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Preparation of dendritic molecule-modified hydrophilic immunomagnetic beads and application of hydrophilic immunomagnetic beads to rapid and efficient cell capture

An immunomagnetic sphere and dendritic technology, applied in animal cells, tumor/cancer cells, vertebrate cells, etc., can solve problems such as poor specificity and sensitivity, limit practical application, etc., to enhance the amount of modification and have great potential for clinical application. , the effect of quick identification

Inactive Publication Date: 2016-12-07
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

According to different separation mechanisms, these new technologies and methods can be divided into physical separation and biological affinity separation: the physical separation method to enrich tumor cells mainly uses the size, density and deformability of tumor cells to be different from normal blood cells. It is simple and convenient, but its specificity and sensitivity are poor, which limits its practical application; the bioaffinity separation method enriches tumor cells by introducing antibodies, aptamers and other biomolecules that can specifically interact with the cell membrane surface. This method has high efficiency, strong specificity, less damage to cells, and has more practical application value

Method used

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  • Preparation of dendritic molecule-modified hydrophilic immunomagnetic beads and application of hydrophilic immunomagnetic beads to rapid and efficient cell capture
  • Preparation of dendritic molecule-modified hydrophilic immunomagnetic beads and application of hydrophilic immunomagnetic beads to rapid and efficient cell capture
  • Preparation of dendritic molecule-modified hydrophilic immunomagnetic beads and application of hydrophilic immunomagnetic beads to rapid and efficient cell capture

Examples

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Embodiment 1

[0029] Example 1: Synthesis of Human Epidermal Growth Factor Receptor Antibody Modified Dendrimers Assisted Hydrophilic Magnetic Nanoparticles as Cell Trapping Substrates

[0030] Synthesize magnetic nanoparticles by hydrothermal method, weigh 1.3 g ferric chloride hexahydrate and put it in a clean beaker, then add 75 mL ethylene glycol into the beaker, stir magnetically until completely dissolved, add 3.6 g acetic acid to the obtained solution Sodium, stir until dissolved, and continue to stir for 30 min, transfer the mixed liquid to the reaction kettle, place it in a muffle furnace at 200°C and heat it for 10-16 h, after cooling, wash the material alternately with water and ethanol, and dry it in vacuum at 50°C Dry in the box for subsequent use to obtain magnetic nanoparticles;

[0031] Weigh 300 mg of magnetic nanoparticles synthesized by the hydrothermal method and disperse them in 50 mL of isopropanol and 8 mL of distilled water, then add 2 mL of tetraethyl orthosilicate ...

Embodiment 2

[0032] Example 2: Application of superhydrophilic immunomagnetic sphere materials in the rapid and efficient capture of human glioma cell U251

[0033] Take 200 μg of the magnetic material co-modified by antibodies and dendrimers and add it to one well of a 12-well plate as a group, add 1 mL of human glioma cell U251 suspension to each well, and the cell density is 10 5 , after incubating in the incubator for 15 min, the supernatant was aspirated and washed three times with PBS, and the cell capture efficiency was calculated. Add 10 μg / mL AO dye to the captured cells, wash them after staining at room temperature for 15 min, and observe with a fluorescence microscope.

[0034] 10, 20, 50, and 100 U251 cells prestained with AO fluorescent dye were respectively selected and added to 1 mL of whole blood, and then 300 μg of dendrimer-modified hydrophilic immunomagnetic beads were added to it for targeted cell capture. After incubation for 15 min, the material was soaked in PBS thr...

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Abstract

The invention discloses preparation of dendritic molecule-modifiedhydrophilic immunomagnetic beads and application of the hydrophilic immunomagnetic beads to rapid and efficient cell capture. The preparation method comprises the following steps: firstly, synthesizing magnetic nanoparticles through a hydrothermal method; hydrolyzing ethyl orthosilicate and modifying with a silylating reagent in order that the surface of a material is rich in amino groups; coupling dendritic molecules to the surface of the material in order that the material is hydrophilic; lastly, fixing an antibody to the surface of the material to prepare a capture substrate capable of recognizing tumor cells specifically. As proved by experiments, the hydrophilic immunomagnetic beads modified by the antibody and the dendritic molecules can shorten the time of combining the material and cells, perform high-specificity recognition and capture tumor cells rapidly, and capturing efficiency of 86+ / -5 percent can be achieved only in 15 minutes; meanwhile, the activity of the cells is kept, and the tumor cells can be successfully separated from whole blood into which a tumor cell standard is added. A template-free, low-cost and low-toxicity material is applied to cell capture, so that the method is novel, convenient, practical and efficient, and has a tremendous clinical application potential.

Description

technical field [0001] The invention belongs to the technical field of separation and analysis of biological macromolecules, and in particular relates to the preparation of a dendrimer-modified hydrophilic immunomagnetic sphere and its application in fast and efficient cell capture. Background technique [0002] In recent years, research on the isolation and analysis of circulating tumor cells has received great attention. Studies have shown that circulating tumor cells can be used as cancer prediction markers, and their detection can reflect the metastasis, recurrence and pathological mechanism of solid tumors, which is conducive to the timely detection, early diagnosis and effective treatment of tumors. At the same time, the content of circulating tumor cells in the blood is extremely low, about 10 9 Only 1-10 circulating tumor cells are present in each blood cell. Therefore, developing a high-efficiency and high-sensitivity method to isolate tumor cells has become a key...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/553G01N33/543C12N5/09
CPCG01N33/574C12N5/0693C12N2509/00G01N33/54326G01N33/54393G01N33/553G01N2400/46
Inventor 高明霞张珮明张祥民
Owner FUDAN UNIV
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