Dendritic macromolecule-copolymer cell capturing material as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomacromolecule separation and analysis and particularly relates to a dendritic macromolecule-copolymer cell capturing material as well as a preparation method and an application thereof. Firstly, a copolymer substrate of acrylamide and methyl methacrylate is synthesized, polyamide-amine type dendritic macromolecules are modified on the substrate, finally, the dendritic macromolecules are used for coupling of phenylboronic acid, and a functional material used for cell capturing can be obtained. An experiment proves that a three-dimensional network structure of the copolymer substrate and dendritic molecules can increase the phenylboronic acid modification amount, and the functional material can capture 87% plus or minus 5% of cells in 45 minutes; the copolymer substrate and the dendritic molecules are both super-hydrophilic compounds, and the captured cells keep high activity. Template-free, low-cost and non-toxic materials are adopted, and the dendritic macromolecule-copolymer cell capturing material is applied to cell capturing, is novel, convenient, practical, efficient and high in stability and has wide application prospect.

Description

technical field

[0001] The invention belongs to the technical field of separation and analysis of biological macromolecules, and in particular relates to a dendrimer-copolymer cell capture material and its preparation method and application. technical background

[0002] In recent years, the study of tumor cell isolation has received great attention. Studies have shown that different types of tumor cells can be used as predictive markers, and their detection can reflect the metastasis, recurrence and progression of solid tumors, which is conducive to the timely detection, early diagnosis and effective treatment of tumors, so the development of high Efficient and high-sensitivity methods to separate tumor cells have become a key step, which provides unlimited possibilities for subsequent detection.

[0003] With the continuous deepening of research on tumor cell isolation, a wide variety of new technologies have emerged for capturing and detecting tumor cells. According to ...

Examples

Embodiment 1

[0021] Example 1 : Synthesis of three-dimensional network-like superhydrophilic dendrimers with aminophenylboronic acid as "bait"-copolymer cell capture material

[0022] Dissolve acrylamide, methylene acrylamide, and PEG-6000 in PBS solution at a mass ratio of 1:1:1.5, add 50 μL of the above solution, 0.5-5.0 μL of methyl methacrylate, and tetramethylmethacrylate to each well of a 96-well plate. 0.6 μL of ethylenediamine, 2.5 μL of 10% ammonium persulfate solution, 50 ° C, shaking reaction for 1 h, a milky white colloidal solid was obtained, which was washed with PBS solution. The copolymer base material was irradiated with ultraviolet rays for 10 minutes, and then washed with PBS, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added to the material with a mass ratio of 1:1. ) and N-hydroxysuccinimide (NHS) in PBS, after activation for 0.5h, add 0.5-10.0μL of PAMAM dendrimers (ethylenediamine core), react at 37°C for 2-6h, wash with PBS net. Ami...

Embodiment 2

[0023] Example 2 : Application of superhydrophilic dendrimers-copolymer materials with three-dimensional network structure in capturing human glioma cells U251

[0024] Add 100 μL of human glioma cell U251 suspension to the 96-well plate containing aminophenylboronic acid-modified dendrimer-copolymer material, and the cell density is 10 5 -10 6 , in an incubator (37°C, 5% CO 2 ) after incubating for 5-90 min, the supernatant was sucked and washed three times with PBS, and the cell capture efficiency was calculated. Add 10 μg / mL DAPI dye to the captured cells, wash them after staining at room temperature for 15 min, and observe with a fluorescence microscope.

technical field

[0001] The invention belongs to the technical field of separation and analysis of biological macromolecules, and in particular relates to a dendrimer-copolymer cell capture material and its preparation method and application. technical background

[0002] In recent years, the study of tumor cell isolation has received great attention. Studies have shown that different types of tumor cells can be used as predictive markers, and their detection can reflect the metastasis, recurrence and progression of solid tumors, which is conducive to the timely detection, early diagnosis and effective treatment of tumors, so the development of high Efficient and high-sensitivity methods to separate tumor cells have become a key step, which provides unlimited possibilities for subsequent detection.

[0003] With the continuous deepening of research on tumor cell isolation, a wide variety of new technologies have emerged for capturing and detecting tumor cells. According to ...

Examples

Embodiment 1

[0021] Example 1 : Synthesis of three-dimensional network-like superhydrophilic dendrimers with aminophenylboronic acid as "bait"-copolymer cell capture material

[0022] Dissolve acrylamide, methylene acrylamide, and PEG-6000 in PBS solution at a mass ratio of 1:1:1.5, add 50 μL of the above solution, 0.5-5.0 μL of methyl methacrylate, and tetramethylmethacrylate to each well of a 96-well plate. 0.6 μL of ethylenediamine, 2.5 μL of 10% ammonium persulfate solution, 50 ° C, shaking reaction for 1 h, a milky white colloidal solid was obtained, which was washed with PBS solution. The copolymer base material was irradiated with ultraviolet rays for 10 minutes, and then washed with PBS, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added to the material with a mass ratio of 1:1. ) and N-hydroxysuccinimide (NHS) in PBS, after activation for 0.5h, add 0.5-10.0μL of PAMAM dendrimers (ethylenediamine core), react at 37°C for 2-6h, wash with PBS net. Ami...

Embodiment 2

[0023] Example 2 : Application of superhydrophilic dendrimers-copolymer materials with three-dimensional network structure in capturing human glioma cells U251

[0024] Add 100 μL of human glioma cell U251 suspension to the 96-well plate containing aminophenylboronic acid-modified dendrimer-copolymer material, and the cell density is 10 5 -10 6 , in an incubator (37°C, 5% CO 2 ) after incubating for 5-90 min, the supernatant was sucked and washed three times with PBS, and the cell capture efficiency was calculated. Add 10 μg / mL DAPI dye to the captured cells, wash them after staining at room temperature for 15 min, and observe with a fluorescence microscope.

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