CN1274740C - Molecular surface imprinting polymer preparing method - Google Patents
Molecular surface imprinting polymer preparing method Download PDFInfo
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- CN1274740C CN1274740C CN 200410084542 CN200410084542A CN1274740C CN 1274740 C CN1274740 C CN 1274740C CN 200410084542 CN200410084542 CN 200410084542 CN 200410084542 A CN200410084542 A CN 200410084542A CN 1274740 C CN1274740 C CN 1274740C
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Abstract
The present invention relates to a preparation method for molecular surface imprinting polymer. The preparation method comprises the following steps: template molecules are fixed to the surface of a carrier, uncombined template molecules are washed with a buffer solution, and the carrier is aired; synthesized monomers, a cross linking reagent, functional monomers and the template molecules fixed to the surface of the carrier are hatched; the template protein molecules and the functional monomers are combined form a composition, which leads that the functional monomers are distributed on the surfaces of the template protein molecules; an initiator is added to initiate polyreaction, and the composition formed from the template molecules and the functional monomers is fixed to the surface of a polymer; the imprinting polymer is washed, the template molecules in the imprinting polymer are removed, and the polymer is obtained. The present invention makes the molecule imprinting prepared on the surface of a material, the disadvantage that the template molecules are embedded in the polymer and are difficult to wash is overcome, and the template molecules can be easily in contact with the molecular imprinting on the surface of the material; therefore, the kinetic properties of identifying and combining with a substance to be measured are greatly improved.
Description
Technical field
The present invention relates to a kind of preparation method of molecular surface imprinting polymer, specifically is a kind of preparation method of surface imprinted polymer that can the recognition template molecule.Be used for molecular recognition, detection and separation and purification field.
Background technology
Molecular imprinting is meant that the character with the shape of specific molecular and the electric charge, hydrophobicity, hydrogen bond receptor and the donor that distribute complementally prints to the process in the polymkeric substance on molecular surface, this polymkeric substance can keep by the shape of seal molecule and the trace of molecular surface character.Trace in the polymkeric substance with had special corresponding relation by the molecule of trace, have the effect of memory template shape of molecule, thus make the imprinted polymer material have identification specific molecular performance.
In recent years, the report of relevant molecular imprinting is a lot, but the report document of relevant biomacromolecule trace preparation is fewer, and satisfied test-results still less.Major cause is that polyreaction is usually carried out at the organic solvent environment, organic solvent destroys the hydrogen bond of biomacromolecule identification on the one hand, make biomacromolecule material generation sex change easily, the volume ratio of biomacromolecule is bigger on the other hand, even unchangeability can be prepared into imprinted polymer smoothly, biomacromolecule is not easy to wash out from polymkeric substance inside, and biomacromolecule also is not easy to diffuse into the inside of small-bore imprinted polymer, can't further develop.At the characteristics of protein molecule fragility, some investigators have carried out the research of the imprinted polymer of preparation identification biomacromolecule.At the bulky characteristics of protein molecule, some investigators adopt surface imprinted strategy to prepare polymkeric substance simultaneously.
Find " the template imprinting nanostructured surface of recognition protein " (Template-imprinted nanostructured surfaces forprotein recognition Nature Vol.398 1999 593-597) that Buddy D.Ratner etc. delivers by prior art documents on " nature ".Method in this paper is earlier the template protein adsorption to be dewatered on mica, use again radio frequency glow discharge plasma deposition method with the disaccharides molecule deposition on the protein molecular surface, and then deposition hexafluoroethylene polymkeric substance is with protein molecular surface two glycopexis.After removing albumen, the trace that is fixed on the disaccharides formation in the hexafluoroethylene polymkeric substance to a certain extent can recognition template albumen.Handiness owing to the protein molecule surface group in this method is lost after dehydration substantially, the complementary functional group can not be deposited near the functional group of electric charge, hydrophobicity, hydrogen bond receptor and donor of protein surface, be difficult on the one hand also be difficult to the mass preparation imprinted material on the other hand by introducing the performance that functional group improves the molecular recognition material.Therefore, the performance of fundamentally improving imprinted polymer is a challenge on the Science and Technology.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of preparation method of molecular surface imprinting polymer is provided, the preparation method of especially protein-based macromolecular surface imprinted polymer, make the wash-out of template molecule and again identification more effective and quick.The function monomer of integrating in the polymkeric substance has improved the interaction with template molecule, has improved the efficient that template molecule discerns again and the performance of imprinted polymer.
The present invention is achieved by the following technical solutions, comprises the following steps:
(1) fixed die plate protein molecular
Template molecule is fixed in carrier surface by physics or chemical method, and carrier includes but is not limited to enzyme plate micropore, mica or glass etc.Rinse unconjugated template molecule with damping fluid, dry carrier.
(2) function monomer and immobilization albumen are hatched the formation mixture
Polymerization single polymerization monomer, linking agent and function monomer and the template molecule that is fixed in carrier surface are hatched, and the template protein molecular combines with function monomer and forms mixture, makes function monomer be distributed in template protein molecular surface.
(3) cause copolymerization fixed function monomer
Add the initiator initiated polymerization, the mixture that template molecule and function monomer are formed is fixed in polymer surfaces.
(4) template molecule on rinsing imprinted polymer surface
With elute soln rinsing imprinted polymer 3-6 time, remove the template molecule in the imprinted polymer, promptly obtain the surface and have polymer materials with the hole of template molecule shape complementarity.Distributing in the hole can with template molecule bonded chemical functional group.
The performance evaluation of the imprinted polymer of gained of the present invention: by detect the template protein molecular on polymkeric substance residual, to the absorption again of template molecule and other protein molecular competition and selective adsorption situation to template molecule, measure the performance of the imprinted polymer of the inventive method preparation.
Theoretical basis of the present invention is that the hole of nanoscale of polymer surfaces and template molecule are complementary in shape, play the effect of spatially screening template molecule during identification, carry out non-covalent interaction from electric charge, hydrogen bond, hydrophobic interaction and Van der Waals force etc. aspect several at the functional group on specific position fixed function monomer and template molecule surface in the nanometer hole, further identification is screened to template molecule, strengthens the affinity interaction to correct molecule.The residual mensuration of template molecule, adsorption test and the competitive adsorption evidence when having other molecule to exist again, the imprinted polymer of the present invention's preparation is low to the template residual quantity, can discern again and, the template molecule in the mixture is had than higher selection recognition capability in conjunction with template molecule.Find that by afm scan there is the hole of the nanoscale that template molecule stays in the imprinted polymer surface.
The imprinted polymer of the present invention preparation with the template molecule trace at polymer surfaces, not only overcome the template molecule embedding and stayed the shortcoming that polymkeric substance inside is difficult to wash-out, also overcome template molecule and need diffuse into the bonded restriction again of polymer materials inside, the present invention has also overcome the fragile character of protein molecule, has prepared the western blotting polymeric material in the water-soluble polymers system.The prepared imprinted polymer of the present invention can adsorb the template protein molecular again, and when having other protein molecular to exist to selective identification of template protein molecular and binding ability.The functions of use monomer can improve the loading capacity of template protein molecular and select performance during the preparation imprinted polymer.These characteristics make the imprinted polymer of preparation have very big potential using value in the separation and purification and the field of biosensors of material.
Embodiment
Be further elaborated below in conjunction with specific examples:
Embodiment 1: the surface imprinted polyacrylamide that with the hepatitis B virus e antigen is template molecule
Be that the imprinted polymer of template preparation represents that with MIPe the imprinted polymer of preparation is represented with MIPc when existing without any template molecule with HBe antigen among the present invention.Step and result are as follows:
One template protein molecular is fixed
With concentration is 10
-4The hepatitis B virus e antigen of mg/ml (HBe) is added in the 96 hole enzyme plate micropores, and 4 ℃ are spent the night.Rinse unconjugated template molecule with the TWEEN-physiological saline that contains 0.5%, it is stand-by to dry enzyme plate.
Two function monomers and immobilization albumen are hatched the formation mixture
10ml acrylamide (50%), N, N '-methylene bisacrylamide liquid storage (8%), methacrylic acid (6%) mix, add 400ul ammonium persulphate and 200ul N, N, N ', N '-Tetramethyl Ethylene Diamine (TEMED), mix, add 0.4ml and hatched 20-30 minute with the template molecule that is fixed in carrier surface in the enzyme plate micropore that is coated with the HBe molecule, the template protein molecular combines with function monomer and forms mixture, makes function monomer be distributed in template protein molecular surface.
Three cause copolymerization fixed function monomer
After hatching 20-30 minute, the initiator initiated polymerization of adding, the mixture that template molecule and function monomer are formed is fixed in polymer surfaces.After finishing, polyreaction obtains the imprinted material MIPe of HBe.
The rinsing of four imprinted materials
Imprinted material MIPe is placed in the centrifuge tube of 1.5ml, adds 3ml 0.1M NaOH rinsing 2 times, add 3ml 0.1M HAC rinsing 2 times, " add again the 3ml phosphate buffered saline buffer (phosphate buffer saline, PBS), H wherein
3PO
4, H2PO
4 -, HPO
4 2-, PO4
3 -Total concn is 10mM, and the total pH of solution is 7.4, and containing Tween 20 simultaneously is 0.5% (v/v), and containing NaCl is 137mM, and containing KCl is 2.7mM, rinsing 2 times.(10mM, pH7.4) rinsing is 1 time to add 3ml PBS at last.Add during rinsing after the rinsing on shaking table with the 200rpm rotating speed, duration of oscillation was absorbed rinsing liquid after 10 minutes under 30 ℃ of temperature.Imprinted material be immersed in PBS (10mM, pH7.4) standby.The performance evaluation of five imprinted polymers
Adopt the method for enzyme linked immunological (ELISA) to detect competition and the selective adsorption of the absorption again of residual, the template molecule of template protein molecular on polymkeric substance and other protein molecular to template molecule.Substrate reactions solution with reference polymkeric substance (not having template albumen) is done contrast, measures the absorbance value of 450nm.According to absorbancy and HBe normal concentration curve, the residual HBe 14ng/cm of the imprinted material MIPe of preparation
2, the amount of absorption HBe is 193ng/cm
2, the concentration that the amount that makes MIPe adsorb HBe reduces half needed HBs molecule is 2 times, 2 times of the concentration of BSA, 8 times of the concentration of RNase A.
Embodiment 2: the surface imprinted POLYPROPYLENE GLYCOL that with the hepatitis B virus e antigen is template molecule
Step and result are as follows:
One template protein molecular is fixed
With embodiment 1
Two function monomers and immobilization albumen are hatched the formation mixture
10ml vinylcarbinol (20%), add 300ul ammonium persulphate and 100ul N in the vinylbenzene (12%), N, N ', N '-Tetramethyl Ethylene Diamine (TEMED) mixes, add 0.4ml in the enzyme plate micropore that is coated with the HBe molecule, hatched 30 minutes with the template molecule that is fixed in carrier surface, the template protein molecular combines with function monomer vinylbenzene and forms mixture, makes function monomer be distributed in template protein molecular surface.
Three cause copolymerization fixed function monomer
After hatching 30 minutes, polymerization reaction take place obtains the imprinted material MIPe of HBe.
The rinsing of four imprinted materials
With embodiment 1
The performance evaluation of five imprinted polymers
With embodiment 1
The amount of the residual HBe of imprinted material MIPe is 16.7ng/cm
2, the amount of the special absorption of MIPe HBe is 88.48ng/cm
2, the concentration that the amount that makes MIPe adsorb HBe reduces half needed HBs molecule is 1 times, 2 times of the concentration of BSA, 4 times of the concentration of RNase A.
Embodiment 3: the surface imprinted polystyrene that with the hepatitis B virus e antigen is template molecule
Step and result are as follows:
One template protein molecular is fixed
With embodiment 1
Two function monomers and immobilization albumen are hatched the formation mixture
Add 300ul ammonium persulphate 120ul N in 10ml vinylbenzene, the methacrylic acid (5%), N, N ', N '-Tetramethyl Ethylene Diamine (TEMED), mix, add 0.4ml in the enzyme plate micropore that is coated with the HBe molecule, hatched 30 minutes with the template molecule that is fixed in carrier surface, the template protein molecular combines with function monomer and forms mixture, makes function monomer be distributed in template protein molecular surface.
Three cause copolymerization fixed function monomer
After hatching 30 minutes, polymerization reaction take place obtains the imprinted material MIPe of HBe.
The rinsing of four imprinted materials
With embodiment 1
The performance evaluation of five imprinted polymers
With embodiment 1
The amount of the residual HBe of imprinted material MIPe is 26.7ng/cm
2, the amount of the special absorption of MIPe HBe is 81.4ng/cm
2, the concentration that the amount that makes MIPe adsorb HBe reduces half needed HBs molecule is 2 times, 2 times of the concentration of BSA, 4 times of the concentration of RNase A.
Embodiment 4: be the surface imprinted polydimethylsiloxane of template with hepatitis B surface antigen (HBs)
With antigen HBs is that the imprinted polymer that template prepares is represented with MIPs, and the imprinted polymer of preparation is represented with MIPc when existing without any template molecule.Step and result are as follows:
One template protein molecular is fixed
With concentration is 10
-6The HBs of mg/ml drips on the surface of the new strip off of mica, and 4 ℃ are spent the night.TWEEN-physiological saline with 0.5% rinses unconjugated template molecule.
Two function monomers and immobilization albumen are hatched the formation mixture
10ml dimethyl siloxane (host and assistant agent 10: 1), acrylate (8%) mix, it is newly surperficial to be added to the mica that is coated with HBs, hatch with the template molecule that is fixed in carrier surface, the template protein molecular combines with function monomer and forms mixture, makes function monomer be distributed in template protein molecular surface.
Three cause copolymerization fixed function monomer
When template protein molecular and function monomer were hatched, to the polymeric system processing that outgases, polymerization reaction take place obtained the imprinted material MIPs of HBs.
The rinsing of four imprinted materials
3ml phosphate buffered saline buffer PBS (0.5%TWEEN 20 for 10mM, pH7.4,137mM NaCl, 2.7mM KCl) rinsing 2 times.(10mM, pH7.4) rinsing is 1 time to add 3ml PBS at last.
The performance evaluation of five imprinted polymers
With embodiment 1
The amount of the residual HBs of imprinted material MIPs is 13.9ng/cm
2, the amount of the special absorption of MIPe HBe is 61.8ng/cm
2, the concentration that the amount that makes MIPe adsorb HBe reduces half needed HBs molecule is 2 times, 1 times of the concentration of BSA, 2 times of the concentration of RNase A.
Embodiment 5: be the surface imprinted polyacrylamide of template molecule with hepatitis B surface antigen (HBs)
Step and result are as follows:
One template protein molecular is fixed
With concentration is 10
-5The HBs of mg/ml is added in the 96 hole enzyme plate micropores, and 4 ℃ are spent the night.TWEEN-physiological saline with 0.5% rinses unconjugated template molecule, and it is stand-by to dry enzyme plate.
Two function monomers and immobilization albumen are hatched the formation mixture
10ml acrylamide (30%), N, N '-methylene bisacrylamide liquid storage (20%), 4-vinylpridine (8%) mix, add 200ul ammonium persulphate and 100ul N, N, N ', N '-Tetramethyl Ethylene Diamine (TEMED), mix, add 0.4ml and hatched 20-30 minute with the template molecule that is fixed in carrier surface in the enzyme plate micropore that is coated with the HBs molecule, the template protein molecular combines with function monomer and forms mixture, makes function monomer be distributed in template protein molecular surface.
Three cause copolymerization fixed function monomer
With embodiment 1
The rinsing of four imprinted materials
With embodiment 1
The performance evaluation of five imprinted polymers
With embodiment 1
The amount of the residual HBs of imprinted material MIPs is 13ng/cm
2, the amount of the special absorption of MIPs HBs is 230ng/cm
2, the concentration that the amount that makes MIPe adsorb HBe reduces half needed HBe is 4 times, 2 times of the concentration of BSA, 4 times of the concentration of RNase A.
Claims (5)
1, a kind of preparation method of molecular surface imprinting polymer is characterized in that, comprises the following steps:
(1) fixed die plate protein molecular: template molecule is fixed in carrier surface, rinses unconjugated template molecule, dry carrier with damping fluid;
(2) function monomer and immobilization albumen are hatched the formation mixture: polymerization single polymerization monomer, linking agent and function monomer and the template molecule that is fixed in carrier surface are hatched, the template protein molecular combines with function monomer and forms mixture, makes function monomer be distributed in template protein molecular surface;
(3) cause copolymerization fixed function monomer: add the initiator initiated polymerization, the mixture that template molecule and function monomer are formed is fixed in polymer surfaces;
(4) template molecule on rinsing imprinted polymer surface: the rinsing imprinted polymer, remove the template molecule in the imprinted polymer, promptly obtain the surface and have polymer materials with the hole of template molecule shape complementarity.
2, the preparation method of molecular surface imprinting polymer according to claim 1 is characterized in that, in the step (1), by physics or chemical method template molecule is fixed in carrier surface.
3, the preparation method of molecular surface imprinting polymer according to claim 1 is characterized in that, in the step (4), with elute soln rinsing imprinted polymer 3-6 time, distributing in the polymkeric substance hole of gained can with template molecule bonded chemical functional group.
4, the preparation method of molecular surface imprinting polymer according to claim 1 is characterized in that, described carrier comprises enzyme plate micropore, mica or glass.
5, the preparation method of molecular surface imprinting polymer according to claim 1, it is characterized in that, the performance evaluation of the imprinted polymer of gained by detect the template protein molecular on polymkeric substance residual, absorption again and other protein molecular of template molecule are measured the competition and the selective adsorption situation of template molecule.
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| CN100342958C (en) * | 2005-11-02 | 2007-10-17 | 华东理工大学 | Composite fiber membrane with chiral molecular imprint and its prepn and application |
| CN100348633C (en) * | 2005-11-25 | 2007-11-14 | 天津理工大学 | DNA print thermo-sensitive high molecule material and preparation process thereof |
| CN101627073B (en) * | 2007-03-05 | 2014-02-26 | Mip技术股份公司 | Imprinted polymers |
| CN104804134A (en) * | 2014-01-26 | 2015-07-29 | 北京林业大学 | Preparation method and application of isorhamnetin surface molecularly imprinted microsphere based on nanometer titanium dioxide |
| CN106633167B (en) * | 2015-10-30 | 2019-11-12 | 中国科学院大连化学物理研究所 | It is a kind of for the imprinted material of specific recognition cell and its preparation and application |
| CN105801900B (en) * | 2016-03-26 | 2018-06-01 | 吉首大学 | A kind of imprinted polymer preparation method and application of optional enrichment F- ions |
| CN108205061A (en) * | 2018-03-13 | 2018-06-26 | 闽江学院 | A kind of immobilized influenza virus trace fluorescent optical sensor of 96 hole elisa Plates and application |
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