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Cancer monitoring-oriented biological coating for specific whole blood capture of circulating tumor cells and preparation method of biological coating

A biological coating and tumor cell technology, applied in the field of functional material technology and biomedical materials, can solve the problems of complex material preparation process, long enrichment time, and low specificity of cell capture, and achieve efficient capture, high capture efficiency, The effect of efficient and sensitive capture

Active Publication Date: 2019-11-29
NANKAI UNIV +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, many platforms, such as CTCs cancer monitoring technology based on microfluidic chips and immunomagnetic beads, have made great progress. Although these methods have certain CTCs capture effects, there are still various deficiencies. For example, the material preparation process is relatively complicated, the capture specificity of cells is not high, or the enrichment time is long, etc.

Method used

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  • Cancer monitoring-oriented biological coating for specific whole blood capture of circulating tumor cells and preparation method of biological coating
  • Cancer monitoring-oriented biological coating for specific whole blood capture of circulating tumor cells and preparation method of biological coating
  • Cancer monitoring-oriented biological coating for specific whole blood capture of circulating tumor cells and preparation method of biological coating

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] (1) Dehydrate tetra-n-octyl ammonium bromide, add ethoxy ethyl glycidyl ether and triisobutyl aluminum to toluene, react at 0°C for three hours, and then add allyl under ice bath conditions Glycidyl ether and triisobutylaluminum were stirred overnight at room temperature. Add water to terminate the reaction, dry the polymer product with sodium sulfate, remove toluene, and purify by dialysis to obtain the polymer (PEEGE-b-PAGE).

[0041] (2) Add the polymer (PEEGE-b-PAGE) obtained in step (1) into tetrahydrofuran together with 37% hydrochloric acid aqueous solution, stir and react at room temperature for 10 hours, wash the polymer precipitate with tetrahydrofuran, and dialyze with methanol to obtain the Acetal protected polymer (PG-b-PAGE).

[0042] (3) Dissolve the polymer (PG-b-PAGE) obtained in step (2) in dry N,N-dimethylformamide, add triethylamine under ice bath conditions, and then add p-toluene Sulfonyl chloride was reacted overnight with stirring at room tempe...

Embodiment 2

[0058] Step (1)-step (13) is identical with embodiment 1.

[0059] (14) Use Accutase to digest MCF7 / T47D / MDA-MB-231 / A549 / HeLa from the cell culture flask, and use CellTrace TM Cells were pre-stained with violet dye for 20 minutes, and the stained cells were mixed into serum-free RPMI-1640 medium at a concentration of 5000 cells per milliliter to make a cancer cell suspension.

[0060] (15) Place the Anti-EpCAM-PG-CatPh substrate obtained in step (13) face up in an eight-hole plate, add 1 ml of breast cancer cell MCF7 suspension with a concentration of 5000 per ml, and place in cell culture In the case (preferred reaction time is 90 minutes), breast cancer cell MCF7 suspension is removed, the bio-substrate is fully rinsed in phosphate buffer, and photographed respectively under 10 times with a Zeiss fluorescence microscope (at least 3 substrates each time) , each substrate selects 10 different positions in the middle), and counts the breast cancer cells MCF7 captured on the b...

Embodiment 3

[0067] Step (1)-step (13) is identical with embodiment 1.

[0068] (14) Use Accutase to digest MCF7 / A549 / MDA-MB-231 / HeLa from the cell culture flask, and use CellTrace TM Cells were prestained with violet dye for 20 minutes. Whole blood from healthy donors was incubated with erythrocyte lysate on ice for 10 minutes to lyse red blood cells, and then the pre-stained MCF7 cells were mixed into healthy donor blood at a concentration of 1000 cells per milliliter to make artificial breast cancer patient blood samples / Artificial blood samples of lung cancer patients / Artificial blood samples of cervical cancer patients.

[0069](15) Place the Anti-EpCAM-PG-CatPh substrate obtained in step (13) face up in an eight-hole plate, add 1 milliliter of artificial breast cancer patient blood samples with a concentration of 1000 per milliliter, and place in a cell culture incubator (preferred reaction time is 90 minutes), remove the artificial breast cancer patient blood sample, the bio-sub...

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Abstract

The invention provides a cancer monitoring-oriented biological coating for the specific whole blood capture of circulating tumor cells and a preparation method of the biological coating and belongs tothe field of biotechnology and clinics. According to the coating, polyglycidyl ether (PG) or polyethylene glycol (PEG) is adopted, the adhesion bionic chemical modification of mussels is used in combination, and therefore, the specific capture of the circulating tumor cells can be performed. According to the biological coating, and the preparation method thereof, the surface of a substrate is modified with a functionalized polymer; the adhesion of non-specific cells to other blood components is prevented; a specific antibody is fixed on the surface of the polymer; a to-be-detected sample (blood) is dropwise added to the surface of the coating; the synergistic effect of specific adsorption and non-specific adhesion resistance is achieved through the synergistic effect of the specific antibody for identifying the circulating tumor cells and the non-specific adhesion resistance polymer, so that the circulating tumor cells in the to-be-detected sample can be efficiently and accurately captured. With the method provided by the invention adopted, the circulating tumor cells can be efficiently captured. The method has the advantages of low cost, simple operation and extremely high sensitivity, and can be used for clinical detection.

Description

technical field [0001] The invention belongs to the field of functional material technology and biomedical materials, and in particular relates to a specific biological coating for enrichment and detection of circulating tumor cells and a preparation method thereof. Background technique [0002] Malignant tumors have a tendency to metastasize and recur, and are the main cause of death in cancer patients. Tumor metastasis is when tumor cells break away from the primary lesion, invade the extracellular matrix, invade the circulatory system, effectively evade the body's immune clearance system, and become circulating tumor cells (CTCs). Although the number of CTCs in blood is very small (several to hundreds per milliliter) mixed with a large number of blood cells (10 9 per milliliter), but it is of great significance in examining cancer metastasis and monitoring the effect of treatment. That is to say, CTCs can not only replace biopsy to find cancer metastasis, but also the n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N1/34
CPCG01N1/2813G01N1/34
Inventor 欧来良陈杰余雷晓李文忠雷纳·哈格
Owner NANKAI UNIV
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