CN1390860A - Magnetic compound microsphere of surface blot gel for biological macromolecular template and its reverse-phase suspension polymerization for preparing its seed - Google Patents
Magnetic compound microsphere of surface blot gel for biological macromolecular template and its reverse-phase suspension polymerization for preparing its seed Download PDFInfo
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- CN1390860A CN1390860A CN 02121486 CN02121486A CN1390860A CN 1390860 A CN1390860 A CN 1390860A CN 02121486 CN02121486 CN 02121486 CN 02121486 A CN02121486 A CN 02121486A CN 1390860 A CN1390860 A CN 1390860A
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Abstract
A composite magnetic gel microsphere for surficial biological macromolecular template blot, based on template blot (molecular blot) technique, is prepared from the different biological macromoleculae as template molecule and different metals (or their oxides) as magnetic component through reverse-phase seed suspension polymerization. Its advantages are less consumption of template moleculae and high adsorption capacity. It can be used for separating biological macromoleculae with more precision and simple process.
Description
Technical field
The invention belongs to Materials Science and Engineering and bioseparation engineering field, more precisely relate to a kind of surface blot gel for biological macromolecular template magnetic composite microsphere and reverse-phase suspension polymerization for preparing its seed thereof.
Background technology
Molecular recognition and recognition specificity are whole biology ubiquity and distinctive characteristics, and it is special and important to play a part in organic evolution.Template imprinting just is based on biological bionical, adopts the manual method preparation that specific molecular (template molecule or microsphere) is had the technology of the polymkeric substance of recognition specificity.Recently, demonstrate application prospects at aspects such as separating purification, immunoassay, analogue enztme and biosensor, become one of novel material of tool potentiality of new millennium with the molecularly imprinted polymer (MIPs) of this technology preparation.
At present, although the template imprinting technology in a lot of fields, obtained extensive and deep research such as a lot of aspects such as the separation of analogue compounds, antibodies simulation, enzyme simulation, biosimulation transmitter, but the selection of template molecule concentrates on the small molecules mainly.The volatility owing to the big also biologically active of molecular weight of biomacromolecule, therefore, its trace all is very difficult with identification, the breadth and depth of research also can not show a candle to small molecules.
At present, biological macromolecular template trace method mainly contains entrapping method, microsphere surface blotting, planar surface blotting and antigenic determinant method etc.Entrapping method (Hjerten SJ, Liao JL, Nakazato K, WangY, Zamaratskaia G, Zhang H-X.Gels mimicking antibodies in theirselective recognition of protein, Chromatography, 1997,44:227-234) preparation process is simple, but last handling process is loaded down with trivial details, must just can finish through processes such as the wash-out of template molecule, grinding, screenings, and cause the segment template molecule because of being embedded in the inner problem that can't wash-out of microballoon; Planar surface blotting (Shi H, Tsai W-B, Ferrari S, Ratner BD.Template imprintednanostructural surfaces for protein, Narure, 1999,398:593-597.) can't obtain the trace microballoon, can only obtain sheet material (or film material) and be used for medically diagnosis and monitoring; Antigenic determinant method (Rachkov A, Minoura N.Towards molecularly imprinted polymersselective to peptides and protein.The epitope approach, Biochimica etBiophysica Acta, 2001,1544:255-266) adopt mass polymerization because of it, its essence also is entrapping method, and only is applicable to the separation of the diverse biomacromolecule of segment structure of the trace of the biomacromolecule that the segment structure of exposure is perfectly clear and exposure; Microsphere surface blotting (Glad M, Narrlow O, Sellergren B, Siegbahn N, Mosbach K.Use of silane monomers for molecularimprinting and enzyme entrapment in polysiloxane-coated porous silica, J Chromatogr, 1985,347:11-23) be a kind of more promising trace method, but can't not carry out simply and separate easily with magnetic field because it does not contain magnetic component, only be fit to be used as the stratographic stationary phase and carry out static separation.In addition, the imprinted polymer that this method obtains is a rigid polymer, for non-rigid, bulky template biomacromolecule from imprinted polymer wash-out and the recognition process template biomacromolecule to reenter " in conjunction with the hole " obviously be unfavorable and difficult.
When imbedding the magnetic responsiveness material in polymer microballoon inside, during as iron, cobalt, nickel or its oxide compound, complex microsphere then has and is adding easy under the action of a magnetic field, magnetic stalling characteristic (C Bor Fuh fast, S Y Chen.Magnetic split-flow thin fractionation:new technique for separation ofmagnetically susceptible particles, Journal of Chromatography A, 1998,813:313-324).After in the template imprinting polymkeric substance, imbedding the magnetic responsiveness material, just can be easy to it is separated with back the adding under the action of a magnetic field of identification in its " initiatively " absorption of finishing template molecule.
Adopt inverse suspension polymerization can directly prepare gel micro-ball.But the gel micro-ball of prior art for preparing is owing to can't separate accurately without trace, and owing to do not contain magnetic component and can't carry out simple and dynamic separation easily with magnetic field, can only carry out static separation (D Wistuba, V Schurig.Enantiomerseparation of chiral pharmaceuticals by capillary electrochromatography, Journal of Chromatography A, 2000,875:255-276); If carry out dynamic separation, then need to separate the template imprinting polymer microballoon through process such as centrifugal, operating process is loaded down with trivial details, complicated.
Summary of the invention
The present invention is intended to by seed inverse suspension polymerization method the template imprinting technology be combined with magnetic susceptibility, prepare nonrigid surface blot gel for biological macromolecular template magnetic composite microsphere (being called for short the surface blot gel complex microsphere), and give its certain magnetic responsiveness, make the surface blot gel complex microsphere that makes finish " initiatively " absorption to the template biomacromolecule and adding under the action of a magnetic field with specificity identification back and be easy to it is separated, reach initiatively identification and convenient isolating purpose at it; Simultaneously, solve inverse suspension method and be embedded in the inner problem that can't wash-out of microballoon, save the consumption of expensive biological macromolecular template because of the segment template molecule.
The present invention need solve the compatibility problem of metal (or its oxide compound) powder and polymkeric substance, realizes the embedding of polymer gel to inorganics, thereby gives the surface blot gel complex microsphere certain magnetic responsiveness.
The present invention need solve the coating problem of shell monomers on greater particle size, and finishes the trace process to the template biomacromolecule simultaneously, makes the surface blot gel complex microsphere have singleness identity to the template biomacromolecule, reaches the purpose of particular separation.
The present invention need solve the limited swelling problem of surface blot gel complex microsphere, and is relatively stable with shape and the size of guaranteeing trace hole in the trace of biomacromolecule and recognition process.
The present invention need guarantee that the biological activity of biomacromolecule in the template imprinting process does not change.
The present invention need select to be suitable for the dispersion agent of inverse suspension polymerization, with kind ball and the formation of himself and stable of guaranteeing the surface blot gel complex microsphere.
The present invention is according to the principle of template imprinting technology, with different biomacromolecules as template molecule, different metal (or its oxide compound) is as magnetic component, different macromolecule stabilizers is a dispersion agent, adopts seed inverse suspension polymerization method to make the biomacromolecule microsphere surface magnetic compound microsphere of blot gel that the template biomacromolecule is had singleness identity and has " dynamically " characteristic.
The prepared surface blot gel for biological macromolecular template magnetic composite microsphere of the present invention is characterized in that being compounded with the magnetic response material in the blot gel for biological macromolecular template microballoon, and the biological macromolecular template trace is on the surface of gel magnetic composite microsphere.The invention process method is described below:
1) preparation of seed gel complex microsphere
The non-polar organic solvent adding is had in the reactor of agitator, ventpipe, add dispersion agent, stir and make it dissolving; With acrylamide (AM), N, N '-methylene-bisacrylamide (Bis) adds in the methacrylic acid sodium water solution, and the dissolving back adds the magnetic powder through grinding, and dispersed with stirring evenly back adds in the reactor, continues to stir; Oxidizing and Reducing Agents is added drop-wise in the reactor after soluble in water; Logical nitrogen deoxygenation, normal-temperature reaction 0.5～4h; After reaction finishes, filtering organic solvent part, then with gel micro-ball with acetone rinsing after, behind the water repetitive scrubbing, hydrochloric acid soln with 0.1～2mol/L soaks, elimination hydrochloric acid soln behind immersion 2～24h, and the water repetitive scrubbing is to neutral, be dried to constant weight for 40～90 ℃ in vacuum drying oven then, obtain being used to prepare the kind ball of surface blot gel complex microsphere;
2) preparation of surface blot gel complex microsphere
Template biomacromolecule, Oxidizing and Reducing Agents are added in the entry, and the dissolving back adds the kind ball of dry state, treats to join in the non-polar organic solvent that is dissolved with dispersion agent after kind of the ball swelling, and agitation condition drips the mixing solutions of AM, Bis down; Logical nitrogen deoxygenation, normal-temperature reaction 0.5～4h; After reaction finished, filtering organic solvent part obtained the surface blot gel complex microsphere;
3) wash-out of biomacromolecule
With prepared surface blot gel complex microsphere with acetone rinsing after, wash repeatedly with distilled water; With NaOH solution soaking and the vibration of 0.1～2m/L, elimination solution washes repeatedly with distilled water behind 2～24h then; Hydrochloric acid soln with 0.1～2m/L soaks again, and elimination solution behind immersion 2～24h washes to neutrality repeatedly with distilled water, is dried to constant weight in 40～90 ℃ with vacuum drying oven then.Wherein:
Non-polar organic solvent is: benzene, toluene, kerosene, gasoline, hexanaphthene, solvent oil;
Dispersion agent is: methylcellulose gum, ethyl cellulose, propyl cellulose;
Magneticsubstance is: Fe, Co, Ni or its oxide compound, alloy;
Oxygenant is: Potassium Persulphate, Sodium Persulfate, ammonium persulphate;
Reductive agent is: S-WAT, sodium bisulfite;
The template biomacromolecule is: protein, polypeptide, nucleic acid, cell;
Dispersion agent is: methylcellulose gum, ethyl cellulose.
Innovative point of the present invention is:
1. magnetic susceptibility (magnetic responsiveness) is combined with the template imprinting technology and prepare magnetic compound microsphere of blot gel for biological macromolecular template, can realize biomacromolecule is separated purpose accurately and rapidly.
2. acrylamide and N, N '-methylene-bisacrylamide multipolymer and metal (or its oxide compound) powder has good consistency, has the characteristics such as easy, easy to prepare that coat.
3. acrylamide and N, the copolymerization conditions gentleness of N '-methylene-bisacrylamide can keep the biological activity of biomacromolecule and unchangeability.
The present invention adopt seed inverse suspension polymerization method because trace occurs in microsphere surface, avoided the segment template molecule because of being embedded in the inner problem that can't wash-out of microballoon, it is few to make that this legal system is equipped with the consumption of the more common inverse suspension polymerization method template molecule of surface blot gel complex microsphere, and loading capacity is big.
5. prepared surface blot gel complex microsphere because of its " in conjunction with hole " can change according to the change of environment, promptly has stimulating responsive, makes that template biomacromolecule wash-out is more or less freely.
6. prepared surface blot gel complex microsphere because of the main master plate biomacromolecule of its identification and " combining the hole " to biomacromolecule size and coincideing mutually in shape, the different biomacromolecule of shape (space structure) so this blot gel complex microsphere separable molecular weight is identical, also separable shape identical (or close) but the different biomacromolecule of molecular weight.
Surface blot gel complex microsphere of the present invention combines the accurate specificity evident characteristics of template imprinting polymkeric substance and magnetic composite microsphere foreign field stalling characteristic dual-use function easily, will be in the bioseparation field, biomacromolecule particularly, aspects such as separation as protein, polypeptide, cell have broad application prospects, and loaded down with trivial details in the past sepn process (as high speed centrifugation etc.) is simplified greatly.Existing be used for the template imprinting polymer microballoon in bioseparation engineering field owing to do not contain magnetic component and can't carry out simply and separate easily with magnetic field, and be all rigid polymer, for non-rigid, bulky template biomacromolecule from imprinted polymer wash-out and recognition process the template biomacromolecule to reenter " in conjunction with the hole " all be unfavorable and difficult; And existing magnetic composite microsphere can not be realized accurate separation owing to do not have the bio-identification specificity without the biomolecules trace.
Selected acrylamide of the present invention and N, N '-methylene-bisacrylamide multipolymer and metal (or its oxide compound) powder has good consistency, have and coat characteristics such as easy, easy to prepare, and the preparation condition gentleness, can keep the biological activity of biomacromolecule and unchangeability.
The present invention adopt seed inverse suspension polymerization method because trace occurs in microsphere surface, avoided the segment template molecule because of being embedded in the inner problem that can't wash-out of microballoon, the consumption that makes this legal system be equipped with the more common inverse suspension polymerization method template molecule of surface blot gel complex microsphere reduces, and loading capacity but increases.
Identical but the different biomacromolecule of shape (space structure) of the prepared separable molecular weight of surface blot gel complex microsphere of the present invention, also separable shape identical (or close) but the different biomacromolecule of molecular weight, the scope of application is wider.
The surface blot gel complex microsphere that the present invention is prepared because of its " in conjunction with hole " can change according to the change of environment, promptly has stimulating responsive, makes that template biomacromolecule wash-out is more or less freely.
Description of drawings Fig. 1: embodiment 1 prepared surface blot gel for biological macromolecular template magnetic composite microsphere single particle
The ESEM photo (hygrometric state, the SEM photo of about 360 μ m Fig. 2: the embodiment of median size 1 prepared biomacromolecule magnetic compound microsphere of surface blot gel (is done
Attitude, the about 300 μ m of median size, Fe
3O
4Content 2.06%)
The preparation of embodiment embodiment 1. bovine serum albumins (BSA) template surface blot gel complex microsphere
(1) preparation of kind ball
100ml toluene is added in the there-necked flask of the 250ml that has agitator, ventpipe, add the 0.2g ethyl cellulose, stir and make it dissolving.The 1g methacrylic acid is joined in the NaOH solution of 11.6ml 1mol/L, add 8gAM, 1g Bis and 20ml water behind the stirring 5min, the dissolving back adds the magnetic Fe of 0.2g through grinding
3O
4Powder, dispersed with stirring evenly back add in the there-necked flask, continue to stir; Be added drop-wise in the there-necked flask after being dissolved in 0.1g Potassium Persulphate and 0.05g sodium bisulfite in the 10ml water.Logical nitrogen deoxygenation, normal-temperature reaction 2h.After reaction finishes, with 180 eye mesh screen filtering organic solvent parts, then with gel micro-ball with acetone rinsing after, behind the water repetitive scrubbing, soak with the hydrochloric acid soln 50ml of 1mol/L, soak elimination hydrochloric acid soln after a few hours, the water repetitive scrubbing is to neutral, be dried to constant weight for 80 ℃ in vacuum drying oven then, obtain being used to prepare the kind ball of surface blot gel complex microsphere.
(2) preparation of BSA template surface blot gel complex microsphere
0.0330g BSA, 0.05g Potassium Persulphate and 0.03g sodium bisulfite are added in the 20ml water, the dissolving back adds the kind ball of 5g dry state, treat to join in the 100ml toluene that is dissolved with the 0.2g ethyl cellulose after the abundant swelling of kind of ball, agitation condition drips the mixing solutions 10ml of 0.9gAM, 0.1gBis down.Logical nitrogen deoxygenation, normal-temperature reaction 2h.Reaction with 180 eye mesh screen filtering organic solvent parts, obtains the surface blot gel complex microsphere after finishing.
(3) wash-out of BSA
With prepared surface blot gel complex microsphere with acetone rinsing after, wash repeatedly with distilled water.NaOH solution 50ml with 0.1m/L soaks and vibration then, and elimination solution washes repeatedly with distilled water behind the 24h; Hydrochloric acid soln 50ml with 0.1m/L soaks again, and elimination solution behind the immersion 6h washes to neutrality repeatedly with distilled water, is dried to constant weight in 80 ℃ with vacuum drying oven then.
Prepared magnetic compound microsphere of blot gel for biological macromolecular template, be in bovine serum albumin mold plate blot gel complex microsphere, to be compounded with the Fe3O4 magneticsubstance, Fe3O4 content is 2.06%, the trace hole that can discern biomacromolecule is contained on its surface, serve as the contrast molecule with N,O-Diacetylmuramidase and Protalbinic acid respectively, it is to the separation factor 3.72 and 3.45 of bovine serum albumin.The preparation of embodiment 2. N,O-Diacetylmuramidase surface blot gel complex microspheres
(1) preparation of kind ball
100ml toluene is added in the there-necked flask of the 250ml that has agitator, ventpipe, add the 0.25g ethyl cellulose, stir and make it dissolving.The 2g methacrylic acid is joined in the NaOH solution of 23.2ml 1mol/L, add 7gAM, 1g Bis and 10ml water behind the stirring 5min, the dissolving back adds the magnetic Fe of 0.3g through grinding
3O
4Powder, dispersed with stirring evenly back add in the there-necked flask, continue to stir; Be added drop-wise in the there-necked flask after being dissolved in 0.1g Potassium Persulphate and 0.05g sodium bisulfite in the 10ml water.Logical nitrogen deoxygenation, normal-temperature reaction 2h.After reaction finishes, with 180 eye mesh screen filtering organic solvent parts, then with gel micro-ball with acetone rinsing after, behind the water repetitive scrubbing, soak with the hydrochloric acid soln 50ml of 1mol/L, soak elimination hydrochloric acid soln after a few hours, the water repetitive scrubbing is to neutral, be dried to constant weight for 80 ℃ in vacuum drying oven then, obtain being used to prepare the kind ball of surface blot gel complex microsphere.
(2) preparation of N,O-Diacetylmuramidase template surface blot gel complex microsphere
0.0144g N,O-Diacetylmuramidase, 0.05g Potassium Persulphate and 0.03g sodium bisulfite are added in the 20ml water, the dissolving back adds the kind ball of 5g dry state, treat to join in the 100ml toluene that is dissolved with the 0.2g ethyl cellulose after the abundant swelling of kind of ball, agitation condition drips the mixing solutions 10ml of 0.9gAM, 0.1gBis down.Logical nitrogen deoxygenation, normal-temperature reaction 2h.Reaction with 180 eye mesh screen filtering organic solvent parts, obtains the surface blot gel complex microsphere after finishing.
(3) wash-out of N,O-Diacetylmuramidase
With prepared surface blot gel complex microsphere with acetone rinsing after, wash repeatedly with distilled water.NaOH solution 50ml with 0.1m/L soaks and vibration then, and elimination solution washes repeatedly with distilled water behind the 24h; Hydrochloric acid soln 50ml with 0.1m/L soaks again, and elimination solution behind the immersion 6h washes to neutrality repeatedly with distilled water, is dried to constant weight in 80 ℃ with vacuum drying oven then.
Prepared magnetic compound microsphere of blot gel for biological macromolecular template is to be compounded with Fe in N,O-Diacetylmuramidase template imprinting gel complex microsphere
3O
4Magneticsubstance, Fe
3O
4Content is 2.45%, and the trace hole that can discern biomacromolecule is contained on its surface, serves as the contrast molecule with BSA and Protalbinic acid respectively, and it is to the separation factor 5.07 and 4.15 of N,O-Diacetylmuramidase.The preparation of embodiment 3. Protalbinic acid template surface blot gel complex microspheres
(1) preparation of kind ball
120ml benzene is added in the there-necked flask of the 250ml that has agitator, ventpipe, add the 0.25g methylcellulose gum, stir and make it dissolving.The 1g methacrylic acid is joined in the NaOH solution of 11.6ml 1mol/L, add 8gAM, 1g Bis and 20ml water behind the stirring 5min, the dissolving back adds the magnetic Fe of 0.4g through grinding
2O
3Powder, dispersed with stirring evenly back add in the there-necked flask, continue to stir; Be added drop-wise in the there-necked flask after being dissolved in 0.1g ammonium persulfate and 0.05g S-WAT in the 10ml water.Logical nitrogen deoxygenation, normal-temperature reaction 2h.After reaction finishes, with 180 eye mesh screen filtering organic solvent parts, then with gel micro-ball with acetone rinsing after, behind the water repetitive scrubbing, soak with the hydrochloric acid soln 50ml of 1mol/L, soak elimination hydrochloric acid soln after a few hours, the water repetitive scrubbing is to neutral, be dried to constant weight for 80 ℃ in vacuum drying oven then, obtain being used to prepare the kind ball of surface blot gel complex microsphere.
(2) preparation of Protalbinic acid template surface blot gel complex microsphere
0.0215g Protalbinic acid, 0.05g ammonium persulfate and 0.03g S-WAT are added in the 20ml water, the dissolving back adds the kind ball of 5g dry state, treat to join in the 100ml toluene that is dissolved with the 0.2g ethyl cellulose after the abundant swelling of kind of ball, agitation condition drips the mixing solutions 10ml of 0.9gAM, 0.1gBis down.Logical nitrogen deoxygenation, normal-temperature reaction 2h.Reaction with 180 eye mesh screen filtering organic solvent parts, obtains the surface blot gel complex microsphere after finishing.
(3) wash-out of Protalbinic acid
With prepared surface blot gel complex microsphere with acetone rinsing after, wash repeatedly with distilled water.NaOH solution 50ml with 0.1m/L soaks and vibration then, and elimination solution washes repeatedly with distilled water behind the 24h; Hydrochloric acid soln 50ml with 0.1m/L soaks again, and elimination solution behind the immersion 6h washes to neutrality repeatedly with distilled water, is dried to constant weight in 80 ℃ with vacuum drying oven then.
Prepared magnetic compound microsphere of blot gel for biological macromolecular template is to be compounded with Fe in Protalbinic acid template imprinting gel complex microsphere
2O
3Magneticsubstance, Fe
2O
3Content is 1.95%, and the trace hole that can discern biomacromolecule is contained on its surface, serves as the contrast molecule with BSA and N,O-Diacetylmuramidase respectively, and it is to the separation factor 4.07 and 3.26 of Protalbinic acid.
Claims (9)
1. a surface blot gel for biological macromolecular template magnetic composite microsphere is characterized in that being compounded with the magnetic response material in the blot gel for biological macromolecular template microballoon, and the biological macromolecular template trace is on the surface of gel magnetic composite microsphere.
2. a kind of magnetic compound microsphere of blot gel for biological macromolecular template as claimed in claim 1, it is characterized in that in bovine serum albumin mold plate blot gel complex microsphere, being compounded with the Fe3O4 magneticsubstance, Fe3O4 content is 2.06%, the trace hole that can discern biomacromolecule is contained on its surface, serve as the contrast molecule with N,O-Diacetylmuramidase and Protalbinic acid respectively, it is to the separation factor 3.72 and 3.45 of bovine serum albumin.
3. a kind of magnetic compound microsphere of blot gel for biological macromolecular template as claimed in claim 1 is characterized in that being compounded with Fe in N,O-Diacetylmuramidase template imprinting gel complex microsphere
3O
4Magneticsubstance, Fe
3O
4Content is 2.45%, and the trace hole that can discern biomacromolecule is contained on its surface, serves as the contrast molecule with BSA and Protalbinic acid respectively, and it is to the separation factor 5.07 and 4.15 of N,O-Diacetylmuramidase.
4. a kind of magnetic compound microsphere of blot gel for biological macromolecular template as claimed in claim 1 is characterized in that being compounded with Fe in Protalbinic acid template imprinting gel complex microsphere
2O
3Magneticsubstance, Fe
2O
3Content is 1.95%, and the trace hole that can discern biomacromolecule is contained on its surface, serves as the contrast molecule with BSA and N,O-Diacetylmuramidase respectively, and it is to the separation factor 4.07 and 3.26 of Protalbinic acid.
5. surface blot gel for biological macromolecular template magnetic composite microsphere and reverse-phase suspension polymerization for preparing its seed thereof may further comprise the steps:
1) preparation of seed gel complex microsphere
The non-polar organic solvent adding is had in the reactor of agitator, ventpipe, add dispersion agent, stir and make it dissolving; With acrylamide (AM), N, N '-methylene-bisacrylamide (Bis) adds in the methacrylic acid sodium water solution, and the dissolving back adds the magnetic powder through grinding, and dispersed with stirring evenly back adds in the reactor, continues to stir; Oxidizing and Reducing Agents is added drop-wise in the reactor after soluble in water; Logical nitrogen deoxygenation, normal-temperature reaction 0.5～4h; After reaction finishes, filtering organic solvent part, then with gel micro-ball with acetone rinsing after, behind the water repetitive scrubbing, hydrochloric acid soln with 0.1～2mol/L soaks, elimination hydrochloric acid soln behind immersion 2～24h, and the water repetitive scrubbing is to neutral, be dried to constant weight for 40～90 ℃ in vacuum drying oven then, obtain being used to prepare the kind ball of surface blot gel complex microsphere;
2) preparation of surface blot gel complex microsphere
Template biomacromolecule, Oxidizing and Reducing Agents are added in the entry, and the dissolving back adds the kind ball of dry state, treats to join in the non-polar organic solvent that is dissolved with dispersion agent after kind of the ball swelling, and agitation condition drips the mixing solutions of AM, Bis down; Logical nitrogen deoxygenation, normal-temperature reaction 0.5～4h; After reaction finished, filtering organic solvent part obtained the surface blot gel complex microsphere;
3) wash-out of biomacromolecule
With prepared surface blot gel complex microsphere with acetone rinsing after, wash repeatedly with distilled water; With NaOH solution soaking and the vibration of 0.1～2m/L, elimination solution washes repeatedly with distilled water behind 2～24h then; Hydrochloric acid soln with 0.1～2m/L soaks again, and elimination solution behind immersion 2～24h washes to neutrality repeatedly with distilled water, is dried to constant weight in 40～90 ℃ with vacuum drying oven then.
6. a kind of surface blot gel for biological macromolecular template magnetic composite microsphere as claimed in claim 5 and reverse-phase suspension polymerization for preparing its seed thereof, it is characterized by described: non-polar organic solvent is: benzene, toluene, kerosene, gasoline, hexanaphthene, solvent oil; Dispersion agent is: methylcellulose gum, ethyl cellulose, propyl cellulose; Magneticsubstance is: Fe, Co, Ni or its oxide compound, alloy; Oxygenant is: Potassium Persulphate, Sodium Persulfate, ammonium persulphate; Reductive agent is: S-WAT, sodium bisulfite; The template biomacromolecule is: protein, polypeptide, nucleic acid, cell; Dispersion agent is: methylcellulose gum, ethyl cellulose.
7. a kind of surface blot gel for biological macromolecular template magnetic composite microsphere as claimed in claim 5 and reverse-phase suspension polymerization for preparing its seed thereof may further comprise the steps:
1) preparation of kind ball
100ml toluene is added in the there-necked flask of the 250ml that has agitator, ventpipe, add the 0.2g ethyl cellulose, stir and make it dissolving; The 1g methacrylic acid is joined in the NaOH solution of 11.6ml 1mol/L, add 8gAM, 1g Bis and 20ml water behind the stirring 5min, the dissolving back adds the magnetic Fe of 0.2g through grinding
3O
4Powder, dispersed with stirring evenly back add in the there-necked flask, continue to stir; Be added drop-wise in the there-necked flask after being dissolved in 0.1g Potassium Persulphate and 0.05g sodium bisulfite in the 10ml water; Logical nitrogen deoxygenation, normal-temperature reaction 2h; After reaction finishes, with 180 eye mesh screen filtering organic solvent parts, then with gel micro-ball with acetone rinsing after, behind the water repetitive scrubbing, soak with the hydrochloric acid soln 50ml of 1mol/L, soak elimination hydrochloric acid soln after a few hours, the water repetitive scrubbing is to neutral, be dried to constant weight for 80 ℃ in vacuum drying oven then, obtain being used to prepare the kind ball of surface blot gel complex microsphere;
2) preparation of template surface blot gel complex microsphere
0.0330g BSA, 0.05g Potassium Persulphate and 0.03g sodium bisulfite are added in the 20ml water, the dissolving back adds the kind ball of 5g dry state, treat to join in the 100ml toluene that is dissolved with the 0.2g ethyl cellulose after the abundant swelling of kind of ball, agitation condition drips the mixing solutions 10ml of 0.9gAM, 0.1g Bis down; Logical nitrogen deoxygenation, normal-temperature reaction 2h; Reaction with 180 eye mesh screen filtering organic solvent parts, obtains the surface blot gel complex microsphere after finishing;
3) wash-out of biomacromolecule
With prepared surface blot gel complex microsphere with acetone rinsing after, wash repeatedly with distilled water; NaOH solution 50ml with 0.1m/L soaks and vibration then, and elimination solution washes repeatedly with distilled water behind the 24h; Hydrochloric acid soln 50ml with 0.1m/L soaks again, and elimination solution behind the immersion 6h washes to neutrality repeatedly with distilled water, is dried to constant weight in 80 ℃ with vacuum drying oven then.
8. a kind of surface blot gel for biological macromolecular template magnetic composite microsphere as claimed in claim 7 and reverse-phase suspension polymerization for preparing its seed thereof is characterized in that described step 1), 2) be:
1) preparation of kind ball
100ml toluene is added in the there-necked flask of the 250ml that has agitator, ventpipe, add the 0.25g ethyl cellulose, stir and make it dissolving; The 2g methacrylic acid is joined in the NaOH solution of 23.2ml 1mol/L, add 7gAM, 1g Bis and 10ml water behind the stirring 5min, the dissolving back adds the magnetic Fe of 0.3g through grinding
3O
4Powder, dispersed with stirring evenly back add in the there-necked flask, continue to stir; Be added drop-wise in the there-necked flask after being dissolved in 0.1g Potassium Persulphate and 0.05g sodium bisulfite in the 10ml water; Logical nitrogen deoxygenation, normal-temperature reaction 2h; After reaction finishes, with 180 eye mesh screen filtering organic solvent parts, then with gel micro-ball with acetone rinsing after, behind the water repetitive scrubbing, soak with the hydrochloric acid soln 50ml of 1mol/L, soak elimination hydrochloric acid soln after a few hours, the water repetitive scrubbing is to neutral, be dried to constant weight for 80 ℃ in vacuum drying oven then, obtain being used to prepare the kind ball of surface blot gel complex microsphere;
2) preparation of N,O-Diacetylmuramidase template surface blot gel complex microsphere
0.0144g N,O-Diacetylmuramidase, 0.05g Potassium Persulphate and 0.03g sodium bisulfite are added in the 20ml water, the dissolving back adds the kind ball of 5g dry state, treat to join in the 100ml toluene that is dissolved with the 0.2g ethyl cellulose after the abundant swelling of kind of ball, agitation condition drips the mixing solutions 10ml of 0.9gAM, 0.1gBis down; Logical nitrogen deoxygenation, normal-temperature reaction 2h; Reaction with 180 eye mesh screen filtering organic solvent parts, obtains the surface blot gel complex microsphere after finishing.
9. a kind of surface blot gel for biological macromolecular template magnetic composite microsphere as claimed in claim 7 and reverse-phase suspension polymerization for preparing its seed thereof is characterized in that described step 1), 2) be:
1) preparation of kind ball
120ml benzene is added in the there-necked flask of the 250ml that has agitator, ventpipe, add the 0.25g methylcellulose gum, stir and make it dissolving; The 1g methacrylic acid is joined in the NaOH solution of 11.6ml 1mol/L, add 8gAM, 1g Bis and 20ml water behind the stirring 5min, the dissolving back adds the magnetic Fe of 0.4g through grinding
2O
3Powder, dispersed with stirring evenly back add in the there-necked flask, continue to stir; Be added drop-wise in the there-necked flask logical nitrogen deoxygenation, normal-temperature reaction 2h after being dissolved in 0.1g ammonium persulfate and 0.05g S-WAT in the 10ml water; After reaction finishes, with 180 eye mesh screen filtering organic solvent parts, then with gel micro-ball with acetone rinsing after, behind the water repetitive scrubbing, soak with the hydrochloric acid soln 50ml of 1mol/L, soak elimination hydrochloric acid soln after a few hours, the water repetitive scrubbing is to neutral, be dried to constant weight for 80 ℃ in vacuum drying oven then, obtain being used to prepare the kind ball of surface blot gel complex microsphere;
2) preparation of Protalbinic acid template surface blot gel complex microsphere
0.0215g Protalbinic acid, 0.05g ammonium persulfate and 0.03g S-WAT are added in the 20ml water, the dissolving back adds the kind ball of 5g dry state, treat to join in the 100ml toluene that is dissolved with the 0.2g ethyl cellulose after the abundant swelling of kind of ball, agitation condition drips the mixing solutions 10ml of 0.9gAM, 0.1g Bis down; Logical nitrogen deoxygenation, normal-temperature reaction 2h; Reaction with 180 eye mesh screen filtering organic solvent parts, obtains the surface blot gel complex microsphere after finishing.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100371458C (en) * | 2005-11-25 | 2008-02-27 | 天津理工大学 | RNA blot electro-response hydrogel and its preparing method |
| CN101747473A (en) * | 2008-12-12 | 2010-06-23 | 南开大学 | Surface-functionalized molecularly imprinted polymer microsphere and preparation method thereof |
| CN101778870B (en) * | 2007-08-02 | 2012-11-07 | 巴斯夫欧洲公司 | Aqueous polymer dispersion based on n,n-diethylaminoethyl methacrylate, its preparation and use |
| CN102145279B (en) * | 2010-02-05 | 2012-11-21 | 华中科技大学 | Method for preparing lysozyme molecular imprinting nano particles with magnetic responsiveness and extremely high adsorption capacity |
| CN113368837A (en) * | 2021-05-26 | 2021-09-10 | 扬州大学 | Molecularly imprinted gel material with magnetic response performance, preparation method and application thereof |
-
2002
- 2002-06-26 CN CN 02121486 patent/CN1226309C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100371458C (en) * | 2005-11-25 | 2008-02-27 | 天津理工大学 | RNA blot electro-response hydrogel and its preparing method |
| CN101778870B (en) * | 2007-08-02 | 2012-11-07 | 巴斯夫欧洲公司 | Aqueous polymer dispersion based on n,n-diethylaminoethyl methacrylate, its preparation and use |
| CN101747473A (en) * | 2008-12-12 | 2010-06-23 | 南开大学 | Surface-functionalized molecularly imprinted polymer microsphere and preparation method thereof |
| CN101747473B (en) * | 2008-12-12 | 2014-04-02 | 南开大学 | Surface-functionalized molecularly imprinted polymer microsphere and preparation method thereof |
| CN102145279B (en) * | 2010-02-05 | 2012-11-21 | 华中科技大学 | Method for preparing lysozyme molecular imprinting nano particles with magnetic responsiveness and extremely high adsorption capacity |
| CN113368837A (en) * | 2021-05-26 | 2021-09-10 | 扬州大学 | Molecularly imprinted gel material with magnetic response performance, preparation method and application thereof |
| CN113368837B (en) * | 2021-05-26 | 2022-05-17 | 扬州大学 | Molecularly imprinted gel material with magnetic response performance, preparation method and application thereof |
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| Publication number | Publication date |
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| CN1226309C (en) | 2005-11-09 |
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