CN104069836A - Molecularly imprinted microsphere material based on polymer phase inversion self assembly and application thereof - Google Patents
Molecularly imprinted microsphere material based on polymer phase inversion self assembly and application thereof Download PDFInfo
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- CN104069836A CN104069836A CN201310106745.1A CN201310106745A CN104069836A CN 104069836 A CN104069836 A CN 104069836A CN 201310106745 A CN201310106745 A CN 201310106745A CN 104069836 A CN104069836 A CN 104069836A
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Abstract
The invention relates to a molecularly imprinted microsphere material for selective recognition of target protein and a preparation process thereof. According to the molecularly imprinted microsphere material, a high molecular material is used as a base material, a peptide fragment on the target protein is taken as a template molecule, a polymer phase inversion self assembly mode is adopted to prepare a microsphere, and the prepared material is used for selective recognition of protein in a complex sample.
Description
Technical field
The invention belongs to Polymer materialspreparation technology and the application in protein identification thereof, a kind of based on polymer phase reversal self assembly molecule trace micro-sphere material specifically.
Background technology
Molecular imprinting is that preparation has the technology of the material of high selectivity recognition capability to target molecule by imitating the molecular recognition process in life process, adopting artificial synthesis.After Klaus Mosbach in 1993 etc. deliver the report about utilizing the synthetic theophylline molecularly imprinted polymer of non-covalent bond on " Nature ", molecular engram material enters rapidly global numerous scientists' the visual field as a kind of artificial synthetic high selectivity material.At present, obtained huge progress for the trace work of organic molecule and metal ion, experimental technique and evaluation criterion are gradually ripe, and field (Chen LX, Chemical Society Reviews, 40 such as start to be applied to clinical analysis, catalyze and synthesize, 2011,2922-2942).In these areas, molecular engram material is compared with natural molecule recognition system, not only has efficient selective, and various physics and chemistry influence factors are had to very high tolerance, but also can reuse, make up natural identification system many deficiencies in use.Meanwhile, in the time that the research of little molecular engram is ripe gradually, the research of molecular engram starts to turn to large biological molecule (especially protein) trace.Dream when this foundes at first to it, " artificial synthetic antibody ", steps and goes a step further again.The protein-imprinted polymer preparation method of bibliographical information mainly comprises (the Yang KG et.al. such as 3D molecular imprinting method (investment), 2D molecular imprinting method (surface imprinted method), metal-chelating trace method, Langmuir monofilm blotting, surperficial micro-contact trace method at present, Analytical and Bioanalytical Chemistry, 403,2012,2173-83.).But current these molecular engram materials all form as basis the polymeric material that is printed on protein identification site taking polymerisation.Owing to usually relating to heat, ultraviolet lighting, strong redox reaction etc. in polymerisation easily by the condition of protein molecule sex change, thereby reduce the specific recognition of imprinted polymer.In addition, in these traces usually taking protein as template molecule, but due to protein template molecule be difficult to obtain, expensive, therefore use (Sellergren, B.Nature Chemisty, 2 of protein imprinted material have been limited, 2010,7).
Therefore, we are taking the continuous amino acid sequence on epitope peptide section or protein sequence on protein as reference, artificial synthetic identical polypeptide, and as template, by the polymer phase reversal self-assembling technique of mild condition, at normal temperatures for the preparation of the selective molecular engram microsphere material of identifying of protein.
Summary of the invention
Employing polymer is host material, taking one section of peptide section sequence on protein sequence as template molecule, prepare the micro polymer ball material of peptide section trace by polymer phase reversal self-assembling technique, and the selective identification for the corresponding protein of peptide section by the micro polymer ball material of this peptide section trace.For achieving the above object, the technical solution used in the present invention is:
(1) prepare the ternary dispersion that polymer-template molecule-organic solution forms, wherein polymer accounts for the 1-50% of ternary system; Template molecule accounts for the 0.5-25% of ternary system; Organic solution accounts for the 10-90% of ternary system.
(2) at room temperature, the non-solvent that solution is dropwise splashed into polymer by syringe needle by the ternary dispersion of polymer-template molecule-organic solution composition by 75 μ m-10mm mutually in, because polymer is insoluble in mutually at non-solvent, the organic solution of non-solvent and dissolve polymer can be dissolved each other, and therefore balling-up is solidified in polymer and template molecule phase reversal.
(3) microballoon making is put into water to remove wherein residual organic solvent.
(4) and with alcohol solution/acetic acid (alcohols percentage by volume is 40%-100%) by template, from solidified microsphere, wash-out is out, and replacing leaching liquor, until on ultraviolet-visible spectrophotometer, can't detect template molecule, to obtain trace microballoon (MIP).With identical step, in pre-assembled system, do not add template, make non-trace microballoon (NIP).
(5) identification for standard form and corresponding protein by this micro-sphere material, and for the selective identification of the corresponding protein of actual sample template peptide section.
Tool of the present invention has the following advantages:
1. the present invention adopts the phase reversal self-assembling technique of mild condition to prepare molecular engram material, is conducive to keep the conformation of biomolecule.
2. the present invention adopts artificial synthesis peptide's section, instead of albumen, as the material of template molecule preparation identification albumen, has solved template molecule in Western blotting and has been difficult to acquisition, expensive problem.
3. phase reversal self-assembling technique is prepared peptide section trace micro-sphere material, and preparation process is simple and easy, favorable reproducibility, be conducive to extensive preparation and the use of protein molecule engram material.
4. the host material using polymer such as polyether sulfone, polysulfones, polyethersulfone ketones as self assembly, is not only conducive to the formation of self-assembly microspheres; More, because of its excellent biocompatibility, be conducive to reduce the non-specific adsorption of host material to protein.
Brief description of the drawings
Fig. 1. the electromicroscopic photograph of polymer phase reversal self assembly molecule trace micro-sphere material (MIP) (a, b, c) and non-trace micro-sphere material (NIP) (d, e, f);
Fig. 2. polymer phase reversal self assembly molecule trace micro-sphere material (MIP) and non-trace micro-sphere material (NIP) curve of adsorption kinetics to template (a) and transferrins (b).Detailed description of the invention
Embodiment
(1) preparation based on polymer phase reversal self assembly molecule trace micro-sphere material
Prepare in the process of epitope trace polyether sulfone (PES) microballoon at self-assembly method, 200mg PES, 30mg transferrins epitope (MRLAVGALL), the magnetic ball that 23mg particle diameter is 20nm, is scattered in and in 800mgDMAc, forms mixed solution.Then, at room temperature, the pre-assembled system of PES-transferrins epitope-DMAc is dropwise splashed into middle self assembly balling-up in water.Afterwards, the microballoon making is put into water to remove wherein residual DMAc.Then, template molecule is extracted from microballoon with methyl alcohol/acetic acid solution (alcohols percentage by volume is 90%), and often change leaching liquor, until at ultraviolet-visible spectrophotometer (Cary60, Agilent company) on can't detect template molecule till, to obtain trace microballoon (MIP).With identical step, in pre-assembled system, do not add template, make non-trace microballoon (NIP).Fig. 1 (a, b, c) is the stereoscan photograph of MIP cross section, in figure (a), has seen the loose structure in PES inside, and many large pore size distributions therebetween.In figure (b), microballoon edge is exaggerated 1000 times, sees the cortex of PES, and finger below cortex, and this is the typical PES self-assembled structures that is separated.In Fig. 1 (c), microballoon inside is exaggerated 5000 times, and we have seen the loose structure of microballoon microcosmic.Fig. 1 (d, e, f) is the stereoscan photograph of NIP, and finger and microcosmic loose structure are separated under corresponding its cross section macropore, cortex respectively.
(2) self assembly molecule trace micro-sphere material is investigated the recognition performance of epitope and corresponding protein
Self assembly trace microballoon is tested at 25 ° of C the identification of transferrins epitope and transferrins, in water, carries out.In experiment, the trace microballoon of 15mg and blank microballoon are joined respectively to 3mL, in the transferrins epitope and transferrins solution of 0.25mg/ml, at different time, detect corresponding molecular concentration in solution.As Fig. 2 has shown the recognition capability of self assembly MIP to epitope (a) and corresponding albumen (b), hatching after 70 hours, can reach 4.12 to the recognition factor of epitope, 2.95, MIP can be reached to the recognition factor of transferrins and 20.62mg/g can be reached to the adsorption capacity of albumen.
(3) selective in mixed protein of self assembly molecule trace micro-sphere material
Containing in transferrins (TRF), ribonuclease B (RNB), cromoci (CYC) and beta lactoglobulin (β-LACT) mixed aqueous solution (concentration of various albumen is 0.25mg/m), adsorb 90 hours, verify the selective of molecular engram microsphere.Experimental result shows (as shown in table 1): self assembly trace microballoon is to TRF, RNB, the migration index of CYC and β-LACT respectively: 0.14,0.05,2.43,1.25.The maximum migration index of imprinted material to TRF, has illustrated by the site in epitope site selective to respective egg white matter.
(4) self assembly molecule trace micro-sphere material identification to target protein in actual sample
Self assembly molecule trace micro-sphere material (MIP) and non-trace micro-sphere material (NIP) have been diluted to the human plasma protein fraction of 50 times with 3ml respectively hatches 72 hours jointly.Get respectively 1mL and hatch the supernatant of end, first by 90 ° of C thermal denaturations, dithiothreitol (DTT) reduction subsequently, iodo-acetamide alkylation, again, add trypsase according to the ratio of albumen/enzyme 25:1, spend the night after enzymolysis at 37 ° of C, peptide hydrolysis is carried out to desalination, with RPLC-ESI-MS/MS, peptide hydrolysis is separated to qualification subsequently.Subsequently, use the abundance method for expressing based on secondary spectral strength, SI
n, the protein identifying is carried out without scalar quantity.As shown in table 2, by front ten kinds of high-abundance proteins matter in supernatant are carried out to quantitative analysis, find that imprinted material has target protein in actual sample system significant selective, the target protein SI that imprinted material causes
nvariation is the target protein SI that blank material causes
nchange 61.6 times, far above other protein in blood plasma.
Table 1. polymer phase reversal self assembly molecule trace micro-sphere material (MIP) and the selective absorption of non-trace micro-sphere material (NIP) in protein mixed solution
* protein content in adsorbance=material adhesion protein amount/original solution
Table 2. polymer phase reversal self assembly molecule trace micro-sphere material (MIP) and non-trace micro-sphere material (NIP)
For the identification of the corresponding protein of actual sample (human plasma) template molecule (transferrins), and the variation of the amount of front ten kinds of high-abundance proteins matter (secondary spectral strength (SI
n) represent).
Claims (8)
1. based on a polymer phase reversal self assembly molecule trace micro-sphere material, it is characterized in that:
Employing polymer is host material, to contain 2-40 amino acid whose peptide section as template molecule, prepares the micro polymer ball material of peptide section trace by polymer phase reversal self assembly, and for the selective identification of the protein containing 2-40 amino acid whose peptide section correspondence.
2. according to molecular engram microsphere material claimed in claim 1, it is characterized in that: the polymer as host material is: one or two or more kinds in polyether sulfone, polysulfones, polyethersulfone ketone.
3. according to molecular engram microsphere material claimed in claim 1, it is characterized in that: be one section of sequence on naturally occurring protein or be the epitope peptide section sequence on naturally occurring protein as 2-40 amino acid whose peptide section of template molecule.
4. according to molecular engram microsphere material claimed in claim 1, it is characterized in that:
Polymer phase reversal self assembling process, is scattered in polymer, template molecule in organic solution, the non-solvent that solution is dropwise splashed into polymer by the syringe needle by 75 μ m-10mm mutually in, and then play the effect of polymer, template molecule being solidified to balling-up;
Water cements out organic solution subsequently, and with alcohols or alcohols and acetic acid mixed solution (alcohols percentage by volume is 40%-100%) from solidified microsphere template molecule wash-out out, and then made molecular engram material.
5. according to molecular engram microsphere material claimed in claim 4, it is characterized in that: described organic solution is the organic solution that the solubility of polymer is greater than to 1%; And the organic solution that can dissolve each other with the non-solvent arbitrary proportion of polymer;
Organic solution is: dimethyl sulfoxide (DMSO), DMF, DMA, two kinds of 1-METHYLPYRROLIDONE and above materials and multiple mixture.
6. according to molecular engram microsphere material claimed in claim 4, it is characterized in that: in the ternary system of described polymer, template molecule and organic solution composition, polymer accounts for the 1-50% of ternary system by mass; Template molecule accounts for the 0.5-25% of ternary system; Organic solution accounts for the 30-90% of ternary system.
7. according to molecular engram microsphere material claimed in claim 4, it is characterized in that: described non-solvent phase character is: can be also mixed solution for single solution, and can dissolve each other by arbitrary proportion with the non-solvent of polymer.
The non-solvent of polymer is: water, methyl alcohol, ethanol, propyl alcohol, isopropyl alcohol, butanols, a kind of or and the two or more mixture of above material in BDO.
8. a selective enrichment that is used for biological sample target protein based on polymer phase reversal self assembly molecule trace micro-sphere material claimed in claim 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109721759A (en) * | 2017-10-30 | 2019-05-07 | 中国科学院大连化学物理研究所 | A kind of acetylglucosamine imprinted material and its identification for acetylglucosamine and acetylglucosamine modification peptide fragment |
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| CN1390861A (en) * | 2002-06-26 | 2003-01-15 | 天津大学 | Magnetic composite microsphere of molecular blot polymer and its preparing process by combination of reverse-phase emulsion polymerization with suspension polymerization |
| WO2011010304A2 (en) * | 2009-07-23 | 2011-01-27 | Infigo Diagnostics Ltd. | Method for preparing molecularly imprinted polymers and uses thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1390861A (en) * | 2002-06-26 | 2003-01-15 | 天津大学 | Magnetic composite microsphere of molecular blot polymer and its preparing process by combination of reverse-phase emulsion polymerization with suspension polymerization |
| WO2011010304A2 (en) * | 2009-07-23 | 2011-01-27 | Infigo Diagnostics Ltd. | Method for preparing molecularly imprinted polymers and uses thereof |
Non-Patent Citations (5)
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| JEAN-MARIE LEHN: ""Toward Self-Organization and Complex Matter"", 《SCIENCE》 * |
| KAIGUANG YANG ET AL: ""Fabrication of Epitope Imprinted Polyethersulfone Particles through Self-assembly for Protein Recogintion"", 《37TH INTERNATIONAL SYMPOSIUM ON HIGH-PERFORMANCE LIQUID PHASE SEPARATIONS AND RELATED TECHNIQUES》, 31 December 2012 (2012-12-31), pages 476 * |
| KAIGUANG YANG ET AL: ""Molecularly imprinted polyethersulfone microspheres for the binding and recognition of bisphenol A"", 《ANALYTICA CHIMICA ACTA》, vol. 546, 8 June 2005 (2005-06-08), pages 2 - 2 * |
| M. MUTHUKUMAR ET AL: ""Competing Interactions and Levels of Ordering in Self-Organizing Polymeric Materials"", 《SCIENCE》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109721759A (en) * | 2017-10-30 | 2019-05-07 | 中国科学院大连化学物理研究所 | A kind of acetylglucosamine imprinted material and its identification for acetylglucosamine and acetylglucosamine modification peptide fragment |
| CN109721759B (en) * | 2017-10-30 | 2021-08-03 | 中国科学院大连化学物理研究所 | Acetyl glucosamine imprinted material and application thereof in recognition of acetyl glucosamine and acetyl glucosamine modified peptide fragment |
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