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A composite magnetic nanoparticle fe3o4/mps/paa/nta-ni2+ and its preparation method and its application in the separation and purification of histidine-tagged proteins

A nanoparticle and composite magnetic technology, which is applied in the field of nanomagnetic materials and biological analysis, can solve the problems of low magnetic response of magnetic microspheres, difficulty in fast adsorption and movement, cumbersome synthesis process, etc., and achieve efficient chelation of metal ions Ability to ensure high-efficiency enrichment and purification efficiency, and the effect of simple separation and purification process

Inactive Publication Date: 2018-06-26
HUBEI UNIV OF TECH
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Problems solved by technology

Xu Bing et al. used NTA to modify the surface of platinum-iron alloy nanoparticles, and then chelated nickel ions for the separation and purification of histidine-tagged recombinant proteins. The results showed that the purification efficiency was greatly improved compared with commercially available commercial magnetic microspheres (XuC, Xu K, Gu H, Zhong X, Guo Z, etal.Nitrilotriacetic Acid-Modified Magnetic Nanoparticles as a General Agent to Bind Histidine-Tagged Proteins.J.Am.Chem.Soc.2004, 126,3392.), but due to the synthesis of platinum Ferroalloy nanoparticles are only 10nm in size and have low magnetic responsiveness. It is difficult to achieve rapid adsorption and movement under the control of an external magnetic field, which is limited to a certain extent.
U.S. Patent (USP4654267) discloses a polymer magnetic microsphere applied to protein separation and purification, but the synthesis process is cumbersome, and the magnetic response of the obtained magnetic microsphere is low
A Korean group synthesized magnetic nanoparticles modified with nickel ions, which were successfully applied to the separation and purification of histidine-tagged proteins, but its magnetic response is very weak (2emu / g), which is not conducive to improving the separation and purification efficiency of proteins
In addition, Xie et al. used PEI as a cross-linking agent to coat gold shells on the surface of magnetic nanoparticles, and then modified them with NTA and combined with Ni 2+ Chelation, the resulting nanocomplexes can rapidly and selectively separate histidine-tagged proteins (XieH.-Y., Zhen R., et al. Fe 3 o 4 / Au Core / Shell Nanoparticles Modified with Ni 2+ -Nitrilotriacetic Acid Specific to Histidine-Tagged Proteins, J.Phys.Chem.C2010,114,4825–4830.), however, the surface monolayer of the nanocomposite binds histidine-tagged proteins, and the adsorption capacity is not high

Method used

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  • A composite magnetic nanoparticle fe3o4/mps/paa/nta-ni2+ and its preparation method and its application in the separation and purification of histidine-tagged proteins
  • A composite magnetic nanoparticle fe3o4/mps/paa/nta-ni2+ and its preparation method and its application in the separation and purification of histidine-tagged proteins
  • A composite magnetic nanoparticle fe3o4/mps/paa/nta-ni2+ and its preparation method and its application in the separation and purification of histidine-tagged proteins

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Embodiment 1

[0040] A kind of composite magnetic nanoparticle Fe of the present embodiment 3 o 4 / MPS / PAA / NTA-Ni 2+ The average hydrated particle size is 247.9nm.

[0041] The above-mentioned composite magnetic nanoparticles Fe 3 o 4 / MPS / PAA / NTA-Ni 2+ Prepared by the following method, comprising the steps of:

[0042] Step 1: Preparation of magnetic Fe by solvothermal method 3 o 4 Nanoparticles, the specific steps are as follows: Weigh 0.2889g ferric chloride hexahydrate (FeCl 3 ·6H 2 O), 0.8248g ammonium acetate (NH 4 AC) and 0.0856g sodium citrate in a round bottom flask, add 15ml ethylene glycol, dissolve, stir vigorously at 170°C for 1h, transfer to a polytetrafluoroethylene reactor after cooling, heat from room temperature to 200°C, and react 8h, then cooled to room temperature, and centrifuged to obtain 0.2g magnetic core Fe 3 o 4 Nanoparticles, the prepared product is washed with absolute ethanol for 3 to 4 times, and finally dispersed in absolute ethanol;

[0043] The...

Embodiment 2

[0050] A kind of composite magnetic nanoparticle Fe of the present embodiment 3 o 4 / MPS / PAA / NTA-Ni 2+ The average hydrated particle size is 338nm.

[0051] The above-mentioned composite magnetic nanoparticles Fe 3 o 4 / MPS / PAA / NTA-Ni 2+ Prepared by the following method, comprising the steps of:

[0052] Step 1: Preparation of magnetic Fe by solvothermal method 3 o 4 Nanoparticles, the specific steps are as follows: Weigh 0.2889g ferric chloride hexahydrate (FeCl 3 ·6H 2 O), 0.8248g ammonium acetate (NH 4 AC) and 0.0856g sodium citrate in a round bottom flask, add 15ml ethylene glycol, dissolve, stir vigorously at 170°C for 1h, transfer to a polytetrafluoroethylene reactor after cooling, heat from room temperature to 200°C, and react 10h, then cooled to room temperature, and centrifuged to obtain 0.2g magnetic core Fe 3 o 4 Nanoparticles, the prepared product is washed with absolute ethanol for 3 to 4 times, and finally dispersed in absolute ethanol;

[0053] The ...

Embodiment 3

[0060] A kind of composite magnetic nanoparticle Fe of the present embodiment 3 o 4 / MPS / PAA / NTA-Ni 2+ The average hydrated particle size is 328.5nm.

[0061] The above-mentioned composite magnetic nanoparticles Fe 3 o 4 / MPS / PAA / NTA-Ni 2+ Prepared by the following method, comprising the steps of:

[0062] Step 1: Preparation of magnetic Fe by solvothermal method 3 o 4 Nanoparticles, the specific steps are as follows: Weigh 0.2889g ferric chloride hexahydrate (FeCl 3 ·6H 2 O), 0.8248g ammonium acetate (NH 4 AC) and 0.0856g sodium citrate in a round bottom flask, add 15ml ethylene glycol, dissolve, stir vigorously at 170°C for 1h, transfer to a polytetrafluoroethylene reactor after cooling, heat from room temperature to 200°C, and react 12h, then cooled to room temperature, and centrifuged to obtain 0.2g magnetic core Fe 3 o 4 Nanoparticles, the prepared product is washed with absolute ethanol for 3 to 4 times, and finally dispersed in absolute ethanol;

[0063] Th...

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Abstract

The invention relates to composite magnetic nanoparticles Fe3O4 / MPS / PAA / NTA-Ni<2+> and a preparation method and an application thereof in separation and purification of histidine-tagged proteins, and belongs to the technical fields of nano magnetic materials and biological analysis. Fe3O4 magnetic nanoparticles with an average hydration particle size of 100-400 nm are used as a core, MPS surface modification is adopted for making the surface rich in double bonds, polycarboxyl-structure magnetic nanoparticles with an average hydration particle size of 100-600 nm are obtained through PAA coating, and then furthermore, the polycarboxyl-structure magnetic nanoparticles are coupled with NTA-Ni<2+> to obtain the composite magnetic nanoparticles Fe3O4 / MPS / PAA / NTA-Ni<2+> having fast magnetic field response. The prepared composite magnetic nanoparticles enable the thickness of a coating layer to be controlled in nanometer scale; the prepared composite magnetic nanoparticles have the advantages of uniform size distribution, strong magnetic and low cost, have higher ability of enrichment and separation purification of the histidine-tagged proteins, and have the purification efficiency of the histidine-tagged proteins as high as 40-70 [mu]g / mg composite magnetic nanoparticles; and the composite magnetic nanoparticles can be repeatedly used.

Description

technical field [0001] The invention belongs to the technical field of nanometer magnetic materials and biological analysis, in particular to a composite magnetic nanoparticle Fe 3 o 4 / MPS / PAA / NTA-Ni 2+ Its preparation method and its application in the separation and purification of histidine tag protein. Background technique [0002] Traditional protein separation and purification methods include electrophoresis, chromatography, centrifugation, dialysis, filtration, etc., among which metal chelate affinity chromatography is considered to be one of the most effective methods. Although metal chelation affinity chromatography has many advantages in the separation and purification of histidine-tagged proteins, it still has disadvantages such as low efficiency, high cost, and cumbersome separation and purification steps, which make it impossible to meet the separation requirements. The combination of metal chelate affinity chromatography and magnetic separation technology to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08F292/00C08F220/06C08F222/38C08F8/44C07K1/22
CPCC07K1/22C08F8/44C08F292/00C08F2800/20C08F220/06C08F222/385
Inventor 郭惠玲
Owner HUBEI UNIV OF TECH
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