CN101265332A - Macro molecule engram polyvinylpyrrolidone/calcium alginate polymer micro-sphere and preparation method thereof - Google Patents
Macro molecule engram polyvinylpyrrolidone/calcium alginate polymer micro-sphere and preparation method thereof Download PDFInfo
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- CN101265332A CN101265332A CNA2008100524660A CN200810052466A CN101265332A CN 101265332 A CN101265332 A CN 101265332A CN A2008100524660 A CNA2008100524660 A CN A2008100524660A CN 200810052466 A CN200810052466 A CN 200810052466A CN 101265332 A CN101265332 A CN 101265332A
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Abstract
The invention relates to a protein macromolecular imprinting polyvinylpyrrolidone/calcium alginate polymer microsphere and a preparation method thereof. The components and the contents by weight percentages of the protein macromolecular imprinting polyvinylpyrrolidone/calcium alginate polymer microsphere are as follows: 10 percent to 50 percent sodium alginate, 0.1 percent to 16 percent polyvinylpyrrolidone, 20 percent to 70 percent calcium chloride and 0.1 percent to 15 percent protein. The experimental results of the protein re-bonding test of the polyvinylpyrrolidone/calcium alginate polymer microsphere which is prepared by the invention show that the imprinting microsphere is 2 to 5 times of the re-bonding amount of the non-imprinting microsphere. The method for preparing the invention has the advantages of simple and easy operation, easy control of the reaction process and so on, which solves the shortcomings of low re-bonding amount and unstable performance of the single calcium alginate microsphere, thus having wide application prospect.
Description
Technical field
The invention belongs to polymer science and technical field, particularly a kind of macromolecule blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere and preparation method thereof.
Background technology
Sodium alginate is a kind of negatively charged ion saccharan that derives from brown alga, and it is made up of D-manuronic and L-glucosidelinkages.The key character of alginates is can produce gel in the presence of divalent-metal ion, as calcium ion, and zine ion etc.Sodium alginate is a feasible and suitable protein molecule engram base material, yet the physical strength of alginate calcium is not high, thereby its swelling usually makes the imprinted sites distortion reduce the trace efficient of protein molecule.Many researchers is attempted the modification alginate calcium or with alginates and the compound application performance that improves of other polymkeric substance for this reason.
Zhang Fengju etc. have reported the calcium alginate gel bead of BSA trace, and Natvosol is used to form interpenetrating(polymer)networks to increase the physical strength of microballoon with sodium alginate (SA), has improved the trace effect to a certain extent.[Cheng Guoxiang, Zhang Fengju, English twilight, double-template method hydroxyethyl cellulose modified alginate microsphere and preparation method, Chinese invention patent, application number 200510015832.1].Polyvinyl alcohol (PVA) and calcium alginate compounded research also there is report [S.Hertzberg.E.Moen。C.Vogelsang.Mixed?photo-cross-linked?polyvinyl?alcohol?and?calcium-alginate?gels?for?cellentrapment.Appl?Microbiol?Biotechnol。1995。43：10-17】。[Chao Liu, Xiujie Ji, Kongyin Zhao, et al.Preparation of Hydroxyapatite/Ca-Alginate Composite Microspheres via Inverse SuspensionCrosslinked Method.Journal of Applied Polymer Science, 104 (2007) 2034-2038] also set forth the compound of calcium polyphosphate and alginate calcium.
The PVP gel has unreactiveness and biocompatibility, strong chemical reaction can not take place with protein in itself, do not make protein denaturation yet, it is had a good application prospect, also be studied [GX cheng and carry out western blotting with the soft wet gel of crosslinked pvp, C W Li, Study on the preparation of BSA-imprinted crosslinked PVP microsphers and therebinding property via suspension polymerization, prepared].
And the present invention is that alginate calcium and two kinds of compositions of PVP is compound, thereby provides a kind of new base material for macromolecule blot.
Summary of the invention
The invention provides the preparation method and the trace method of macro-molecular protein blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere.
Macromolecule blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere of the present invention, its component and weight percent are as follows:
Sodium alginate 10%-50%
Polyvinylpyrolidone (PVP) 0.1%-16%
Calcium chloride 20%-70%
Protein 0.1%-15%.
Described protein comprises bovine serum albumin, Protalbinic acid or N,O-Diacetylmuramidase.
Described polymer microballoon is that protein embedding blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere size is 100-600 μ m..
The preparation method of macromolecule blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere of the present invention is characterized in that preparation method's step of described protein embedding blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere is as follows:
A) get PROTEIN B SA solution, add sodium alginate, stir it is fully dissolved, leaving standstill the cancellation bubble becomes water in the experiment;
B) by 1 of water volume: 1-4: 1 measures organic phase, pours in the container, and to the ethyl cellulose of emulsifying agent that wherein adds 1%-5% and 0.1%-15%, mechanical stirring is fully dissolved it;
C) mixed aqueous solution is joined under magnetic agitation in the container that fills the machine oil phase, stirring at normal temperature makes the heavy-gravity mixing solutions be dispersed into microballoon; Under magnetic agitation, in system, add CaCl
2Solution carries out gelation reaction;
D) leave standstill and make calcium alginate gel bead be deposited to container bottom, slowly remove supernatant liquid, use the deionized water rinsing microballoon, obtain the Polyvinylpyrolidone (PVP)/calcium alginate polymer micro-sphere of protein macromolecule embedding trace after the wash-out template is handled.
Described organic phase density is 0.75-0.95g/mL, and by soybean oil with butylacetate is composite forms, emulsifying agent is Tween80, and its quality accounts for the 1%-5% of oil phase quality, and ethyl cellulose accounts for the 0.1%-15% of sodium alginate quality.
The preparation method of described macromolecule blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere, the volume ratio that it is characterized in that organic phase and water is 1: 1-4: 1.
Among the preparation method of macromolecule blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere of the present invention, described organic phase is the organic phase that adopts usually, requiring the density of organic phase is 0.75-0.95g/ml, wherein dissolves emulsifying agent or dispersion agent that mass percent is 1%-5%.But, can adopt following organic phase for environmental protection needs:
Organic phase 1: soybean oil and butylacetate are according to 4: 1-1: 1 volume ratio is mixed, and emulsifying agent is Tween80, and its quality accounts for the 1%-5% of oil phase quality.
Organic phase 2: salad oil and butylacetate are according to 4: 1-1: 1 volume ratio is mixed, and the dispersion agent ethyl cellulose accounts for the 0.1%-15% of sodium alginate quality.
Polyvinylpyrolidone (PVP)/calcium alginate polymer micro-sphere that the present invention is prepared shows that in conjunction with test result the heavy binding capacity of trace microballoon is non-trace microballoon through protein is heavy, 2-5 times of several microballoons that do not add the protein preparation.Preparation method of the present invention is simple, reaction process is easy to advantages such as control, and the heavy binding capacity that has solved single calcium alginate microsphere is low, and the deficiency of unstable properties is with a wide range of applications.
Description of drawings
Fig. 1: bovine serum albumin blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere photo;
Fig. 2: non-blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere photo;
Fig. 3: embedding blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere is to the heavy binding curve of bovine serum albumin;
Fig. 4: embedding blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere is to bovine serum albumin trace efficiency curve.
Embodiment
Embodiment 1.
1. get the 20ml deionized water and add 0.005gBSA wiring solution-forming solution, add the sodium alginate of 0.5g and the PVP of 0.005g, stirring is fully dissolved it, leaves standstill the cancellation bubble;
2. measure soybean oil by 4: 1 volume ratio and butylacetate amounts to 80ml, pour in the beaker of 250ml, to wherein adding a certain amount of 0.1g tween80 and 0.06g ethyl cellulose, stirring at normal temperature 10min fully dissolves it;
3. mixed aqueous solution is joined under magnetic agitation in the beaker that fills the machine oil phase, stirring at normal temperature 10min makes the heavy-gravity mixing solutions be dispersed into microballoon; Under magnetic agitation, in system, add 4% CaCl
2Solution 20ml carries out gelation reaction, continues to stir 2 hours.
4. left standstill 30 minutes, and made calcium alginate gel bead be deposited to beaker bottom, slowly remove supernatant liquid, use deionized water rinsing microballoon 3 times.Carry out next step wash-out template and handle, obtain the Polyvinylpyrolidone (PVP)/calcium alginate polymer micro-sphere of bovine serum albumin embedding trace.
The optical microscope photograph of the Polyvinylpyrolidone (PVP)/calcium alginate polymer micro-sphere of embedding trace is seen Fig. 1, and it is heavy in conjunction with seeing Fig. 3 to bovine serum albumin. see that from Fig. 1 the microballoon pattern is better, microsphere features smooth surface.As seen from Figure 3, surface imprinted microballoon to the heavy binding capacity of bovine serum albumin obviously greater than the dash binding capacity of non-trace microballoon to bovine serum albumin, heavy binding capacity is 4.091 times of the heavy binding capacity of non-trace microballoon during balance, and the trace efficient that also draws the trace microballoon from Fig. 4 as can be seen is also higher.The polyalcohol microspherulite diameter size that obtains is 100-600 μ m.
Embodiment 2
1. get sodium alginate and 0.16gPVP that the 20ml deionized water adds 0.5g, stirring is fully dissolved it, leaves standstill the cancellation bubble;
2. measure salad oil by 1: 1 volume ratio and butylacetate amounts to 80ml, pour in the beaker of 250ml, to wherein adding a certain amount of 0.1gtween80 and 0.06g ethyl cellulose, stirring at normal temperature 10min fully dissolves it;
3. mixed aqueous solution is joined under magnetic agitation in the beaker that fills the machine oil phase, stirring at normal temperature 10min makes the heavy-gravity mixing solutions be dispersed into microballoon; Under magnetic agitation, in system, add 6% CaCl
2Solution 20ml carries out gelation reaction, continues to stir 2 hours.
4. left standstill 30 minutes, and made calcium alginate gel bead be deposited to beaker bottom, slowly remove supernatant liquid, use deionized water rinsing microballoon 3 times.Carry out next step wash-out template and handle, obtain the Polyvinylpyrolidone (PVP)/calcium alginate polymer micro-sphere of BSA embedding trace.
The non-trace microballoon that draws is compared not too big difference in shape as shown in Figure 2 with the trace microballoon.
Embodiment 3
1. get the 20ml deionized water and add 0.06g Protalbinic acid wiring solution-forming solution, add 0.4g sodium alginate and 0.08gPVP, stirring is fully dissolved it, leaves standstill the cancellation bubble;
2. measure salad oil by 3: 1 volume ratio and butylacetate amounts to 70ml, pour in the beaker of 250ml, to wherein adding a certain amount of 0.05gtween80 and 0.04g ethyl cellulose, stirring at normal temperature 10min fully dissolves it;
3. mixed aqueous solution is joined under magnetic agitation in the beaker that fills the machine oil phase, stirring at normal temperature 10min makes the heavy-gravity mixing solutions be dispersed into microballoon; Under magnetic agitation, in system, add 3% CaCl
2Solution 30ml carries out gelation reaction, continues to stir 1 hour.
4. left standstill 30 minutes, and made calcium alginate gel bead be deposited to beaker bottom, slowly remove supernatant liquid, use deionized water rinsing microballoon 3 times.Carry out next step wash-out template and handle, obtain the Polyvinylpyrolidone (PVP)/calcium alginate polymer micro-sphere of BSA embedding trace.
Embodiment 4
1. get the 20ml deionized water and add 0.13g N,O-Diacetylmuramidase wiring solution-forming solution, add sodium alginate and the 0.01gPVP of 0.5g, stirring is fully dissolved it, leaves standstill the cancellation bubble;
2. measure soybean oil by 2: 1 volume ratio and butylacetate amounts to 85ml, pour in the beaker of 250ml, to wherein adding a certain amount of 0.2gtween80 and 0.03g ethyl cellulose, stirring at normal temperature 10min fully dissolves it;
3. mixed aqueous solution is joined under magnetic agitation in the beaker that fills the machine oil phase, stirring at normal temperature 10min makes the heavy-gravity mixing solutions be dispersed into microballoon; Under magnetic agitation, in system, add 5% CaCl
2Solution 30ml carries out gelation reaction, continues to stir 1 hour.
4. left standstill 30 minutes, and made calcium alginate gel bead be deposited to beaker bottom, slowly remove supernatant liquid, use deionized water rinsing microballoon 3 times.Carry out next step wash-out template and handle, obtain the Polyvinylpyrolidone (PVP)/calcium alginate polymer micro-sphere of BSA embedding trace.
Macromolecule blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere that the present invention proposes and preparation method thereof, be described by embodiment, person skilled obviously can be changed or suitably change and combination content as herein described in not breaking away from content of the present invention, spirit and scope, realizes the present invention.Special needs to be pointed out is, the replacement that all are similar and change apparent to those skilled in the artly, they are regarded as being included in spirit of the present invention, scope and the content.
Claims (7)
1. macromolecule blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere is characterized in that component and weight percent are as follows:
Sodium alginate 10%-50%
Polyvinylpyrolidone (PVP) 0.1%-16%
Calcium chloride 20%-70%
Protein 0.1%-15%.
2. as right 1 described a kind of macromolecule blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere, the described microspherulite diameter size of its feature is 100-600 μ m.
3. as right 1 described a kind of macromolecule blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere, the described protein of its feature comprises bovine serum albumin, Protalbinic acid or N,O-Diacetylmuramidase.
4. macromolecule blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere as claimed in claim 1 is characterized in that described polymer microballoon is protein embedding blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere.
5. the preparation method of macromolecule blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere as claimed in claim 1 is characterized in that preparation method's step of described protein embedding blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere is as follows:
A) get PROTEIN B SA solution, add sodium alginate, stir it is fully dissolved, leaving standstill the cancellation bubble becomes water in the experiment;
B) by 1 of water volume: 1-4: 1 measures organic phase, pours in the container, and to the ethyl cellulose of emulsifying agent that wherein adds 1%-5% and 0.1%-15%, mechanical stirring is fully dissolved it;
C) mixed aqueous solution is joined under magnetic agitation in the container that fills the machine oil phase, stirring at normal temperature makes the heavy-gravity mixing solutions be dispersed into microballoon; Under magnetic agitation, in system, add CaCl
2Solution carries out gelation reaction;
D) leave standstill and make calcium alginate gel bead be deposited to container bottom, slowly remove supernatant liquid, use the deionized water rinsing microballoon, obtain the Polyvinylpyrolidone (PVP)/calcium alginate polymer micro-sphere of protein macromolecule embedding trace after the wash-out template is handled.
6. the preparation method of macromolecule blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere as claimed in claim 5, it is characterized in that described organic phase density is 0.75-0.95g/mL, by soybean oil with butylacetate is composite forms, emulsifying agent is Tween80, its quality accounts for the 1%-5% of oil phase quality, and ethyl cellulose accounts for the 0.1%-15% of sodium alginate quality.
7. the preparation method of macromolecule blotting polythene based pyrrolidone/calcium alginate polymer micro-sphere as claimed in claim 5, the volume ratio that it is characterized in that organic phase and water is 1: 1-4: 1.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101857698A (en) * | 2010-06-10 | 2010-10-13 | 厦门大学 | Polystyrene/calcium alginate composite gel microsphere in nuclear shell structure and preparation method thereof |
| CN101962464A (en) * | 2010-09-27 | 2011-02-02 | 青岛大学 | Method for preparing calcium alginate microspheres with temperature response performance |
| CN104142361A (en) * | 2014-07-31 | 2014-11-12 | 中南民族大学 | Protein molecular imprinting polyion liquid membrane electrochemical transducer |
| CN104888226A (en) * | 2014-03-06 | 2015-09-09 | 中国科学院大连化学物理研究所 | Protein and/or polypeptide substance preparation |
-
2008
- 2008-03-18 CN CNA2008100524660A patent/CN101265332A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101857698A (en) * | 2010-06-10 | 2010-10-13 | 厦门大学 | Polystyrene/calcium alginate composite gel microsphere in nuclear shell structure and preparation method thereof |
| CN101857698B (en) * | 2010-06-10 | 2012-06-27 | 厦门大学 | Polystyrene/calcium alginate composite gel microsphere in nuclear shell structure and preparation method thereof |
| CN101962464A (en) * | 2010-09-27 | 2011-02-02 | 青岛大学 | Method for preparing calcium alginate microspheres with temperature response performance |
| CN104888226A (en) * | 2014-03-06 | 2015-09-09 | 中国科学院大连化学物理研究所 | Protein and/or polypeptide substance preparation |
| CN104142361A (en) * | 2014-07-31 | 2014-11-12 | 中南民族大学 | Protein molecular imprinting polyion liquid membrane electrochemical transducer |
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Open date: 20080917 |