CN111948589A

CN111948589A - Method for centrifugally separating ferritin nanoparticles by sucrose density gradient

Info

Publication number: CN111948589A
Application number: CN201910416453.5A
Authority: CN
Inventors: 张瑜; 周金花; 胡迎澳; 董亦馨; 王飞; 李迅
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2019-05-17
Filing date: 2019-05-17
Publication date: 2020-11-17

Abstract

The invention relates to a method for centrifugally separating ferritin nanoparticles by sucrose density gradient. The method comprises the following specific steps: firstly, dissolving sucrose in a buffer solution, preparing sucrose solutions with the mass percentage concentrations of 20%, 35%, 50% and 65%, and slowly dripping a certain amount of sucrose solution in sequence by adopting a layer-laying method; then slowly adding the ferritin sample loaded with ferroferric oxide into the sucrose gradient solution; performing horizontal centrifugation at 50,000rpm for 5h at 4 deg.C; and finally, slowly and equally sucking out the mixed solution, and respectively measuring the protein content and the iron content of the sucrose solution layer by using an ultraviolet spectrophotometer under the wavelengths of 280nm and 410 nm. Finally, ferritin loaded with ferroferric oxide in the ferritin cavity is obtained through separation, separation whether the ferroferric oxide is loaded in the ferritin cavity or not is achieved, and another simple and feasible method except magnetic field separation is provided for separating the ferritin.

Description

Method for centrifugally separating ferritin nanoparticles by sucrose density gradient

Technical Field

The invention relates to a method for separating ferritin particles, in particular to a method for separating ferritin internally loaded with ferroferric oxide particles by sucrose density gradient centrifugation.

Background

With the continuous improvement of scientific research and technical means, the research, utilization and development of the ferritin nanoshells in vitro have made rapid development. In the research and application of biomagnetism, biomedicine and nano material science, ferritin has a very important position as a special nano material.

The ferritin structure is spherical and consists of two parts, namely an iron core and a protein shell, wherein the former exists in the protein shell in the form of ferric hydroxide and phosphate, and the latter is an octahedral highly symmetrical structure consisting of 24 subunit monomers. The hydrated iron oxide inner core in the ferritin has uniform grain size, and researches show that under the anaerobic condition, the ferritin iron core after redox reaction can be removed to obtain the empty shell ferritin; by utilizing the self-assembly property of the ferritin structure, metal particles or metal oxide particles are packed into ferritin to form particles with special physicochemical property. When metal oxide particles such as ferroferric oxide are synthesized in ferritin, the diameter of an inner cavity of the ferritin is only 8nm, the nanoparticles have superparamagnetism, and the ferritin has tumor targeting property, so the Magnetic Resonance Imaging (MRI) research is commonly used.

Ferritin is the first choice in the study of the synthesis of iron oxide-containing nanoparticles. By reaction of 3Fe²⁺+H₂O₂+H₂O→Fe₃O₄+6H⁺And synthesizing ferroferric oxide particles in the ferritin. Because some ferritin cavities do not contain ferroferric oxide particles, in order to be better applied to subsequent research, ferroferric oxide ferritin particles with uniform particle size and single component are necessary to be obtained. The current methods for separating and removing the non-synthesized ferroferric oxide protein particles are mainly based on the magnetic properties, but the magnetic separation method is not suitable for the ferritin particles of other metals or oxides.

Disclosure of Invention

The invention aims to provide a method for centrifugally separating ferritin containing ferroferric oxide particles by utilizing sucrose density gradient. The method is characterized by comprising the following specific steps:

(1) concentrating the ferritin sample loaded with ferroferric oxide;

(2) preparing sucrose solutions with different concentrations according to the mass percentages of 20%, 35%, 50% and 65% of sucrose respectively, and preparing sucrose gradient solutions from equal volumes of the sucrose solutions with different concentrations in sequence;

(3) slowly adding the ferritin sample treated in the step (1) into the sucrose gradient solution treated in the step (2);

(4) performing horizontal centrifugation at 50,000rpm for 5h at 4 ℃;

(5) slowly and equally sucking out the mixed solution; finally obtaining ferritin containing ferroferric oxide inside.

The invention has the beneficial effects that: the method for purifying the ferroferric oxide-containing particles in the ferritin by using the centrifugal method is simple, convenient and feasible to operate; (2) the separation method is not limited to the ferroferric oxide particles of the ferritin, is also applicable to separation and purification of other particles for synthesizing metal or metal oxide in the ferritin, and has wide application range. Therefore, the invention has wide application prospect in the fields of analytical chemistry and nano materials.

Drawings

FIG. 1 is a comparison of sucrose density gradient after centrifugation;

FIG. 2 is a graph showing the result of analysis by an ultraviolet spectrophotometer;

FIG. 3 is a TEM image of the sample before sucrose density gradient centrifugation (scale 50 nm);

FIG. 4 is a TEM image of the sample after sucrose density gradient centrifugation (scale 50 nm).

Detailed Description

The invention will be described with reference to specific embodiments:

the first embodiment is as follows:

(1) firstly, concentrating a sample after reaction by using an ultrafiltration tube, centrifuging for 10min at 10,000g, and removing insoluble substances in the solution;

(2) manufacturing a density gradient: accurately weighing sucrose by using an electronic balance, and preparing sucrose solutions with the mass percentages of 20%, 35%, 50% and 65% respectively; sequentially taking 600 mu L of sucrose solution from 20% and slowly adding the sucrose solution into a centrifuge tube to prepare a gradient solution;

(3) slowly adding the concentrated solution to the gradient solution along the wall of the centrifugal tube;

(4) performing horizontal centrifugation at 50,000rpm for 5h at 4 deg.C;

(5) and slowly sucking the mixed solution in equal parts, wherein each equal part is 200 mu L, slowly sucking the mixed solution in equal parts from the top by using a pipette, each equal part is 200 mu L, slowly sucking the mixed solution from the top by using the pipette, and finally analyzing to obtain the ferritin containing the ferroferric oxide particles inside.

Claims

1. A method for centrifugally separating ferritin nanoparticles containing metal or oxides thereof from the interior by sucrose density gradient is characterized by comprising the following specific steps:

(1) concentrating the ferritin sample loaded with ferroferric oxide;

(4) performing horizontal centrifugation at 50,000rpm for 5h at 4 ℃;

(5) slowly and equally sucking out the mixed solution; finally obtaining the ferritin with the iron oxide loaded inside.

2. The method of sucrose density gradient centrifugation of ferritin nanoparticles as claimed in claim 1 wherein the ferritin particles comprise ferriferrous oxide ferritin particles.