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Nitrocellulose-membrane-based chip for conveniently capturing cancer cells

A nitrocellulose membrane and cancer cell technology, applied in the field of circulating tumor cell (CTC) capture chip, can solve the problems of complex chip preparation process, difficulty in large-scale production, hindering prospects, etc., and achieve easy optical detection and good bio-affinity Sexuality and high efficiency

Inactive Publication Date: 2013-08-07
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The latest research results on CTC chips reported so far have a high capture efficiency for CTCs and good control of false positives. However, most of the chip preparation processes are too complicated, and the repeatability is poor, making it difficult to produce on a large scale. Seriously hampers its clinical application prospects; In addition, due to the size limitation of the chip channel, the analysis of the patient's blood takes a long time, and the captured cancer cells are not convenient for subsequent biochemical analysis such as re-cultivation

Method used

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  • Nitrocellulose-membrane-based chip for conveniently capturing cancer cells
  • Nitrocellulose-membrane-based chip for conveniently capturing cancer cells
  • Nitrocellulose-membrane-based chip for conveniently capturing cancer cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Preparation of CTC capture chip

[0023] Commercial nitrocellulose membranes were cut into small pieces of 1 cm × 1 cm, then soaked in pure ethanol for 10 s to soften and transparent, and spread on a pre-cut PMMA flat plate to dry naturally, and then assembled into a chip base. Before the antibody is adsorbed, it needs to be activated by soaking in PBS buffer containing 10% acetonitrile for one hour; then dissolve 20 μL of antibody (0.5 μg / μL) in 200 μL of 1 mM PBS buffer containing 10% acetonitrile, and drop evenly Add it on the CTC chip and react at 37 °C for 0.5 h, then wash off excess antibody with PBS; add 50 μL of BSA solution (0.5%) to block for half an hour to reduce its non-specificity to cancer cells adsorption. The prepared CTC chips were stored in a refrigerator at 4 °C for future use.

Embodiment 2

[0024] Example 2 CTC chip captures cancer cells in PBS buffer

[0025] Non-small lung cancer cells NCI-H1650 were cultured in RPMI1640 medium supplemented with 10% (v / v) fetal bovine serum in a constant temperature incubator at 37 °C containing 5% (v / v) carbon dioxide. After the cells matured, they were incubated with 0.25 % (w / w) trypsin-EDTA was digested by incubation for 3 min, and washed 3 times with PBS buffer to prepare a certain concentration of cell suspension. Add the cell liquid onto the CTC chip, put it in a 37°C incubator and incubate for a certain period of time, wash off the uncaptured cells with PBS, and then perform microscope imaging counting and subsequent detection.

Embodiment 3

[0026] Example 3 Adjust the contact incubation time between the CTC chip and the cancer cells as 5 min, 15 min, 30 min, 45 min and 1 h, and then count the captured cancer cells. The results are shown in Figure 4 shown.

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Abstract

The invention belongs to the technical field of bioanalysis and detection, and in particular relates to a circulating tumor cell (CTC) capturing chip as well as a manufacturing method and an application thereof. A nitrocellulose membrane is used as a main body of a CTC chip, an antibody is fixed by using the ultrastrong adsorption capacity of the nitrocellulose membrane for proteins, and further cancer cells in blood are specifically captured. Experiment results show that the CTC chip has very high capturing efficiency for non-small lung cancer cells NCI-H1650 and successfully separates and detects micro cancer cells in human whole blood. The CTC capturing chip is novel and convenient, practical and high-efficient and great in clinical popularization and application potentials.

Description

technical field [0001] The invention belongs to the technical field of biological analysis and detection, in particular to a circulating tumor cell (CTC) capture chip. Background technique [0002] Circulating tumor cells (CTCs) are cancer cells that break off from tumor tissue and enter the systemic circulation in the blood. It is transformed from ordinary cancer cells, and can grow tumor tissue again through reverse differentiation at any time, so it is closely related to the spread and metastasis of cancer, and is a weathervane that reflects the development of cancer. Detailed research on it is essential for early diagnosis of cancer decisive for treatment. The content of CTCs in human peripheral blood is very low, generally not more than 100 / mL, while the background interfering cells such as red blood cells and white blood cells are as high as 10 9 This is a great challenge to the existing separation and analysis methods. In general, the current research urgently need...

Claims

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Application Information

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IPC IPC(8): G01N33/574G01N33/531
Inventor 高明霞张祥民张鹏
Owner FUDAN UNIV
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