CN1631945A - Preparation method of molecular surface imprinted polymer - Google Patents

Abstract

A method for preparing molecule surface engram polymer has the steps as follows: fix the pattern molecule on the carrier surface, wash that not being combined by buffer solution, dry the carrier; hatch the synthesized monomer, cross linking reagent and functional monomerand the pattern molecule fixed on the carrier surface for combining pattern protein molecule and function monomer to be compound and distributing function monomer on the surface of pattern protein; add initiator to cause polymerization, fix the compound formed by pattern molecule and function monomer on the polymer surface; wash the engram polymer for clearing away the pattern molecule remarked in it, finally acquire the product. The invention makes the engram of molecule on the surface of material, which eliminates hard washing of pattern molecule embedded in polymer as well as makes the pattern molecule engram, and greatly advances the recognition to substance and dynamic performance.

Description

Preparation method of molecular surface imprinted polymer

technical field

The invention relates to a method for preparing a molecular surface imprinted polymer, in particular to a method for preparing a surface imprinted polymer capable of recognizing template molecules. It is used in the field of molecular recognition, detection and separation and purification.

Background technique

Molecular imprinting refers to the process of complementary imprinting the shape of specific molecules and the properties of charges, hydrophobicity, hydrogen bond acceptors and donors distributed on the surface of molecules into polymers, which can retain the properties of the printed molecules. Imprinting of shape and molecular surface properties. The imprint in the polymer has a specific corresponding relationship with the imprinted molecules, and has the effect of memory template molecular shape, so that the imprinted polymer material has the ability to recognize specific molecules.

In recent years, there have been many reports on molecular imprinting, but there are relatively few reports on the preparation of biomacromolecular imprinting, and there are even fewer satisfactory test results. The main reason is that the polymerization reaction is often carried out in an organic solvent environment. On the one hand, organic solvents destroy the hydrogen bonds recognized by biomacromolecules, which easily denatures biomacromolecules. Prepared into imprinted polymers, biomacromolecules are not easy to wash out from the interior of the polymer, and biomacromolecules are not easy to diffuse into the interior of small-pore imprinted polymers, which cannot be further developed and utilized. In view of the fragility of protein molecules, some researchers have carried out research on the preparation of imprinted polymers that recognize biomacromolecules. At the same time, in view of the large size of protein molecules, some researchers have adopted the strategy of surface imprinting to prepare polymers.

After searching the prior art documents, it was found that Buddy D. Ratner et al. published "Template-imprinted nanostructured surfaces for protein recognition Nature Vol. 398 1999 593-597" in "Nature". The method in this paper is to first adsorb the template protein on mica for dehydration, then use radio frequency glow discharge plasma deposition method to deposit disaccharide molecules on the surface of protein molecules, and then deposit hexafluoroethylene polymer to fix the disaccharide on the surface of protein molecules . After protein removal, imprinted disaccharides immobilized in hexafluoroethylene polymers can partially recognize template proteins. In this method, since the flexibility of the surface groups of protein molecules is basically lost after dehydration, complementary functional groups cannot be deposited near the functional groups of charge, hydrophobicity, hydrogen bond acceptors and donors on the protein surface. It is difficult to improve the performance of molecular recognition materials by introducing functional groups, and on the other hand, it is also difficult to prepare imprinted materials on a large scale. Therefore, fundamentally improving the performance of imprinted polymers is a scientific and technological challenge.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing molecular surface imprinted polymers, especially a method for preparing surface imprinted polymers of protein macromolecules, so that template molecules can be eluted and re-recognized more efficient and faster. The integrated functional monomer in the polymer improves the interaction with the template molecule, improves the efficiency of template molecule re-recognition, and the performance of the imprinted polymer.

The present invention is achieved through the following technical solutions, comprising the following steps:

(1) immobilized template protein molecules

The template molecules are immobilized on the surface of the carrier by physical or chemical methods, and the carrier includes (but not limited to) the microwell of the microplate, mica, glass and the like. Rinse off unbound template molecules with buffer, and dry the carrier.

(2) Incubation of functional monomers and immobilized proteins to form complexes

Polymerized monomers, cross-linking agents and functional monomers are incubated with the template molecules immobilized on the surface of the carrier, and the template protein molecules combine with the functional monomers to form a complex, so that the functional monomers are distributed on the surface of the template protein molecules.

(3) Initiate copolymerization of fixed functional monomers

The polymerization reaction is initiated by adding an initiator, and the complex formed by template molecules and functional monomers is fixed on the surface of the polymer.

(4) Rinse the template molecules on the imprinted polymer surface

Rinse the imprinted polymer for 3-6 times with the eluting solution to remove the template molecules in the imprinted polymer to obtain a polymer material with holes on the surface that are complementary in shape to the template molecules. Chemical functional groups that can combine with template molecules are distributed in the holes.

Performance evaluation of the imprinted polymer obtained in the present invention: by detecting the residue of the template protein molecule on the polymer, the re-adsorption of the template molecule, and the competition and selective adsorption of other protein molecules to the template molecule, the imprinted polymer prepared by the method of the present invention is determined. Properties of imprinted polymers.

The theoretical basis of the present invention is that the nanoscale pores on the surface of the polymer are complementary in shape to the template molecules, and play a role in spatially screening the template molecules during recognition. The functional groups interact non-covalently from the aspects of charge, hydrogen bond, hydrophobic interaction and van der Waals force, and further screen the template molecular recognition to enhance the affinity for the correct molecule. Template molecule residue determination, re-adsorption test and competitive adsorption test when other molecules exist prove that the imprinted polymer prepared by the present invention has a low amount of template residue, can re-recognize and bind template molecules, and has a relatively high effect on template molecules in the mixture. selection recognition ability. Scanning by atomic force microscopy revealed that there were nanoscale holes left by template molecules on the surface of the imprinted polymer.

The imprinted polymer prepared by the present invention imprints the template molecules on the surface of the polymer, which not only overcomes the disadvantage that the template molecules are embedded in the polymer and is difficult to elute, but also overcomes the limitation that the template molecules need to diffuse into the polymer material to recombine , the present invention also overcomes the fragility of protein molecules, and prepares a western imprinting polymer material in a water-soluble polymer system. The imprinted polymer prepared by the invention can re-adsorb the template protein molecule, and has selective recognition and binding ability to the template protein molecule in the presence of other protein molecules. The use of functional monomers in the preparation of imprinted polymers can improve the adsorption capacity and selectivity of template protein molecules. These characteristics make the prepared imprinted polymer have great potential application value in the field of separation and purification of substances and biosensors.

Detailed ways

The following is a further elaboration with specific examples:

Example 1: Surface imprinted polyacrylamide with hepatitis B e antigen as template molecule

In the present invention, the imprinted polymer prepared with HBe antigen as a template is represented by MIPe, and the imprinted polymer prepared without any template molecule is represented by MIPc. The steps and results are as follows:

Immobilization of a template protein molecule

Hepatitis B e antigen (HBe) at a concentration of 10 ⁴ mg/ml was added to the microwells of the 96-well microtiter plate, and left overnight at 4°C. Rinse off unbound template molecules with 0.5% TWEEN-normal saline, and dry the microtiter plate for later use.

Incubation of bifunctional monomers with immobilized proteins to form complexes

10ml of acrylamide (50%), N,N'-methylenebisacrylamide stock solution (8%), methacrylic acid (6%) were mixed evenly, and 400ul of ammonium persulfate and 200ul of N,N,N',N '-Tetramethylethylenediamine (TEMED), mix evenly, add 0.4ml to the microwell of the microplate plate coated with HBe molecules and incubate with the template molecules immobilized on the surface of the carrier for 20-30 minutes, the template protein molecules and The functional monomers combine to form a complex, so that the functional monomers are distributed on the surface of the template protein molecule.

Tri-initiated copolymerization of fixed functional monomers

After incubation for 20-30 minutes, the added initiator initiates the polymerization reaction, and the complex formed by the template molecule and the functional monomer is fixed on the surface of the polymer. After the polymerization reaction is completed, the imprinted material MIPe of HBe is obtained.

4. Rinsing of Blotted Material

Put the imprinted material MIPe in a 1.5ml centrifuge tube, add 3ml 0.1M NaOH to rinse twice, add 3ml 0.1M HAC to rinse twice, then add 3ml phosphate buffered saline PBS (10mM, pH7.4 containing 0.5% TWEEN20 , 137mM NaCl, 2.7mM KCl) rinsed twice. Finally, add 3ml of PBS (10mM, pH7.4) to rinse once. After rinsing, add rinsing solution on the shaker at 200 rpm, shaking at 30°C for 10 minutes, then suck out the rinsing liquid. The imprinted material was soaked in PBS (10 mM, pH 7.4) for later use.

Performance Evaluation of Penta-imprinted Polymers

The enzyme-linked immunosorbent assay (ELISA) method is used to detect the residue of the template protein molecule on the polymer, the re-adsorption of the template molecule, and the competition and selective adsorption of other protein molecules to the template molecule. A substrate reaction solution of a reference polymer (without template protein) was used as a control, and the light absorption value at 450 nm was measured. According to the curve of absorbance and HBe standard concentration, the prepared imprinting material MIPe has residual HBe of 14ng/cm ² and the amount of adsorbed HBe is 193ng/cm ² . The concentration of HBs molecules required to reduce the amount of HBe adsorbed by MIPe by half is twice that of BSA. 2 times the concentration of RNase A and 8 times the concentration of RNase A.

Example 2: Surface-imprinted poly(propylene alcohol) using hepatitis B e antigen as a template molecule

The steps and results are as follows:

Immobilization of a template protein molecule

Same as Example 1

Incubation of bifunctional monomers with immobilized proteins to form complexes

Add 300ul ammonium persulfate and 100ul N,N,N',N'-tetramethylethylenediamine (TEMED) to 10ml propenyl alcohol (20%), styrene (12%), mix well, add 0.4ml to the bag In the microwells of the ELISA plate with HBe molecules, incubate with the template molecules immobilized on the surface of the carrier for 30 minutes, and the template protein molecules combine with the functional monomer styrene to form a complex, so that the functional monomers are distributed on the surface of the template protein molecules.

Tri-initiated copolymerization of fixed functional monomers

After incubation for 30 minutes, the polymerization reaction occurred, and the imprinted material MIPe of HBe was obtained.

4. Rinsing of Blotted Material

Same as Example 1

Performance Evaluation of Penta-imprinted Polymers

Same as Example 1

The amount of residual HBe in the imprinted material MIPe is 16.7ng/cm ² , and the amount of HBe specifically adsorbed by MIPe is 88.48ng/cm ² . The concentration of HBs molecules required to reduce the amount of HBe adsorbed by MIPe by half is 1 times, and the concentration of BSA is 2 times, the concentration of RNase A was 4 times.

Example 3: Surface imprinted polystyrene with hepatitis B c antigen as template molecule

The steps and results are as follows:

Immobilization of a template protein molecule

Same as Example 1

Incubation of bifunctional monomers with immobilized proteins to form complexes

Add 300ul ammonium persulfate and 120ul N, N, N', N'-tetramethylethylenediamine (TEMED) to 10ml styrene and methacrylic acid (5%), mix well, add 0.4ml to the coated HBe molecule Incubate with the template molecules immobilized on the surface of the carrier for 30 minutes in the microwells of the microtiter plate, and the template protein molecules combine with the functional monomers to form a complex, so that the functional monomers are distributed on the surface of the template protein molecules.

Tri-initiated copolymerization of fixed functional monomers

After incubation for 30 minutes, the polymerization reaction occurred, and the imprinted material MIPe of HBe was obtained.

4. Rinsing of Blotted Material

Same as Example 1

Performance Evaluation of Penta-imprinted Polymers

Same as Example 1

The amount of residual HBe in MIPe of the imprinted material is 26.7ng/cm ² , and the amount of HBe specifically adsorbed by MIPe is 81.4ng/cm ² . The concentration of HBs molecules required to reduce the amount of HBe adsorbed by MIPe by half is 2 times, and the concentration of BSA is 2 times, the concentration of RNase A was 4 times.

Example 4: Surface imprinted polydimethylsiloxane with hepatitis B surface antigen (HBs) as template

The imprinted polymers prepared with antigen HBs as a template are represented by MIPs, and the imprinted polymers prepared without any template molecules are represented by MIPc. The steps and results are as follows:

Immobilization of a template protein molecule

Add HBs at a concentration of 10 ^-6 mg/ml dropwise on the freshly peeled surface of mica, overnight at 4°C. Unbound template molecules were washed away with 0.5% TWEEN-saline.

Incubation of bifunctional monomers with immobilized proteins to form complexes

Mix 10ml of dimethylsiloxane (main agent and auxiliary agent 10:1) and acrylate (8%) evenly, add it to the new surface of mica coated with HBs, and incubate with the template molecules fixed on the surface of the carrier, the template The protein molecule combines with the functional monomer to form a complex, so that the functional monomer is distributed on the surface of the template protein molecule.

Tri-initiated copolymerization of fixed functional monomers

While the template protein molecule is incubated with the functional monomer, the polymer system is degassed to undergo a polymerization reaction to obtain the imprinted material MIPs of HBs.

4. Rinsing of Blotted Material

3ml 0.1M NaOH was rinsed 4 times, and 3ml was rinsed 2 times with phosphate buffered saline PBS (10mM, pH7.4 containing 0.5% TWEEN20, 137mM NaCl, 2.7mM KCl). Finally, add 3ml of PBS (10mM, pH7.4) to rinse once.

Performance Evaluation of Penta-imprinted Polymers

Same as Example 1

The amount of residual HBs in MIPs of the imprinted material is 13.9ng/cm ² , the amount of HBe specifically adsorbed by MIPe is 61.8ng/cm ² , the concentration of HBs molecules required to reduce the amount of HBe adsorbed by MIPe by half is 2 times, and the concentration of BSA is 1 times, the concentration of RNase A was 2 times.

Embodiment 5: take hepatitis B surface antigen (HBs) as the surface imprinted polyacrylamide of template molecule

The steps and results are as follows:

Immobilization of a template protein molecule

Add HBs at a concentration of 10 ^-5 mg/ml into the microwells of a 96-well enzyme plate, and overnight at 4°C. Rinse off unbound template molecules with 0.5% TWEEN-normal saline, and dry the microtiter plate for later use.

Incubation of bifunctional monomers with immobilized proteins to form complexes

Mix 10ml of acrylamide (30%), N,N'-methylenebisacrylamide stock solution (20%), and 4-vinylpyridine (8%), add 200ul ammonium persulfate and 100ul N,N,N' , N'-tetramethylethylenediamine (TEMED), mix well, add 0.4ml to the microwell of the microplate plate coated with HBs molecules and incubate with the template molecules immobilized on the surface of the carrier for 20-30 minutes, the template protein The molecule combines with the functional monomer to form a complex, so that the functional monomer is distributed on the surface of the template protein molecule.

Tri-initiated copolymerization of fixed functional monomers

Same as Example 1

4. Rinsing of Blotted Material

Same as Example 1

Performance Evaluation of Penta-imprinted Polymers

Same as Example 1

The amount of residual HBs in MIPs of the imprinted material is 13ng/cm ² , and the amount of HBs specifically adsorbed by MIPs is 230ng/cm ² . The concentration of HBe required to reduce the amount of HBe adsorbed by MIPe by half is 4 times, the concentration of BSA is 2 times, and the concentration of RNase The concentration of A is 4 times.

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, coupling 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, 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.