CN104277176B - Preparation method for fluorescent western-blotting magnetic composite microballoon - Google Patents
Preparation method for fluorescent western-blotting magnetic composite microballoon Download PDFInfo
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Abstract
The invention belongs to the field of material science and engineering and bioseparation engineering, and concretely relates to a preparation method for a fluorescent western-blotting magnetic composite microballoon. A miniemulsion polymerization method is employed to take phycocyanin as a molecular imprinting template and combine with a magnetically-sensitive fluorescent material, so that the phycocyanin molecular-imprinted-polymer magnetic fluorescent composite microballoon is obtained. By using the miniemulsion polymerization method, the molecular-imprinted-polymer microballoon integrates the magnetic and fluorescence double-responsive material, has specific recognition and autonomous adsorption on phycocyanin, is good in selectivity, high in adsorption efficiency, fast in speed, convenient and rapid for separation under the effect of an external magnetic field, and good in repeated usage performance, and is capable of realizing fluorescence real-time detection and fluorescence imaging at a relatively wide pH scope. Also the obtained magnetic core-shell blotting microballoon is regular in morphology and uniform in particle size. The preparation method has the advantages of low cost, simple and convenient operation, good reappearance and the like, and has wide application prospect.
Description
Technical field
The invention belongs to Materials Science and Engineering and bio-separation engineering field, a kind of specifically fluorescin trace
The preparation method of magnetic composite microsphere.
Background technology
Molecular imprinting refers to that preparing the polymer having specific selectivity to a certain specific target molecules is molecule print
Mark polymer（Molecularly Imprinted Polymer, MIP）Program, often visually be depicted as manufacture identification " point
The technology of " the artificial lock " of sub- key ".Because protein structure is flexible, conformation is complicated, it is highly prone to the environmental factors such as temperature
Impact, in addition high-crosslinking-degree material can stop protide macromolecular from freeing in and out polymer network, on the one hand leads to template molecule
It is difficult to remove, on the other hand cause template molecule to be difficult to enter imprinted sites a series of problems, such as recombine therewith, these are all
Lead to the exploitation of protein molecule engram material hindered.
Phase early 1970s, Ugelstad, El-Aasser and Vanderhoff of Lehigh university of the U.S. etc. are a collection of
Scholar, it is proposed that new particle nucleation mechanism causes nucleation in sub-micro monomer droplet, develops miniemulsion
（miniemulsion）Polymerization new technology.Mini-emulsion polymerization has most of advantage of conventional emulsion polymerization concurrently, and has following excellent
Gesture：System stability is high, is conducive to industrial enforcement；The particle diameter of product latex is larger, and by the use of assistant for emulsifying agent
Amount is easily controlled；Rate of polymerization is moderate, produces easily controllable；Introduce assistant for emulsifying agent in system, and employ microemulsified
Technique, so makes originally larger monomer droplet be dispersed into less monomer submicron droplets.
Mostly it is by static separation for the molecular blotting polymer microsphere without magnetic component for the separation engineering field
Or Dynamic Separation, need to separate through processes such as centrifugations, operating process is loaded down with trivial details, complicated.And imbed magnetic response inside polymer microballoon
During property material, complex microsphere then has easy under additional magnetic fields, quick Magneto separate characteristic.When burying in complex microsphere
After entering magnetic responsiveness material, just can after it completes to " actively " of template molecule absorption and identification under additional magnetic fields very
Easily it is separated, reach autonomous classification and separate easy purpose.
Phycocyanin is to exist only in the very rare natural nutrient of one of blue-green algae.Fluorescent phycocyanin is from spiral shell
Novel fluorescence label isolating and purifying in rotation algae, having unique optical properties, has excellent property：Compared with wide spectrum model
There is higher absorption coefficient in enclosing；There is higher fluorescent yield in the range of wider pH；Their fluorescence is not because of other biological points
Son presence and disappear；There is preferable water solubility；All extremely stable in the presence of liquid or solid state, and when can preserve longer
Between.Phycocyanin thus receive significant attention.
Content of the invention
It is an object of the invention to provide a kind of prepare fluorescin trace magnetic composite microsphere using mini-emulsion polymerization
Method.
The technical solution used in the present invention is for achieving the above object：
A kind of preparation method of fluorescin trace magnetic composite microsphere, with fine emulsion polymerization will using phycocyanin as
The molecular engram of template is combined with magnetic susceptibility and fluorescent material, that is, obtain phycocyanin molecular engram polymer magnetic and
Epipolic complex microsphere.
The preparation of described fluorescin trace magnetic composite microsphere：
a.Fe3O4Prepared by magnetic material：By FeCl3·6H2O is dissolved in ethylene glycol, and using sodium acetate as reducing agent, gathers
Ethylene glycol is that macromolecule stabilizer dissolves through sonic oscillation, and reacts in airtight hydrothermal reaction kettle, that is, obtain black Fe3O4
Magnetic material；
B. a step miniemulsion polymerization synthesis superparamagnetism carrier particle：Above-mentioned particle wraps up oleic acid, adds methacrylic acid
Methyl esters, Ethylene-glycol-dimethacrylate mix, and are added in dodecyl sodium sulfate and hexadecanol solution system after mixing, are formed
Miniemulsion, miniemulsion drops in sodium dodecyl sulfate solution, and deoxygenation then adds ammonium persulfate, carries out polymerisation,
It is dried after washing and collect, stand-by；
C. carrier particle surface modification：
（1）Amination：Above-mentioned gained superparamagnetism carrier particle is dispersed in DMF, adds reacting ethylenediamine, wash
Wash；
（2）Aldehyde-functionalized：Above-mentioned amidized particle is added the cushioning liquid of acetic acid and sodium acetate, deoxygenation, remove slow
Rush liquid, add in glutaraldehyde, reaction, washing；
D. fixed form molecule：The aldehyde-functionalized polymer beads of above-mentioned preparation, are washed with phosphate buffer, add
Phycocyanin template solution is reacted, washing；
E. two step miniemulsion polymerization synthesis outer shell：Above-mentioned particle adds methyl methacrylate, dimethacrylate second
Diester mixes, and is added in dodecyl sodium sulfate and hexadecanol solution system, forms miniemulsion, and miniemulsion drops to dodecane
In base sodium sulfonate solution, deoxygenation, add ammonium persulfate and sodium hydrogensulfite, reaction, washing；
F. template molecule elution：Above-mentioned particle is added in NaOH solution, hydrolysis, washs and be dried.
Further, described step a.Fe3O4Prepared by magnetic material：Weigh 3.00g FeCl3·6H2O is substantially soluble in
In 80-100mL ethylene glycol, add 7.2g sodium acetate, ultrasonic and mechanical agitation is uniformly dissolved, and is subsequently adding the poly- second of 2.0-4.0g two
Alcohol, is slowly stirred to dissolving at 60 DEG C, is transferred completely into yellow liquid airtight with polytetrafluoroethylliner liner after solvent heat
In hydrothermal reaction kettle, react 8-12h at 190-210 DEG C, be cooled to room temperature after reaction, that is, obtain magnetic black Fe3O4Magnetic material
Material.
Described magnetic black Fe3O4Magnetic material is respectively washed 4-8 time with ultra-pure water and absolute ethyl alcohol successively, by products therefrom
12-24h is dried, collects dry Fe3O4Magnetic material.
Further, described step b. mono- step miniemulsion polymerization synthesis superparamagnetism carrier particle：Take 1.0g above-mentioned
Fe3O4Magnetic material and 1mL oleic acid are sufficiently mixed the viscogel obtaining black, add in the viscogel of gained black by
Mol ratio is 1:The methyl methacrylate of 4-6 mixing and Ethylene-glycol-dimethacrylate（Preferably, the viscogel of gained black
In add 1.28mL methyl methacrylate and 9.05mL Ethylene-glycol-dimethacrylate）, be sufficiently mixed uniformly, after mixing
Ultrasonically treated 90-100s under 65% power；
It is added dropwise to 0.01mol/L lauryl sodium sulfate and the 0.03mol/L hexadecanol of 50mL after sonicated
Mixed solution in, acutely rock after dropping, after mixing, form miniemulsion through ultrasonic 90-100s under 65% power again；
By above-mentioned gained miniemulsion by 0.05% dodecyl sodium sulfate being added dropwise to 600mL, after dropping at 65 DEG C nitrogen
10-15min is swept in air-blowing, adds 0.5g ammonium sulfate, initiated polymerization, be warming up to 70 after polymerisation 1-1.5h after nitrogen treatment
DEG C continue reaction 24h, obtain final product mini-emulsion polymerization synthesis superparamagnetism carrier particle.
Described polymerization resulting polymers carrier particle milli-Q water 3-5 time, is washed 2-3 time with 50% ethanol, then with surpassing
Pure water is washed 2-3 time, spontaneously dries 24h, stand-by.
Further, described step c. carrier particle surface modification：
With DMF, the 1g polymer support particle of above-mentioned preparation is washed and evenly spread in the DMF of 20mL, subsequently, to
The ethylenediamine of 20mL is added, with mechanical agitation back flow reaction under the rotating speed of 200-400r/min at 100-120 DEG C in mixture
12h；Product milli-Q water 1-2 time, is washed 2-3 time with 50% ethanol, then with ultrapure washing 2-3 time；
（2）Aldehyde-functionalized：The polymer support processing through amination particle 1g is immersed in the cushioning liquid of 10mL, and
Deaerate 10-15min at room temperature, then removes buffer solution, particle is dispersed in 5% glutaraldehyde of brand-new, so that mixture is existed
12h is reacted under the rotating speed mechanical agitation of room temperature 200-400r/min；Milli-Q water 2-3 time；Described cushioning liquid is 5 for pH value
± 0.1 acetic acid and sodium acetate buffer solution.
Described state cushioning liquid for pH value be 5 ± 0.1 acetic acid and sodium acetate buffer solution be by volume be 3：6.8-
The 0.2mol/L acetic acid of 7.2 mixing and 0.2mol/L sodium acetate.
Further, described step e. two step miniemulsion polymerization synthesis outer shell：By 1.28mL methyl methacrylate,
The surface modification of 9.05mL Ethylene-glycol-dimethacrylate and 1.0g superparamagnetism nuclear particle mixing, then by mixture with
40-50% power ultrasonic 90-100s, after being sufficiently mixed uniformly, gained mixture is added dropwise to the 0.01mol/L's of 50mL
The mixed solution of the hexadecanol of lauryl sodium sulfate and 0.03mol/L, mixture is acutely rocked, ultrasonic under 65% power
90s forms miniemulsion, and miniemulsion is dropwise added in the dodecyl sodium sulfate of the 0.05% of 600mL, and then at 40 DEG C, nitrogen blows
Sweep 10-15min, will be 1 in mass ratio:The ammonium persulfate of 1 mixing and sodium hydrogensulfite are added in this mixture, cause polymerization
Reaction, reacts 16-24h, after the completion of, washing and drying is stand-by.
Advantage for present invention：
Instant invention overcomes a variety of difficulties of protein molecule engram and mini-emulsion polymerization, the complex microsphere pair finally giving
The repeatability of the specific adsorption efficiency highest of template protein and complex microsphere is strong.Gained magnetic core-shell structure copolymer trace microballoon pattern
Rule, uniform particle diameter, the method that the present invention specifically applies mini-emulsion polymerization, molecular blotting polymer microsphere is compounded with magnetic
Property and the double responsive materials of fluorescence, have specific adsorption to phycocyanin.
Particularly the present invention passes through fine emulsion polymerization by proteins molecularly imprinted technique selectivity and magnetic susceptibility and fluorescence
Property combine, give the certain magnetic of molecular blotting polymer microsphere and fluorescent dual response characteristic, reach protein molecule print
Mark polymer magnetic complex microsphere specific recognition is adsorbed with autonomous, and convenient, quick separating under additional magnetic fields, uses
The purpose that the fluorescent instruments such as fluorescence spectrum easily detect in real time.
In gained molecular blotting polymer microsphere of the present invention, the compound double responsive materials with fluorescence that are magnetic, have to phycocyanin
Specific adsorption, selectively good, adsorption efficiency is high, and mass transfer rate is fast, and repeat performance is good.Wherein being compounded with magnetic intensity is
10.770emu/g Fe3O4Magnetic Nano material；Template protein fluorescence property is stable simultaneously, has higher in compared with wide spectral range
Absorption coefficient, has higher fluorescent yield, good water solubility in the range of wider pH, measures analysis beneficial to quick, and be used for it
Its mass signatures.
Brief description
Fig. 1 is nucleocapsid trace magnetic composite microsphere preparation process schematic diagram provided in an embodiment of the present invention.
Fig. 2A is magnetic composite microsphere adhesion protein design sketch provided in an embodiment of the present invention；
Fig. 2 B is magnetic composite microsphere adhesion protein hysteresis curve figure provided in an embodiment of the present invention.
Fig. 3 A is the impact effect figure of the fluorescence to phycocyanin for the pH provided in an embodiment of the present invention；
Fig. 3 B is that the fluorescence to 0.1mg/L-0.7mg/L concentration range phycocyanin solution provided in an embodiment of the present invention rings
Answer collection of illustrative plates.
Fig. 4 is application drawing in SDS-PAGE electrophoresis for the nucleocapsid trace magnetic composite microsphere provided in an embodiment of the present invention,
Wherein No. 1 swimming lane is protein mark swimming lane, and No. 2 is the swimming lane of original protein solution, and No. 3 is micro- through nucleocapsid trace magnetic coupling
Ball absorption after protein solution, No. 4 be through non-nucleocapsid trace magnetic composite microsphere absorption after protein solution.
Fig. 5 adsorbs algae indigo plant egg for nucleocapsid trace magnetic composite microsphere provided in an embodiment of the present invention in HeLa cell environment
The fluoroscopic image of white solution；Wherein A, B, C are respectively HeLa cell, HeLa cell adds phycocyanin, HeLa cell adds algae
Azurin the fluorescence photo with exciting in 559nm after the absorption of nucleocapsid trace magnetic composite microsphere, G, H, I are respectively A, B, C and exist
Photo under light field, D is the synthesising picture of A and G, and E is the synthesising picture of B and H, and F is the synthesising picture of C and I.
Specific embodiment
The present invention is initially with a step miniemulsion polymerization synthesis Fe3O4The superparamagnetism carrier particle of base, is carried out surface
Amination and aldehyde-functionalized modification, fixation has the phycocyanin template molecule of photoluminescent property, then adopts two step miniemulsion to gather
Synthesis outer shell, gained magnetic core-shell trace microballoon pattern rule, uniform particle diameter.The method of application mini-emulsion polymerization, is dividing
It is compounded with magnetic and the double responsive materials of fluorescence in sub- imprinted polymer microballoon, there is to phycocyanin specific recognition and inhale with autonomous
Attached, selectively good, adsorption efficiency is high, and speed is fast, and convenient, quick separating under additional magnetic fields, and repeat performance is good,
And carry out fluorescence real-time detection and fluorescence imaging in the range of wider pH.This law has low cost, simple and efficient to handle, reproduction
The advantages of property is good, is with a wide range of applications.
Embodiment 1
a.Fe3O4Prepared by magnetic material：Weigh the FeCl of 3.00g3·6H2O is dissolved in 90mL ethylene glycol, adds 7.2g vinegar
Sour sodium, ultrasonic and mechanical agitation dissolving.It is subsequently adding 3.9g polyethylene glycol to stir to dissolving with 400r/min at 60 DEG C.Dissolving
Afterwards yellow liquid is transferred completely in the airtight hydrothermal reaction kettle with polytetrafluoroethylliner liner, anti-in 200 DEG C of drying boxes
Answer 10h.After being cooled to room temperature, magnetic capture atrament, use ultra-pure water and absolute ethyl alcohol respectively washing 6 times successively, gained is produced
Thing is dried 24h, collects dry Fe3O4Magnetic material.
B. a step miniemulsion polymerization synthesis superparamagnetism carrier particle：The above-mentioned Fe of 1.0g3O4Magnetic material and the oleic acid of 1mL
It is mixed to get the viscogel of black.Mol ratio is 1:4 methyl methacrylate（1.28mL）With dimethacrylate second two
Ester（9.05mL）Add the magnetic material of Coated with Oleic Acid, and be sufficiently mixed.Then mixture is ultrasonically treated under 65% power
80s.After mixing, gained mixture is added dropwise to the 0.01mol/L dodecyl of 50mL with 2-3 drop/sec of speed
In the mixed solution of the hexadecanol of sodium sulfonate and 0.03mol/L.Mixture is acutely rocked, ultrasonically treated 90s under 65% power
Form miniemulsion.Miniemulsion is added dropwise in 0.05% dodecyl sodium sulfate of 600mL with 2-3 drop/sec of speed.Should
Reactant mixture is transferred in the three neck round bottom of a 1L, and nitrogen purging 15min at 65 DEG C, by ammonium persulfate（0.5g）
It is added in reactant mixture, initiated polymerization, be warming up to 70 DEG C after reaction 1-1.5h and continue reaction, continue 24h.Complete
Afterwards, polymer support particle milli-Q water 3 times, is washed 3 times with 50% ethanol, then with ultrapure washing 3 times.Spontaneously dry
24h, collects.
C. carrier particle surface modification：
（1）Amination：With DMF, the polymer support particle of above-mentioned for 1g preparation is washed twice and be distributed to the DMF of 20mL
In.Subsequently, add the ethylenediamine of 20mL in mixture, with the rotating speed of 400r/min, at 110 DEG C, mechanical agitation backflow is anti-
Answer 12h.Product milli-Q water 1 time, the washing 2 times of the ethanol with 50%, then with ultrapure washing 2 times.
（2）Aldehyde-functionalized：Secure ph is 5 acetic acid and the cushioning liquid of sodium acetate（Be is 3 by volume：7 mixing
0.2mol/L acetic acid and 0.2mol/L sodium acetate）.Polymer support particle above-mentioned for 1g is immersed in the cushioning liquid of 10mL
In, and the 10min that deaerates at room temperature.Then remove this buffer solution, this particle is dispersed in brand-new 10mL 5% glutaraldehyde
In.By this mixture under the rotating speed of room temperature 400r/min mechanic whirl-nett reaction 12h.Then, with milli-Q water 3 times.
D. fixed form molecule：The aldehyde-functionalized polymer beads of above-mentioned preparation, with the phosphate-buffered of 0.01mol/L
Liquid（PBS）Washing 1 time.Phycocyanin solution to 20mL（1mg/mL）The middle addition above-mentioned particle of 1.0g.Gained mixed system is put
In 4 DEG C, mechanical agitation 3h under the rotating speed of 300r/min, carry out coupled reaction.After the completion of reaction, with milli-Q water, this is fixed
Phycocyanin template 3 times.
E. two step miniemulsion polymerization synthesis outer shell：Methyl methacrylate（1.28mL）, Ethylene-glycol-dimethacrylate
（9.05mL）Superparamagnetism nuclear particle mixing with the above-mentioned surface modification being fixed with template molecule of 1.0g.Then by mixture
With 45% power ultrasonic 90s, to guarantee to be thoroughly mixed.After mixing, by gained mixture with 2-3 drop/sec of speed dropwise
It is added in the dodecyl sodium sulfate of the 0.01mol/L of 50mL and the mixed solution of the hexadecanol of 0.03mol/L.By mixture
Acutely rock, under 65% power, ultrasonic 90s forms miniemulsion.Miniemulsion is added dropwise to 600mL's with 2-3 drop/sec of speed
In 0.05% dodecyl sodium sulfate.This reactant mixture is transferred in the three neck round bottom of a 1L, nitrogen at 40 DEG C
15min is swept in air-blowing, by ammonium persulfate（0.25g）And sodium hydrogensulfite（0.25g）It is added in this mixture to cause polymerization anti-
Should, react 24h.After the completion of, the magnetic core-shell structure copolymer microballoon of this polymer milli-Q water 3 times is washed 3 times with 50% ethanol,
Washed 3 times with ultrapure again.Spontaneously dry 24h, collect.
F. template molecule elution, removes fixing phycocyanin template molecule by basic hydrolysis, specially：20mL's
The magnetic core-shell structure copolymer microballoon of 1.0g is added in the NaOH solution of 1.0mol/L.With the rotating speed stirring hydrolysis of 300r/min at 35 DEG C
Mixture, reacts 5h.By these surface imprinted particles milli-Q water 3 times, spontaneously dry 24h, obtain final product fluorescin print
Mark magnetic composite microsphere（Referring to Fig. 1）.
Non- imprinted polymer preparation：According to aforesaid operations code, simply it is not added with outside template molecule phycocyanin, other steps
Suddenly ibid.
Embodiment 2
Weigh phycocyanin 20mg, with redistilled water constant volume in the volumetric flask of 1L, obtain the phycocyanin solution of 20mg/L,
The phycocyanin solution taking 10mL is added in the rib collar bottle of 20mL, adds the fluorescin trace magnetic coupling of 100mg micro-
Ball, vibration absorption 10min, adsorbs 2min with magnet in the side of bottle, you can complex microsphere is adsorbed onto side, is clarified
Solution（Referring to Fig. 2A）.Illustrate that this complex microsphere has strong adsorption capacity to phycocyanin, and separation process be quick, simple,
Higher referring to this microballoon magnetic intensity shown in Fig. 2 B.
Embodiment 3
Take 7 parts of the 100mg/L phycocyanin solution of 200 μ L, be respectively placed in the plastic centrifuge tube of 10mL, then respectively to
The PBS of different pH value is added in each pipe（Na2HPO4And NaH2PO4It is respectively 0.2mol/L, volume ratio 1：1）
9.8mL, dilution obtains the 2mg/L phycocyanin solution that pH value is respectively 5.6,6.0,6.4,6.8,7.2,7.6 and 8.0, with glimmering
Light spectrophotometer measures its corresponding fluorescence intensity respectively, can get the fluorescence intensity when pH value is 6.4 with maximum（Referring to
Fig. 3 A）, and increase with the concentration of phycocyanin, fluorescence intensity is gradually increased（Referring to Fig. 3 B）.
Embodiment 4
Weigh lysozyme（LZM）, chicken serum albumin（CEA）, bovine serum albumin（BSA）And phycocyanin（PC）Each 6mg,
Mixing, is subsequently adding 6mL redistilled water, obtains the mixed protein solution of 1mg/mL.2mL mixed solution is taken to be added to three glass respectively
In glass centrifuge tube, it is numbered 2,3,4.Separately take a glass centrifuge tube to be numbered 1, add mark protein solution.In No. 2 centrifuge tubes
Do not carry out any process, the magnetic blotting microballoon of No. 3 addition 20mg, the non-imprinted polymer of No. 4 addition 20mg, then will be centrifuged
Pipe is respectively placed in vibration absorption 6h, centrifuging and taking supernatant 40 μ L on oscillator, carries out SDS-PAGE electrophoresis experiment.Referring to Fig. 4,4
Number swimming lane compare No. 2 swimming lanes, the band of 4 kinds of albumen is not changed in substantially, and the band of No. 3 swimming lane PC substantially weakens, other
Protein band does not have significant change.Result shows that this trace complex microsphere has high selectivity to phycocyanin, can carry out special
Property absorption.
Embodiment 5
Culture HeLa cell, adds cover glass on the Tissue Culture Plate in 24 holes, takes the cell liquid of 9 parts of 1mL to add respectively
Cultivate on Tissue Culture Plate, and mark No. 1-9, treat cell normal growth on the cover slip, with No. 1-3 as a control group, no
Plus any solution, in taking the phycocyanin solution of the 10mg/mL of 60 μ L to add No. 4-9, cultivate 6h, add 100 toward 7-9 sample
The non-imprinted polymer of the 0.1mg/mL of μ L is adsorbed, slight oscillatory, takes cover glass therein in confocal microscope
Under observed.Referring to Fig. 5, there is no the sample processing, fluorescence intensity is very big in space between cells;Add trace complex microsphere it
Afterwards, in cellular environment, fluorescence intensity substantially weakens, and several fluorescence bright spots of appearance are trace microballoon, and this trace complex microsphere is described
Can in cellular environment specific adsorption phycocyanin.
Claims (8)
1. a kind of preparation method of fluorescin trace magnetic composite microsphere it is characterised in that：Will be with algae with fine emulsion polymerization
Azurin is combined with magnetic susceptibility and fluorescent material as the molecular engram of template, that is, obtain phycocyanin molecular engram and gather
Compound magnetic and epipolic complex microsphere；
The preparation of described fluorescin trace magnetic composite microsphere：
a.Fe3O4Prepared by magnetic material：By FeCl3·6H2O is dissolved in ethylene glycol, and using sodium acetate as reducing agent, polyethylene glycol
For macromolecule stabilizer, it is slowly stirred to dissolving at 60 DEG C, and reacts in airtight hydrothermal reaction kettle, that is, obtain black Fe3O4
Magnetic material；
B. a step miniemulsion polymerization synthesis superparamagnetism carrier particle：Above-mentioned black Fe3O4Magnetic material wraps up oleic acid, adds first
Base methyl acrylate, Ethylene-glycol-dimethacrylate mix, and are added to dodecyl sodium sulfate and hexadecanol solution body after mixing
In system, form miniemulsion, miniemulsion drops in sodium dodecyl sulfate solution, then adds ammonium persulfate, is polymerized
Reaction, is dried after washing and collects, stand-by；
C. carrier particle surface modification：
(1) amination：Above-mentioned gained superparamagnetism carrier particle is dispersed in DMF (DMF), adds
Reacting ethylenediamine, washing；
(2) aldehyde-functionalized：Above-mentioned amidized particle is added the cushioning liquid of acetic acid and sodium acetate, add in glutaraldehyde, instead
Should, washing；
D. fixed form molecule：Step c gained particle, is washed with phosphate buffer, adds the reaction of phycocyanin template solution,
Washing；
E. two step miniemulsion polymerization synthesis outer shell：Step d gained particle adds methyl methacrylate, dimethacrylate second
Diester mixes, and is added in dodecyl sodium sulfate and hexadecanol solution system, forms miniemulsion, and miniemulsion drops to dodecane
In base sodium sulfonate solution, deoxygenation, add ammonium persulfate and sodium hydrogensulfite, reaction, washing；
F. template molecule elution：Above-mentioned particle is added in NaOH solution, hydrolysis, washs and be dried.
2. the fluorescin trace magnetic composite microsphere as described in claim 1 preparation method it is characterised in that：Described step
a.Fe3O4Prepared by magnetic material：Weigh 3.00g FeCl3·6H2O is substantially soluble in 70-100mL ethylene glycol, adds 7.2g acetic acid
Sodium, so as to be uniformly dissolved, is subsequently adding 2.0-4.0g polyethylene glycol, is slowly stirred to dissolving at 60 DEG C, by yellow liquid after dissolving
Body is transferred completely in the airtight hydrothermal reaction kettle with polytetrafluoroethylliner liner, reacts 8-12h, reaction at 190-210 DEG C
After be cooled to room temperature, that is, obtain magnetic black Fe3O4Magnetic material.
3. the fluorescin trace magnetic composite microsphere as described in claim 1 or 2 preparation method it is characterised in that：Described
Magnetic black Fe3O4Magnetic material is respectively washed 4-8 time with ultra-pure water and absolute ethyl alcohol successively, and products therefrom is dried 12-24h, collects
The Fe being dried3O4Magnetic material.
4. the fluorescin trace magnetic composite microsphere as described in claim 1 preparation method it is characterised in that：Described step
B. a step miniemulsion polymerization synthesis superparamagnetism carrier particle：Take above-mentioned Fe3O4Magnetic material and oleic acid are sufficiently mixed and obtain black
Viscogel, add in the viscogel of gained black in molar ratio be 1:The methyl methacrylate and two of 4-6 mixing
Methacrylic acid second diester, is sufficiently mixed uniformly, after mixing under 65% power ultrasonically treated 90-100s；
It is added dropwise in molar ratio 1 after sonicated:3 dodecyl sodium sulfate of ratio mixing and the mixing of hexadecanol
In solution, acutely rock after dropping, after mixing, form miniemulsion through 90-100s ultrasonic under 65% power again；
Above-mentioned gained miniemulsion is added dropwise in 0.05% dodecyl sodium sulfate of 600mL, after dropping at 65 DEG C nitrogen
10-15min is swept in air-blowing, adds ammonium persulfate, initiated polymerization, be warming up to 70 DEG C after polymerisation 1-1.5h after nitrogen treatment
Continue reaction 24h, obtain final product mini-emulsion polymerization synthesis superparamagnetism carrier particle.
5. the fluorescin trace magnetic composite microsphere as described in claim 1 or 4 preparation method it is characterised in that：Described
Polymerization resulting polymers carrier particle milli-Q water 3-5 time, is washed 2-3 time with 50% ethanol, then with ultrapure washing 2-3
Secondary, spontaneously dry 24h, stand-by.
6. the fluorescin trace magnetic composite microsphere as described in claim 1 preparation method it is characterised in that：
Described step c. carrier particle surface modification：
(1) amination：With DMF, above-mentioned gained superparamagnetism carrier particle is washed and evenly spreads in DMF, subsequently, to mixed
Ethylenediamine is added, with mechanical agitation back flow reaction 12h under the rotating speed of 200-400r/min at 100-120 DEG C in compound；Product
With milli-Q water 1-2 time, washed 2-3 time with 50% ethanol, then with ultrapure washing 2-3 time；
(2) aldehyde-functionalized：In the cushioning liquid that the superparamagnetism carrier particle processing through amination is immersed in, and at room temperature
Degassing 10-15min, then removes buffer solution, particle is dispersed in 5% glutaraldehyde of brand-new, makes mixture in room temperature
12h is reacted under the rotating speed mechanical agitation of 200-400r/min；Milli-Q water 2-3 time；Described cushioning liquid for pH value be 5 ±
0.1 acetic acid and sodium acetate buffer solution.
7. the fluorescin trace magnetic composite microsphere as described in claim 6 preparation method it is characterised in that：Described buffering
Solution for pH value be 5 ± 0.1 acetic acid and sodium acetate buffer solution, as by volume be 3：6.8-7.2 the 0.2mol/ of mixing
L acetic acid and 0.2mol/L sodium acetate.
8. the fluorescin trace magnetic composite microsphere as described in claim 1 preparation method it is characterised in that：
Described step e. two step miniemulsion polymerization synthesis outer shell：By 1.28mL methyl methacrylate, 9.05mL dimethyl propylene
Olefin(e) acid second diester and 1.0g step d gained mix particles, are sufficiently mixed after uniformly, gained mixture are added dropwise to mole dense
Degree ratio is 1:In 3 dodecyl sodium sulfate and the mixed solution of hexadecanol, mixture is acutely rocked, super under 65% power
Sound 80-100s forms miniemulsion, and miniemulsion is dropwise added in the dodecyl sodium sulfate of the 0.05% of 600mL, then at 40 DEG C
Lower nitrogen purges 10-15min, will be 1 in mass ratio:The ammonium persulfate of 1 mixing and sodium hydrogensulfite are added in this mixture,
Initiated polymerization, reacts 16-24h, after the completion of, washing and drying is stand-by.
Priority Applications (1)
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| CN105327684B (en) * | 2015-12-03 | 2017-10-31 | 湖北出入境检验检疫局检验检疫技术中心 | Recognize magnetic fluorescence molecular engram material of moxidectin and preparation method thereof |
| CN109021238B (en) * | 2018-06-05 | 2021-02-09 | 济南大学 | Bombay fiber-based bovine hemoglobin imprinted magnetic tubular micromotor and application thereof |
| CN109406473B (en) * | 2018-11-08 | 2019-07-30 | 青岛大学 | A kind of preparation method of phycoerythrin ratio fluorescent sensor |
| CN113671185A (en) * | 2021-08-23 | 2021-11-19 | 安龄(上海)生物科技有限公司 | Exosome recognition device and method |
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Non-Patent Citations (3)
| Title |
|---|
| Monodisperse Magnetic Single-Crystal Ferrite Microspheres;Hong Deng et al.;《Angewandte Chemie International Editon》;20050324;第117卷(第18期);第2842－2845页 * |
| Preparation of Bovine Serum Albumin Surface-Imprinted Submicrometer Particles with Magnetic Susceptibility through Core-Shell Miniemulsion Polymerization;Chau JinTan et al.;《Analytical Chemistry》;20080201;第80卷(第3期);第683－692页 * |
| 仿生荧光免疫分析卵清白蛋白;朱庆枝等;《福州大学学报（自然科学版）》;19990930;第27卷(第S1期);第115－117页 * |
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