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Functional magnetic bead, preparation method and method for specifically capturing circulating tumor cells of colorectal cancer

A colorectal cancer and tumor cell technology, applied in the field of chemical biology, can solve the problems of not being able to mass-produce, low antibody expression, and high price, and achieve the effect of short time, good specificity, and simple preparation method

Inactive Publication Date: 2020-01-07
山东康华生物医疗科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is still subject to the limitations of large-scale production and high price, and the expression of antibodies in some tumors (such as melanoma and sarcoma) is low or almost non-existent

Method used

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  • Functional magnetic bead, preparation method and method for specifically capturing circulating tumor cells of colorectal cancer
  • Functional magnetic bead, preparation method and method for specifically capturing circulating tumor cells of colorectal cancer
  • Functional magnetic bead, preparation method and method for specifically capturing circulating tumor cells of colorectal cancer

Examples

Experimental program
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preparation example Construction

[0037] The present invention also provides a preparation method of functionalized magnetic beads, comprising:

[0038] 1) Wash the MBs, then add EDC and NHS for activation to obtain activated MBs;

[0039] 2), the activated MBs were magnetically sorted to remove the supernatant, washed with HEPES buffer, then added with HEPES buffer containing UEA-I, and reacted at room temperature;

[0040] 3) After the reaction, remove the supernatant by magnetic separation, wash with HEPES buffer to remove unreacted UEA-I, suspend the modified MB-UEA-I in HEPES buffer, and save for future use.

[0041] According to the present invention, first add MBs to the centrifuge tube, wash with MES buffer solution for 3 to 5 times, then add EDC and NHS for activation; the activation temperature is preferably room temperature, and the activation time is preferably 30 to 120 min. The concentration of the MES buffer solution is preferably 100 mmol / L, the pH value is 5.5-6.7, and the mass mg of the MBs:...

Embodiment 1

[0058] Embodiment 1 Preparation of functionalized magnetic beads MB-UEA-I

[0059] Take 100μL of MBs (5mg / mL) into a 2mL centrifuge tube, add 1mL of MES buffer to wash three times, remove the supernatant by magnetic separation each time, then add 100mmol / L EDC and 100mmol / L NHS 500μL each, and place at room temperature Vibrate on a shaker to activate for 1h (160rpm).

[0060] After the activation, wash with HEPES buffer three times, then add 1 mL of HEPES (containing 0.025 mg of UEA-I), and place on a shaker at room temperature for 3 h (160 rpm).

[0061] After the coupling reaction, wash with HEPES buffer three times to remove unreacted lectin, and finally add 1 mL of HEPES buffer to suspend MB-UEA-I, and store at 4°C for future use.

[0062] figure 1 It is the SEM characterization diagram before and after MBs modification of UEA-I in Example 1 of the present invention. (a) is the SEM characterization image of MBs, and (b) is the SEM characterization image of MB-UEA-I. fi...

Embodiment 2

[0063] Example 2 MB-UEA-I efficiently and specifically captures tumor cells

[0064] Experimental group: about 100 SW480 cells were added to 1 mL of complete medium, and then 0.05 mg of the functionalized magnetic beads MB-UEA-I prepared in Example 1 were added.

[0065] Control group: Add about 100 HCT116 cells and NCM460 cells to 1 mL of complete medium, then add 0.05 mg of the functionalized magnetic beads MB-UEA-I prepared in Example 1; add about 100 SW480 cells to 1 mL To the complete medium, add 0.05 mg of unmodified MBs.

[0066] Set up the above four groups of experiments, each with three parallel experiments. After the cells were mixed with MB-UEA-I or MBs, they were incubated on a shaker at 37°C for 45min (150rpm). After the reaction, the captured cells were obtained by magnetic separation, and the supernatant was added to a 48-well plate, and after standing for 20 min, the uncaptured cells in the supernatant were counted using an inverted microscope. In addition,...

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Abstract

The invention provides a functional magnetic bead, a preparation method and a method for specifically capturing circulating tumor cells of colorectal cancer and belongs to the technical field of chemicobiology. The functional magnetic bead is obtained by modifiying a lectin UEA-I capable of being specifically combined with alpha-1,2-fucose on the surface of a carboxylated Fe3O4 magnetic bead MBs by utilizing amidation. The invention also provides a preparation method of the functional magnetic bead. The invention also provides a method for applying the functional magnetic bead to specific capture of the circualting tumor cells of the colorectal cancer. The functional magnetic bead provided by the invention realizes separation of specific circulating tumor cells under the action of an externally applied magnetic field, efficiencies of capturing the circulating tumor cells in a complete medium and blood by the functional magnetic bead respectively reach 94% and 89%, the captured cells can perform normal adherence, proliferation and passage, growth rate of the captured cells has no obvious difference with that of normally cultured cells, and deep study can be conveniently performed onthe captured cells in a later period.

Description

technical field [0001] The invention belongs to the technical field of chemical biology, and in particular relates to a functionalized magnetic bead, a preparation method and a method for specifically capturing colorectal cancer circulating tumor cells. Background technique [0002] Circulating tumor cells (CTCs) refer to tumor cells that are shed from the primary tumor or metastases and enter the blood circulation during tumor formation or progression, and have the potential to develop into metastatic lesions. Therefore, the capture and detection of CTCs has important medical significance in the early diagnosis of major diseases and the evaluation of treatment effects. However, due to high heterogeneity and rarity (1 mL of peripheral blood 10 6 -10 9 There are several to hundreds of CTCs in a blood cell), and it is still a great challenge to enrich CTCs with high efficiency and high purity. [0003] Currently, many techniques and devices based on the physical properties ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N1/30G01N1/34H01F7/02H01F41/02
CPCG01N1/30G01N1/34G01N21/6458H01F7/02H01F41/0253
Inventor 王振新杨致亭田荣荣马立娜杨锋斌范文翠
Owner 山东康华生物医疗科技股份有限公司
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