Protein surface molecular imprinting material based on RAFT (Reversible Addition-Fragmentation Chain Transfer) strategy as well as preparation method and application thereof

A surface molecular imprinting and protein technology, which is applied in chemical instruments and methods, and other chemical processes, can solve the problems of protein spatial conformation changes, difficult imprinting sites, etc., and achieve excellent selective recognition ability, fast mass transfer rate, The effect of good hydrophilicity

Active Publication Date: 2014-12-24
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This may be due to the harsh conditions for the preparation of western imprinted materials. The RAFT strategy currently used in the preparation of small molecule imprinted materials is mainly carried out in the oil phase system using thermal initiation. For proteins, high concentrations of organic solvents and Higher temperature (usually ≥60°C) will cause greater changes in the spatial conformation of the protein, making it difficult to obtain effective imprinting sites

Method used

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  • Protein surface molecular imprinting material based on RAFT (Reversible Addition-Fragmentation Chain Transfer) strategy as well as preparation method and application thereof
  • Protein surface molecular imprinting material based on RAFT (Reversible Addition-Fragmentation Chain Transfer) strategy as well as preparation method and application thereof
  • Protein surface molecular imprinting material based on RAFT (Reversible Addition-Fragmentation Chain Transfer) strategy as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1. Preparation of lysozyme surface imprinted material based on RAFT method

[0045] Such as figure 1 As shown, it is prepared according to the following process:

[0046] 1) Preparation of silica gel particles modified by RAFT reagent: firstly, amino groups are modified on the silica gel surface through silanization reaction of aminopropyltriethoxysilane on the surface of silica gel particles, and 100 mg of silica gel particles and 2.0 mL of methanol are added to each batch, and fully homogenized Afterwards, 50 μL of aminopropyltriethoxysilane was added, shaken and mixed, and reacted for 24 hours. Weigh 46.17mg of EDC, 22.60mg of NHS, 45.50mg of dithioester reagent 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPCP), add 30mL of ethanol to fully dissolve, weigh 400mg of dried amino silicon spheres, homogenate For the sealing reaction, the reaction temperature is 25° C., and the reaction time is 18 hours. After the reaction, wash with acetonitrile, ethanol, and w...

Embodiment 2

[0055] In order to investigate the adsorption capacity and kinetic characteristics of the lysozyme surface imprinted materials prepared above based on the RAFT method, the adsorption capacities of MIP1 and NIP1 materials to template proteins were measured at different adsorption times. The protein concentration in the solution was determined by chromatography. The specific measurement steps are as follows: take MIP1 and NIP1 materials respectively, 10 mg each, add 0.50 mL of 0.40 mg / mL lysozyme, and statically adsorb at 4 ° C for 1, 5, 15, 30 and 60 minutes, and then centrifuge to remove the upper Wash the supernatant solution twice with 1.0mL 40% acetonitrile to remove the remaining monomer and cross-linking agent, and then use 0.5mL eluent (20% formic acid + 40% acetonitrile + 40% water, v / v) to elute the template protein After 10 min, the protein concentration in the eluate was determined by HPLC. Its adsorption kinetic curve is given by Figure 4 shown. The adsorption c...

Embodiment 3

[0057] In order to investigate the selectivity and non-specific adsorption of the lysozyme surface imprinting material MIP1 based on the RAFT method and the control material NIP1 prepared above to the target protein and non-specific adsorption, further interference experiments were carried out.

[0058] 1. Preparation of protein mixture solution

[0059] Prepare 5.0mg / mL phosphate solutions of lysozyme, ribonuclease B, porcine serum albumin and myoglobin (pH=7.4) respectively. The four protein solutions were added in equal volumes, and then diluted with phosphate solution (pH=7.4), so that the final concentration of each protein was 0.40 mg / mL.

[0060] 2. Selective recognition of template proteins

[0061] Take 15 mg of lysozyme surface imprinting material based on the RAFT method prepared in Example 1, add 1.0 mL buffer for washing, and then add 0.4 mL of the protein mixed solution described in 1 for each part, mix well and then statically adsorb for 24 hours. Centrifuge...

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Abstract

The invention relates to a protein surface molecular imprinting material based on an RAFT (Reversible Addition-Fragmentation Chain Transfer) strategy. The protein surface molecular imprinting material is subjected to polymerization reaction by taking a silica gel granule with a dithioester functional group modified as a matrix, a lysozyme as a template protein, MAA (Methyl Acrylic Acid) and HEMA (Hydroxyethyl Methacrylate) as monomers, MBA (Methylene Diacrylamide) as a cross-linking agent and a phosphate water solution as a solvent and adopting a redox initiate polymerization reaction mode; after the polymerization reaction is completed, the template protein formed in a polymer is eluted, and then the dithioester functional group on a material surface is reduced into sulfhydryl, so that a hydrophilic lysozyme molecular imprinting core-shell structure microsphere can be obtained; and the hydrophilic lysozyme molecular imprinting core-shell structure microsphere is applied to the specific identification of the lysozyme. The protein surface molecular imprinting material disclosed by the invention can be used for realizing the controllability of the polymerization reaction and polymer chain growth by virtue of the reversible addition-fragmentation chain transfer strategy, and realizing narrow polymer distribution, controllable chain length and uniform polymer layer distribution so as to form sufficient surface imprinting sites.

Description

technical field [0001] The present invention relates to the selective recognition of protein molecularly imprinted granular materials for target proteins, specifically a lysozyme core-shell structure surface imprinted granular material based on a reversible addition-active chain transfer (RAFT) strategy. The preparation method and the application of the material to the selective identification of lysozyme components in biological samples. Background technique [0002] In nature, molecular recognition plays an important and special role in the evolution of life. The idea of ​​molecular imprinting originated from the concepts of "lock and key model" and "antigen-antibody" in molecular recognition. It is a new method and new technology for selective molecular recognition. This method achieves highly selective recognition of a specific target molecule by preparing a molecularly imprinted polymer (MIP) containing holes complementary to the target molecule. Compared with natural...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08F220/06C08F220/28C08F222/38C08J9/26B01J20/26B01J20/30
Inventor 张丽华李沁然杨开广梁玉江波张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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