CN104277176A - Preparation method for fluorescent western-blotting magnetic composite microballoon - Google Patents
Preparation method for fluorescent western-blotting magnetic composite microballoon Download PDFInfo
- Publication number
- CN104277176A CN104277176A CN201310287723.XA CN201310287723A CN104277176A CN 104277176 A CN104277176 A CN 104277176A CN 201310287723 A CN201310287723 A CN 201310287723A CN 104277176 A CN104277176 A CN 104277176A
- Authority
- CN
- China
- Prior art keywords
- preparation
- miniemulsion
- mixing
- sodium
- washing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Manufacturing Of Micro-Capsules (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
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 bioseparation engineering field, specifically a kind of preparation method of fluorescin trace magnetic composite microsphere.
Background technology
Molecular imprinting refers to that preparation has polymkeric substance and molecularly imprinted polymer (the Molecularly Imprinted Polymer of specific selectivity to a certain specific target molecules, MIP) program, is often depicted as the technology manufacturing " manually locking " of identifying " molecule key " visually.Because protein structure is flexible, conformation is complicated, very easily be subject to the impact of the environmental factorss such as temperature, high-crosslinking-degree material can stop protein macromole to free in and out polymer network in addition, template molecule is caused to be difficult to remove on the one hand, cause on the other hand template molecule to be difficult to enter the imprinted sites series of problems such as recombine with it, these all cause the exploitation of protein molecule engram material to be hindered.
Phase early 1970s, the a collection of scholar such as Ugelstad, El-Aasser and Vanderhoff of Lehigh university of the U.S., propose new particle nucleation mechanism---in sub-micro monomer droplet, cause nucleation, develop miniemulsion (miniemulsion) and be polymerized new technology.Mini-emulsion polymerization has most of advantage of conventional emulsion polymerization concurrently, and has following advantage: system stability is high, is conducive to industrial enforcement; The particle diameter of product latex is comparatively large, and is easy to control by the consumption of assistant for emulsifying agent; Rate of polymerization is moderate, produces and is easy to control; In system, introduce assistant for emulsifying agent, and have employed microemulsified technique, made original larger monomer droplet be dispersed into less monomer submicron droplets like this.
For separating of engineering field is not carry out static separation or dynamic separation containing the molecular blotting polymer microsphere of magnetic component mostly, and need be separated through the process such as centrifugal, operating process is loaded down with trivial details, complicated.And when magnetic responsiveness material is imbedded in polymer microballoon inside, complex microsphere then has easy under additional the action of a magnetic field, Magneto separate characteristic fast.After imbed magnetic responsiveness material in complex microsphere, just can complete at it and " initiatively " of template molecule be adsorbed and is easy to be separated after identification under additional the action of a magnetic field, reach autonomous classification and be separated easy object.
Phycocyanins, C-is a kind of very rare natural nutrient existed only in blue-green algae.Fluorescent phycocyanin separation and purification from spirulina, there is the novel fluorescence marker of unique optical properties, there is excellent character: in comparatively wide spectral range, have higher absorption coefficient; Higher fluorescent yield is had within the scope of wider pH; Their fluorescence does not disappear because of the existence of other biomolecules; Have water-soluble preferably; All very stable when liquid or solid state exists, and can the long period be preserved.Thus Phycocyanins, C-is subject to extensive concern.
Summary of the invention
The object of the present invention is to provide a kind of method utilizing mini-emulsion polymerization to prepare fluorescin trace magnetic composite microsphere.
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, combine using Phycocyanins, C-as the molecular imprinting of template with magnetic susceptibility and fluorescent material with fine emulsion polymerization, namely obtain Phycocyanins, C-molecular engram polymer magnetic and epipolic complex microsphere.
The preparation of described fluorescin trace magnetic composite microsphere:
A.Fe
3o
4prepared by magneticsubstance: by FeCl
36H
2o is dissolved in ethylene glycol, and using sodium-acetate as reductive agent, polyoxyethylene glycol is that macromolecule stabilizer dissolves through sonic oscillation, then reacts in airtight hydrothermal reaction kettle, namely obtains black Fe
3o
4magneticsubstance;
B. a step miniemulsion polymerization synthesis superparamagnetism carrier particle: above-mentioned particle parcel oleic acid, add methyl methacrylate, Ethylene-glycol-dimethacrylate mixing, join in sodium laurylsulfonate and hexadecanol solution system after mixing, form miniemulsion, miniemulsion drops in sodium dodecyl sulfate solution, deoxygenation, then add ammonium persulphate again, carry out polyreaction, after washing, dry collection, stand-by;
C. carrier particle finishing:
(1) amination: above-mentioned gained superparamagnetism carrier particle is dispersed in DMF, then adds reacting ethylenediamine, washing;
(2) aldehyde-functionalized: the buffered soln above-mentioned amidized particle being added acetic acid and sodium acetate, deoxygenation, removing damping fluid, adds in glutaraldehyde, reaction, washing;
D. fixed die plate molecule: the aldehyde-functionalized polymer beads of above-mentioned preparation, with phosphate buffered saline buffer washing, adds the reaction of Phycocyanins, C-template solution, washing;
E. two step miniemulsion polymerization synthesis outer shell: above-mentioned particle adds methyl methacrylate, Ethylene-glycol-dimethacrylate mixing, join in sodium laurylsulfonate and hexadecanol solution system, form miniemulsion, miniemulsion drops in sodium dodecyl sulfate solution, deoxygenation, add ammonium persulphate and sodium bisulfite, reaction, washing;
F. template molecule elution: join in NaOH solution by above-mentioned particle, hydrolysis, washing is also dry.
Further, described step a.Fe
3o
4prepared by magneticsubstance: take 3.00g FeCl
36H
2o is fully dissolved in 80-100mL ethylene glycol, add 7.2g sodium-acetate, ultrasonic and mechanical stirring and dissolving is even, then 2.0-4.0g polyoxyethylene glycol is added, slowly be stirred to dissolving at 60 DEG C, after solvent thermal, yellow liquid all transferred in the airtight hydrothermal reaction kettle with polytetrafluoroethylliner liner, at 190-210 DEG C, react 8-12h, be cooled to room temperature after reaction, namely obtain magnetic black Fe
3o
4magneticsubstance.
Described magnetic black Fe
3o
4magneticsubstance respectively washs 4-8 time with ultrapure water and dehydrated alcohol successively, by dry for products therefrom 12-24h, collects dry Fe
3o
4magneticsubstance.
Further, described step b. mono-step miniemulsion polymerization synthesis superparamagnetism carrier particle: get the above-mentioned Fe of 1.0g
3o
4magneticsubstance and 1mL oleic acid are fully mixed to get the viscous gel of black, add again in the viscous gel of gained black in molar ratio for the methyl methacrylate of 1:4-6 mixing and Ethylene-glycol-dimethacrylate (preferably, 1.28mL methyl methacrylate and 9.05mL Ethylene-glycol-dimethacrylate is added again) in the viscous gel of gained black, fully mix, after mixing under the power of 65% supersound process 90-100s;
Dropwise join after supersound process in the 0.01mol/L sodium lauryl sulphate of 50mL and the mixing solutions of 0.03mol/L hexadecanol, acutely rock after dropping, after mixing, under the power of 65%, ultrasonic 90-100s forms miniemulsion again;
By above-mentioned gained miniemulsion by being added dropwise in 0.05% sodium laurylsulfonate of 600mL, after dropping at 65 DEG C nitrogen purging 10-15min, 0.5g ammonium sulfate is added after nitrogen treatment, initiated polymerization, be warming up to 70 DEG C after polyreaction 1-1.5h and continue reaction 24h, obtain mini-emulsion polymerization synthesis superparamagnetism carrier particle.
Described polymerization resulting polymers carrier particle milli-Q water 3-5 time, washes 2-3 time with the ethanol of 50%, then with ultrapure washing 2-3 time, seasoning 24h, stand-by.
Further, described step c carrier particle finishing:
With DMF, the 1g polymer support particle of above-mentioned preparation washed and evenly spreads in the DMF of 20mL, subsequently, in mixture, adding the quadrol of 20mL, with mechanical stirring back flow reaction 12h under the rotating speed of 200-400r/min at 100-120 DEG C; Product milli-Q water 1-2 time, washes 2-3 time with the ethanol of 50%, then with ultrapure washing 2-3 time;
(2) aldehyde-functionalized: the polymer support particle 1g through amination process is immersed in the buffered soln of 10mL, and at room temperature degassed 10-15min, then damping fluid is removed, by Granular composite in the glutaraldehyde of 5% of brand-new, mixture is made to react 12h under the rotating speed mechanical stirring of room temperature 200-400r/min; Milli-Q water 2-3 time; Described buffered soln to be pH value be 5 ± 0.1 acetic acid and sodium acetate buffer solution.
Described to state buffered soln be pH value be 5 ± 0.1 acetic acid and sodium acetate buffer solution be 0.2mol/L acetic acid and the 0.2mol/L sodium acetate of 3:6.8-7.2 mixing by volume.
Further, described step e. two step miniemulsion polymerization synthesis outer shell: by 1.28mL methyl methacrylate, the superparamagnetism nuclear particle mixing of the surface modification of 9.05mL Ethylene-glycol-dimethacrylate and 1.0g, then by mixture with 40-50% power ultrasonic 90-100s, after fully mixing, gained mixture is dropwise joined the mixing solutions of the sodium lauryl sulphate of the 0.01mol/L of 50mL and the hexadecanol of 0.03mol/L, mixture is acutely rocked, under the power of 65%, ultrasonic 90s forms miniemulsion, miniemulsion is dropwise added in the sodium laurylsulfonate of 0.05% of 600mL, then nitrogen purging 10-15min at 40 DEG C, by in mass ratio for 1:1 mixing ammonium persulphate and sodium bisulfite join in this mixture, initiated polymerization, reaction 16-24h, after completing, washing and drying is stand-by.
The advantage that the present invention has:
Instant invention overcomes all difficulties of protein molecule engram and mini-emulsion polymerization, the specific adsorption of the complex microsphere finally obtained to template protein repeatability that is most effective and complex microsphere is strong.Gained magnetic core-shell structure copolymer trace microballoon pattern rule, uniform particle diameter, the present invention specifically applies the method for mini-emulsion polymerization, is compounded with magnetic and the two responsive materials of fluorescence, has specific adsorption to Phycocyanins, C-in molecular blotting polymer microsphere.
Particularly proteins molecularly imprinted technique selectivity is combined with magnetic susceptibility and fluorescence by fine emulsion polymerization by the present invention, give the certain magnetic of molecular blotting polymer microsphere and fluorescent dual response characteristic, reach protein molecule engram polymer magnetic complex microsphere specific recognition and independently adsorb, and convenient under additional the action of a magnetic field, sharp separation, use the object that the fluorescent instruments such as fluorescence spectrum easily detect in real time.
Be compounded with magnetic and the two responsive materials of fluorescence in gained molecular blotting polymer microsphere of the present invention, have specific adsorption to Phycocyanins, C-, selectivity is good, and adsorption efficiency is high, and rate of mass transfer is fast, and repeat performance is good.Wherein be compounded with the Fe that magnetic intensity is 10.770emu/g
3o
4magnetic Nano material; Simultaneously template protein fluorescence property is stablized, and has higher absorption coefficient, have higher fluorescent yield, good water solubility, be beneficial to Fast Measurement analysis within the scope of wider pH in comparatively wide spectral range, and for other mass signatures.
Accompanying drawing explanation
The nucleocapsid trace magnetic composite microsphere preparation process schematic diagram that Fig. 1 provides for the embodiment of the present invention.
The magnetic composite microsphere adhesion protein design sketch that Fig. 2 A provides for the embodiment of the present invention;
The magnetic composite microsphere adhesion protein magnetic hysteresis loop figure that Fig. 2 B provides for the embodiment of the present invention.
The pH that Fig. 3 A provides for the embodiment of the present invention is to the impact effect figure of the fluorescence of Phycocyanins, C-;
The fluorescence response collection of illustrative plates to 0.1mg/L-0.7mg/L concentration range Phycocyanins, C-solution that Fig. 3 B provides for the embodiment of the present invention.
The application drawing of nucleocapsid trace magnetic composite microsphere in SDS-PAGE electrophoresis that Fig. 4 provides for the embodiment of the present invention, wherein No. 1 swimming lane is protein mark swimming lane, No. 2 is the swimming lane of original protein solution, No. 3 is the protein solution after the absorption of nucleocapsid trace magnetic composite microsphere, and No. 4 is the protein solution after non-nucleocapsid trace magnetic composite microsphere absorption.
Fig. 5 adsorbs the fluoroscopic image of Phycocyanins, C-solution for nucleocapsid trace magnetic composite microsphere that the embodiment of the present invention provides in HeLa cell environment; The fluorescence photo that wherein A, B, C are respectively HeLa cell, HeLa cell adds Phycocyanins, C-, HeLa cell adds Phycocyanins, C-and excite at 559nm after adsorbing with nucleocapsid trace magnetic composite microsphere, G, H, I are respectively A, B, C photo under light field, D is the synthesising picture of A and G, E is the synthesising picture of B and H, and F is the synthesising picture of C and I.
Embodiment
First the present invention adopts a step miniemulsion polymerization synthesis Fe
3o
4the superparamagnetism carrier particle of base, carried out surface amination and aldehyde-functionalized modification, fixedly be there is the Phycocyanins, C-template molecule of photoluminescent property, then adopted two step miniemulsion polymerization synthesis outer shell, gained magnetic core-shell trace microballoon pattern rule, uniform particle diameter.The method of application mini-emulsion polymerization, magnetic and the two responsive materials of fluorescence is compounded with in molecular blotting polymer microsphere, Phycocyanins, C-is had specific recognition and independently adsorbed, selectivity is good, adsorption efficiency is high, and speed soon, and facilitates under additional the action of a magnetic field, sharp separation, repeat performance is good, and within the scope of wider pH, carries out fluorescence detect in real time and fluorescence imaging.This law has that cost is low, simple and efficient to handle, high repeatability and other advantages, is with a wide range of applications.
Embodiment 1
A.Fe
3o
4prepared by magneticsubstance: the FeCl taking 3.00g
36H
2o is dissolved in 90mL ethylene glycol, adds 7.2g sodium-acetate, ultrasonic and mechanical stirring and dissolving.Then add 3.9g polyoxyethylene glycol and be stirred to dissolving with 400r/min at 60 DEG C.After dissolving, yellow liquid is all transferred in the airtight hydrothermal reaction kettle with polytetrafluoroethylliner liner, in 200 DEG C of loft drier, react 10h.After being cooled to room temperature, magnetic capture atrament, respectively washs 6 times with ultrapure water and dehydrated alcohol successively, by dry for products therefrom 24h, collects dry Fe
3o
4magneticsubstance.
B. a step miniemulsion polymerization synthesis superparamagnetism carrier particle: the above-mentioned Fe of 1.0g
3o
4the oleic acid of magneticsubstance and 1mL is mixed to get the viscous gel of black.Mol ratio is the magneticsubstance that the methyl methacrylate (1.28mL) of 1:4 and Ethylene-glycol-dimethacrylate (9.05mL) add Coated with Oleic Acid, and fully mixes.Then by mixture supersound process 80s under the power of 65%.After mixing, gained mixture is dropwise joined in the mixing solutions of the 0.01mol/L sodium laurylsulfonate of 50mL and the hexadecanol of 0.03mol/L with the speed of 2-3 drop/sec.Acutely rocked by mixture, under the power of 65%, supersound process 90s forms miniemulsion.Miniemulsion dropwise joins in 0.05% sodium laurylsulfonate of 600mL with the speed of 2-3 drop/sec.This reaction mixture is transferred in the three neck round-bottomed flasks of a 1L, nitrogen purging 15min at 65 DEG C, ammonium persulphate (0.5g) is joined in reaction mixture, initiated polymerization, be warming up to 70 DEG C after reaction 1-1.5h and continue reaction, continue 24h.After completing, polymer support particle milli-Q water 3 times, the ethanol with 50% washes 3 times, then with ultrapure washing 3 times.Seasoning 24h, collects.
C. carrier particle finishing:
(1) amination: the polymer support particle of above-mentioned for 1g preparation is washed twice and is distributed in the DMF of 20mL with DMF.Subsequently, in mixture, add the quadrol of 20mL, with the rotating speed of 400r/min, at 110 DEG C, mechanical stirring back flow reaction 12h.Product milli-Q water 1 time, the ethanol with 50% wash 2 times, then with ultrapure washing 2 times.
(2) aldehyde-functionalized: secure ph is the acetic acid of 5 and the buffered soln (be is 0.2mol/L acetic acid and the 0.2mol/L sodium acetate of 3:7 mixing by volume) of sodium acetate.Polymer support particle above-mentioned for 1g is immersed in the buffered soln of 10mL, and at room temperature degassed 10min.Then remove this damping fluid, by this Granular composite brand-new 10mL 5% glutaraldehyde in.By this mixture mechanic whirl-nett reaction 12h under the rotating speed of room temperature 400r/min.Then, with milli-Q water 3 times.
D. fixed die plate molecule: the aldehyde-functionalized polymer beads of above-mentioned preparation, washs 1 time with the phosphate buffered saline buffer (PBS) of 0.01mol/L.The above-mentioned particle of 1.0g is added in the Phycocyanins, C-solution (1mg/mL) of 20mL.Gained mixed system is placed in 4 DEG C, mechanical stirring 3h under the rotating speed of 300r/min, carries out coupling reaction.React this fixing Phycocyanins, C-template of rear milli-Q water 3 times.
E. two step miniemulsion polymerization synthesis outer shell: the above-mentioned superparamagnetism nuclear particle mixing being fixed with the surface modification of template molecule of methyl methacrylate (1.28mL), Ethylene-glycol-dimethacrylate (9.05mL) and 1.0g.Then by mixture with 45% power ultrasonic 90s, to guarantee thorough mixing.After mixing, gained mixture is dropwise joined with the speed of 2-3 drop/sec in the mixing solutions of the sodium laurylsulfonate of the 0.01mol/L of 50mL and the hexadecanol of 0.03mol/L.Acutely rocked by mixture, under the power of 65%, ultrasonic 90s forms miniemulsion.Miniemulsion dropwise joins in the sodium laurylsulfonate of 0.05% of 600mL with the speed of 2-3 drop/sec.This reaction mixture is transferred in the three neck round-bottomed flasks of a 1L, nitrogen purging 15min at 40 DEG C, ammonium persulphate (0.25g) and sodium bisulfite (0.25g) are joined with initiated polymerization in this mixture, reaction 24h.After completing, by the magnetic core-shell structure copolymer microballoon of this polymkeric substance milli-Q water 3 times, the ethanol with 50% washes 3 times, then with ultrapure washing 3 times.Seasoning 24h, collects.
F. template molecule elution, namely by the Phycocyanins, C-template molecule that basic hydrolysis removing is fixing, is specially: the magnetic core-shell structure copolymer microballoon adding 1.0g in the NaOH solution of the 1.0mol/L of 20mL.Hydrolysed mix is stirred with the rotating speed of 300r/min, reaction 5h at 35 DEG C.By these surface imprinted particle milli-Q water 3 times, seasoning 24h, obtains fluorescin trace magnetic composite microsphere (see Fig. 1).
Non-imprinted polymer preparation: according to aforesaid operations code, just do not add outside template molecule Phycocyanins, C-, other steps are the same.
Embodiment 2
Take Phycocyanins, C-20mg, with redistilled water constant volume in the volumetric flask of 1L, obtain the Phycocyanins, C-solution of 20mg/L, the Phycocyanins, C-solution getting 10mL joins in the rib collar bottle of 20mL, add the fluorescin trace magnetic composite microsphere of 100mg, vibration absorption 10min, with magnet at the side of bottle absorption 2min, complex microsphere can be adsorbed onto side, obtain clear soln (see Fig. 2 A).Illustrate that this complex microsphere has strong adsorptive power to Phycocyanins, C-, and sepn process is fast, simply, higher see this microballoon magnetic intensity shown in Fig. 2 B.
Embodiment 3
Get the 100mg/L Phycocyanins, C-solution 7 parts of 200 μ L, be placed in the plastic centrifuge tube of 10mL respectively, then respectively to the phosphate buffer soln (Na adding different pH value in each pipe
2hPO
4and NaH
2pO
4be respectively 0.2mol/L, volume ratio 1:1) 9.8mL, dilution obtains the 2mg/L Phycocyanins, C-solution that pH value is respectively 5.6,6.0,6.4,6.8,7.2,7.6 and 8.0, its corresponding fluorescence intensity is measured respectively with spectrophotofluorometer, can obtain that there is when pH value is 6.4 maximum fluorescence intensity (see Fig. 3 A), and increase along with the concentration of Phycocyanins, C-, fluorescence intensity increases gradually (see Fig. 3 B).
Embodiment 4
Take N,O-Diacetylmuramidase (LZM), chicken serum albumin (CEA), bovine serum albumin (BSA) and Phycocyanins, C-(PC) each 6mg, mixing, then adds 6mL redistilled water, obtains the mixed protein solution of 1mg/mL.Getting 2mL mixing solutions respectively joins in three glass centrifuge tube, and label is 2,3,4.Separately getting a glass centrifuge tube label is 1, adds mark protein solution.Do not carry out any process in No. 2 centrifuge tubes, No. 3 magnetic blotting microballoons adding 20mg, No. 4 non-imprinted polymers adding 20mg, are then placed in vibration absorption 6h, centrifuging and taking supernatant liquor 40 μ L on vibrator respectively, carry out SDS-PAGE electrophoresis experiment by centrifuge tube.See Fig. 4, the swimming lane of No. 4 compares No. 2 swimming lanes, and the band of 4 kinds of albumen does not change substantially, and the band of No. 3 swimming lane PC obviously weakens, and other protein bands do not have considerable change.Result shows that this trace complex microsphere has highly selective to Phycocyanins, C-, can carry out specific adsorption.
Embodiment 5
Cultivate HeLa cell, the Tissue Culture Plate in 24 holes adds cover glass, the enchylema getting 9 parts of 1mL respectively joins on Tissue Culture Plate to be cultivated, and mark No. 1-9, treat cell normal growth on the cover slip, with No. 1-3 as a control group, do not add any solution, the Phycocyanins, C-solution getting the 10mg/mL of 60 μ L adds in No. 4-9, cultivate 6h, the non-imprinted polymer adding the 0.1mg/mL of 100 μ L toward 7-9 sample adsorbs, and slight oscillatory, the cover glass got wherein is observed under confocal microscope.See Fig. 5, do not have the sample processed, in intercellular substance, fluorescence intensity is very large; After adding trace complex microsphere, in cellular environment, fluorescence intensity obviously weakens, and several fluorescence bright spots of appearance are trace microballoon, illustrate this trace complex microsphere can in cellular environment specific adsorption Phycocyanins, C-.
Claims (9)
1. the preparation method of a fluorescin trace magnetic composite microsphere, it is characterized in that: combine using Phycocyanins, C-as the molecular imprinting of template with magnetic susceptibility and fluorescent material with fine emulsion polymerization, namely obtain Phycocyanins, C-molecular engram polymer magnetic and epipolic complex microsphere.
2., by the preparation method of fluorescin trace magnetic composite microsphere according to claim 1, it is characterized in that: the preparation of described fluorescin trace magnetic composite microsphere:
A.Fe
3o
4prepared by magneticsubstance: by FeCl
36H
2o is dissolved in ethylene glycol, and using sodium-acetate as reductive agent, polyoxyethylene glycol is that macromolecule stabilizer dissolves through sonic oscillation, then reacts in airtight hydrothermal reaction kettle, namely obtains black Fe
3o
4magneticsubstance;
B. a step miniemulsion polymerization synthesis superparamagnetism carrier particle: above-mentioned particle parcel oleic acid, add methyl methacrylate, Ethylene-glycol-dimethacrylate mixing, join in sodium laurylsulfonate and hexadecanol solution system after mixing, form miniemulsion, miniemulsion drops in sodium dodecyl sulfate solution, then adds ammonium persulphate again, carries out polyreaction, after washing, dry collection, stand-by;
C. carrier particle finishing:
(1) amination: above-mentioned gained superparamagnetism carrier particle is dispersed in DMF (DMF), then adds reacting ethylenediamine, washing;
(2) aldehyde-functionalized: the buffered soln above-mentioned amidized particle being added acetic acid and sodium acetate, adds in glutaraldehyde, reaction, washing;
D. fixed die plate molecule: the aldehyde-functionalized polymer beads of above-mentioned preparation, with phosphate buffered saline buffer washing, adds the reaction of Phycocyanins, C-template solution, washing;
E. two step miniemulsion polymerization synthesis outer shell: above-mentioned particle adds methyl methacrylate, Ethylene-glycol-dimethacrylate mixing, join in sodium laurylsulfonate and hexadecanol solution system, form miniemulsion, miniemulsion drops in sodium dodecyl sulfate solution, deoxygenation, add ammonium persulphate and sodium bisulfite, reaction, washing;
F. template molecule elution: join in NaOH solution by above-mentioned particle, hydrolysis, washing is also dry.
3., by the preparation method of fluorescin trace magnetic composite microsphere according to claim 2, it is characterized in that: described step a.Fe
3o
4prepared by magneticsubstance: take 3.00g FeCl
36H
2o is fully dissolved in 70-100mL ethylene glycol, add 7.2g sodium-acetate, make it be uniformly dissolved, then 2.0-4.0g polyoxyethylene glycol is added, slowly be stirred to dissolving at 60 DEG C, after dissolving, yellow liquid all transferred in the airtight hydrothermal reaction kettle with polytetrafluoroethylliner liner, at 190-210 DEG C, react 8-12h, be cooled to room temperature after reaction, namely obtain magnetic black Fe
3o
4magneticsubstance.
4., by the preparation method of the fluorescin trace magnetic composite microsphere described in Claims 2 or 3, it is characterized in that: described magnetic black Fe
3o
4magneticsubstance respectively washes 4-8 time with ultrapure water and dehydrated alcohol successively, by dry for products therefrom 12-24h, collects dry Fe
3o
4magneticsubstance.
5. by the preparation method of fluorescin trace magnetic composite microsphere according to claim 2, it is characterized in that: described step b. mono-step miniemulsion polymerization synthesis superparamagnetism carrier particle: get above-mentioned Fe
3o
4magneticsubstance and oleic acid are fully mixed to get the viscous gel of black, add in the viscous gel of gained black is methyl methacrylate and the Ethylene-glycol-dimethacrylate of 1:4-6 mixing in molar ratio again, fully mix, after mixing under the power of 65% supersound process 90-100s;
In the mixing solutions of the sodium lauryl sulphate that the ratio dropwise joining 1:3 in molar ratio after supersound process mixes and hexadecanol, acutely rock after dropping, after mixing, under the power of 65%, ultrasonic 90-100s forms miniemulsion again;
Above-mentioned gained miniemulsion is dropwise joined in 0.05% sodium laurylsulfonate of 600mL, after dropping at 65 DEG C nitrogen purging 10-15min, ammonium sulfate is added after nitrogen treatment, initiated polymerization, be warming up to 70 DEG C after polyreaction 1-1.5h and continue reaction 24h, obtain mini-emulsion polymerization synthesis superparamagnetism carrier particle.
6. by the preparation method of the fluorescin trace magnetic composite microsphere described in claim 2 or 5, it is characterized in that: described polymerization resulting polymers carrier particle milli-Q water 3-5 time, wash 2-3 time with the ethanol of 50%, then with ultrapure washing 2-3 time, seasoning 24h, stand-by.
7., by the preparation method of fluorescin trace magnetic composite microsphere according to claim 2, it is characterized in that:
The finishing of described step c carrier particle:
(1) amination: with DMF the polymer support particle of above-mentioned preparation washed and evenly spread in DMF, subsequently, adding quadrol in mixture, with mechanical stirring back flow reaction 12h under the rotating speed of 200-400r/min at 100-120 DEG C; Product milli-Q water 1-2 time, washes 2-3 time with the ethanol of 50%, then with ultrapure washing 2-3 time;
(2) aldehyde-functionalized: in the buffered soln that the polymer support particle through amination process is immersed in, and at room temperature degassed 10-15min, then damping fluid is removed, by Granular composite in the glutaraldehyde of 5% of brand-new, mixture is made to react 12h under the rotating speed mechanical stirring of room temperature 200-400r/min; Milli-Q water 2-3 time; Described buffered soln to be pH value be 5 ± 0.1 acetic acid and sodium acetate buffer solution.
8. by the preparation method of fluorescin trace magnetic composite microsphere according to claim 7, it is characterized in that: described in state buffered soln be pH value be 5 ± 0.1 acetic acid and sodium acetate buffer solution, being is 0.2mol/L acetic acid and the 0.2mol/L sodium acetate of 3:6.8-7.2 mixing by volume.
9., by the preparation method of fluorescin trace magnetic composite microsphere according to claim 2, it is characterized in that:
Described step e. two step miniemulsion polymerization synthesis outer shell: by 1.28mL methyl methacrylate, the superparamagnetism nuclear particle mixing of 9.05mL Ethylene-glycol-dimethacrylate and the above-mentioned surface modification of 1.0g, after fully mixing, gained mixture dropwise being joined molar concentration rate is in the sodium lauryl sulphate of 1:3 and the mixing solutions of hexadecanol, mixture is acutely rocked, under the power of 65%, ultrasonic 80-100s forms miniemulsion, miniemulsion is dropwise added in the sodium laurylsulfonate of 0.05% of 600mL, then nitrogen purging 10-15min at 40 DEG C, by in mass ratio for 1:1 mixing ammonium persulphate and sodium bisulfite join in this mixture, initiated polymerization, reaction 16-24h, after completing, washing and drying is stand-by.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310287723.XA CN104277176B (en) | 2013-07-10 | 2013-07-10 | Preparation method for fluorescent western-blotting magnetic composite microballoon |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310287723.XA CN104277176B (en) | 2013-07-10 | 2013-07-10 | Preparation method for fluorescent western-blotting magnetic composite microballoon |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104277176A true CN104277176A (en) | 2015-01-14 |
| CN104277176B CN104277176B (en) | 2017-02-08 |
Family
ID=52252560
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310287723.XA Active CN104277176B (en) | 2013-07-10 | 2013-07-10 | Preparation method for fluorescent western-blotting magnetic composite microballoon |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104277176B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105032422A (en) * | 2015-06-29 | 2015-11-11 | 中国科学院广州能源研究所 | Fe3O4 nanometer catalyst for Fischer-Tropsch synthesis and preparation method of Fe3O4 nanometer catalyst |
| CN105327684A (en) * | 2015-12-03 | 2016-02-17 | 湖北出入境检验检疫局检验检疫技术中心 | Magnetic fluorescent molecular imprinting material for recognizing moxidectin and preparation method of magnetic fluorescent molecular imprinting material |
| CN109021238A (en) * | 2018-06-05 | 2018-12-18 | 济南大学 | A kind of bovine hemoglobin trace magnetism tubulose micro motor and its application based on bombax cotton |
| CN109406473A (en) * | 2018-11-08 | 2019-03-01 | 青岛大学 | A kind of preparation method of the phycoerythrin ratio fluorescent sensor based on magnetic molecularly imprinted core shell polymer |
| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102977288A (en) * | 2012-12-04 | 2013-03-20 | 温州医学院 | Molecularly imprinted magnetic microsphere, preparation method and application thereof |
-
2013
- 2013-07-10 CN CN201310287723.XA patent/CN104277176B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102977288A (en) * | 2012-12-04 | 2013-03-20 | 温州医学院 | Molecularly imprinted magnetic microsphere, preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| CHAU JINTAN ET AL.: "Preparation of Bovine Serum Albumin Surface-Imprinted Submicrometer Particles with Magnetic Susceptibility through Core-Shell Miniemulsion Polymerization", 《ANALYTICAL CHEMISTRY》 * |
| HONG DENG ET AL.: "Monodisperse Magnetic Single-Crystal Ferrite Microspheres", 《ANGEWANDTE CHEMIE INTERNATIONAL EDITON》 * |
| 朱庆枝等: "仿生荧光免疫分析卵清白蛋白", 《福州大学学报（自然科学版）》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105032422A (en) * | 2015-06-29 | 2015-11-11 | 中国科学院广州能源研究所 | Fe3O4 nanometer catalyst for Fischer-Tropsch synthesis and preparation method of Fe3O4 nanometer catalyst |
| CN105327684A (en) * | 2015-12-03 | 2016-02-17 | 湖北出入境检验检疫局检验检疫技术中心 | Magnetic fluorescent molecular imprinting material for recognizing moxidectin and preparation method of magnetic fluorescent molecular imprinting material |
| CN109021238A (en) * | 2018-06-05 | 2018-12-18 | 济南大学 | A kind of bovine hemoglobin trace magnetism tubulose micro motor and its application based on bombax cotton |
| CN109021238B (en) * | 2018-06-05 | 2021-02-09 | 济南大学 | Bombay fiber-based bovine hemoglobin imprinted magnetic tubular micromotor and application thereof |
| CN109406473A (en) * | 2018-11-08 | 2019-03-01 | 青岛大学 | A kind of preparation method of the phycoerythrin ratio fluorescent sensor based on magnetic molecularly imprinted core shell polymer |
| CN109406473B (en) * | 2018-11-08 | 2019-07-30 | 青岛大学 | A kind of preparation method of phycoerythrin ratio fluorescent sensor |
| WO2020093639A1 (en) * | 2018-11-08 | 2020-05-14 | 青岛大学 | Method for preparing phycoerythrin ratio fluorescence sensor based on magnetic molecular imprinted nucleus/shell polymer |
| US10895536B1 (en) | 2018-11-08 | 2021-01-19 | Qingdao University | Method for preparing a ratiometric fluorescent sensor for phycoerythrin based on a magnetic molecularly imprinted core-shell polymer |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104277176B (en) | 2017-02-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104277176A (en) | Preparation method for fluorescent western-blotting magnetic composite microballoon | |
| Yang et al. | One-pot synthesis of hydrophilic molecularly imprinted nanoparticles | |
| Wei et al. | Ionic liquids skeleton typed magnetic core-shell molecularly imprinted polymers for the specific recognition of lysozyme | |
| CN106188437B (en) | A kind of method that Pickering emulsion polymerizations prepare metal organic frame particle trace adsorbent | |
| CN107200812A (en) | A kind of preparation method of magnetic molecularly imprinted material | |
| Yang et al. | Ultrasensitive detection of bisphenol A in aqueous media using photoresponsive surface molecular imprinting polymer microspheres | |
| CN106540668B (en) | Magnetic hydrophilic molecules trace composite material and preparation method | |
| CN102861541B (en) | Preparation method of surface-modified fluorescent magnetic polymer composite microspheres | |
| CN106423292B (en) | A kind of hybrid microspheres preparation method with temperature/magnetic double responsiveness of loading functional L-PROLINE | |
| CN104479072A (en) | Method for preparing magnetic molecularly-imprinted adsorbent | |
| CN107353372B (en) | A kind of preparation method of the nNOS-PSD-95 uncoupler imprint surface polymer based on magnetic mesoporous molecular sieve | |
| CN102417558B (en) | Magnetic molecularly imprinted polymer for separating atrazine, and preparation method for magnetic molecularly imprinted polymer | |
| CN103833942A (en) | Preparation method and use of diethylstibestrol molecularly-imprinted magnetic microspheres | |
| CN102489273B (en) | Preparation method of magnetic beads of dendritic molecular imprinting polymer on surface of estrogenic nano silica gel | |
| CN106589743B (en) | A kind of preparation method of magnetic fluorescent composite material | |
| CN109160976A (en) | A kind of preparation method of tetracycline carbon dots fluorescent molecule imprinted material | |
| CN105170112A (en) | Preparation method for magnetic carbon microsphere surface cephalexin molecular imprinted adsorbent material | |
| CN101733081A (en) | Method for preparing molecularly imprinted microspheres on surface of polymer carrier | |
| CN107746841A (en) | A kind of amphion magnetic coupling aquogel fixed enzyme carrier and preparation method | |
| CN103613720A (en) | Method for preparing silane coupling agent modified attapulgite surface molecular imprinting material aiming at bisphenol A | |
| Garcia et al. | “On-off” switchable tool for food sample preparation: merging molecularly imprinting technology with stimuli-responsive blocks. Current status, challenges and highlighted applications | |
| CN107999019B (en) | Amphiphilic magnetic nanosphere and preparation method and adsorption application thereof | |
| CN104062275A (en) | MWCNTs-QDs-based nano fluorescence bionic sensor and preparation method thereof | |
| CN106883346B (en) | A kind of preparation method of dexamethasone magnetic molecularly imprinted polymer | |
| CN106565908B (en) | A kind of preparation method of monodispersed large grain-size polymer microballoon |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |