at 90 ~ 150 DEG C；Reaction is divided after terminating with magnetic
From, and washed with absolute ethyl alcohol, obtain the hydroxyl magnetic composite microsphere in surface；
Step (4) operating process：Hydroxyl 0.1 ~ the 5g of magnetic composite microsphere in surface that step (3) is obtained adds 20 ~ 500mL
In acetonitrile, 0.1 ~ 3g DCC, 0.01 ~ 0.5g DMAP, 0.1 ~ 3g RAFT reagents, ultrasonic disperse, in 20 ~ 120 are subsequently adding
DEG C reaction 10 ~ 30 h；Reaction uses Magneto separate after terminating, and is washed with deionized water, and obtaining surface modification has RAFT reagents
Magnetic composite microsphere；
Step (5) operating process：0.1 ~ 5g of magnetic composite microsphere that the surface modification that step (4) is obtained there are RAFT reagents is added
Enter in the dioxane of 10 ~ 500mL, be subsequently adding vinyl monomer of 1 ~ 10mL side chains with carboxyl, 1 ~ 50mg 2,2- azos two are different
Butyronitrile, leads to nitrogen 20-100 min after ultrasonic disperse, then heat to 60 ~ 100 DEG C, and reaction carries out 5 ~ 50h；Reaction uses magnetic after terminating
Separate, and washed with deionized water, obtaining surface modification has the magnetic composite microsphere of PAA chains, obtains final product required product.
3. preparation method according to claim 2, it is characterised in that the acetate described in step (1) is sodium acetate, vinegar
One kind in sour ammonium, potassium acetate, lithium acetate or magnesium acetate, described citrate is sodium citrate.
4. preparation method according to claim 2, it is characterised in that the silane coupler with double bond is described in step (2)
One kind in KH570, VTES or vinyltrimethoxy silane, or it is therein several.
5. preparation method according to claim 2, it is characterised in that the hydroxyl alkenes list of side chain described in step (3)
Body is the one kind in hydroxyethyl methacrylate, hydroxy propyl methacrylate or N hydroxymethyl acrylamide monomer, or therein
It is several.
6. described method is prepared according to claim 2, it is characterised in that the hydroxyl alkenes list of side chain described in step (3)
Body andN, N’The concentration sum of-methylene-bisacrylamide is the wt% of 0.001 wt% to 10.
7. preparation method according to claim 2, it is characterised in that described in step (3)N, N’- di-2-ethylhexylphosphine oxide third
The consumption of acrylamide, withN, N’The percentage of-methylene-bisacrylamide consumption and the hydroxyl vinyl monomer consumption summation of side chain
Ratio is 10 wt of wt % to 50 %.
8. preparation method according to claim 2, it is characterised in that alkenes list of the side chain with carboxyl described in step (5)
Body is the one kind in acrylic or methacrylic acid.
9. the core-shell magnetic composite microsphere containing polyelectrolyte that a kind of preparation method as claimed in claim 1 or 2 is obtained.
10. the core-shell magnetic composite microsphere of polyelectrolyte is contained as claimed in claim 9, in the various albumen of separation and concentration
In application.
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| CN106432644B (en) * | 2016-09-23 | 2018-09-07 | 河南中医药大学 | A kind of hydrophilic polymers functional magnetic nanoparticle and its preparation method and application |
| CN106362703B (en) * | 2016-10-12 | 2018-10-30 | 四川大学 | Modified carragheen-chitosan polyelectrolyte microsphere and its preparation method and application |
| CN109012630B (en) * | 2018-08-30 | 2021-06-08 | 健帆生物科技集团股份有限公司 | Magnetic composite adsorbent for blood perfusion, preparation method thereof and perfusion apparatus |
| CN110237048B (en) * | 2019-04-22 | 2022-11-01 | 天津市泌尿外科研究所 | PEG-modified magnetic/quaternized chitosan nanoparticles with peroxidase activity and preparation method and application thereof |
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| CN101220187A (en) * | 2008-01-31 | 2008-07-16 | 复旦大学 | Magnetic composite microsphere with nucleocapsid structure and method for producing the same |
| CN102432780A (en) * | 2011-09-16 | 2012-05-02 | 复旦大学 | Surface carboxyl functionalized core-shell type magnetic composite microsphere and preparation method thereof |
| EP1802662B1 (en) * | 2004-10-04 | 2013-01-23 | The University Of Sydney | Surface polymerisation process and polymer product using raft agent |
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| EP1802662B1 (en) * | 2004-10-04 | 2013-01-23 | The University Of Sydney | Surface polymerisation process and polymer product using raft agent |
| CN101220187A (en) * | 2008-01-31 | 2008-07-16 | 复旦大学 | Magnetic composite microsphere with nucleocapsid structure and method for producing the same |
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