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Method for synchronously capturing and identifying circulating tumor cells

A tumor cell and leukocyte technology, which is applied in the field of simultaneous capture and identification of circulating tumor cells by nanomaterials, can solve the problems of CTCs loss, time-consuming, and many operation steps, and achieves the avoidance of CTCs loss, simple and time-saving operation, and light stability. Excellent effect

Active Publication Date: 2016-07-27
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the currently developed identification methods for CTCs severely damage the cell viability, which greatly limits the follow-up research on CTCs.
On the other hand, the identification of CTCs is almost always carried out separately from the capture, and there are many steps in the identification process, which makes the entire CTCs detection process cumbersome and time-consuming.
In addition, multi-step operation will bring a large loss of CTCs, which will affect the sensitivity and reliability of the method

Method used

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  • Method for synchronously capturing and identifying circulating tumor cells
  • Method for synchronously capturing and identifying circulating tumor cells
  • Method for synchronously capturing and identifying circulating tumor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] [Example 1] Preparation of immunomagnetic ball and immunofluorescent ball

[0041] The preparation of magnetic nanospheres (MNs) adopts the layer-by-layer assembly method developed in our laboratory. First, EDC / NHS is used to activate the carboxyl groups on the surface of the styrene acrylamide polymer spheres (Pst-AAm-COOH), and the polyethylene imine (PEI) ) Is covalently bonded to the amino group; then use γ-Fe 2 O 3 The coordination of Fe and the primary amine of PEI will transform γ-Fe 2 O 3 Assemble to the surface of the polymer ball; then repeat the assembly of PEI and γ-Fe 2 O 3 Until 5 layers of γ-Fe are assembled 2 O 3 Afterwards, the obtained magnetic nanospheres are treated with tetraethoxysiloxane and (3-aminopropyl)triethoxysiloxane to coat a layer of silicon shell on the surface and introduce amino groups; finally, the last step is obtained The magnetic nanospheres are treated with succinic anhydride to introduce carboxyl groups on their surface. The obtaine...

Embodiment 2

[0044] [Example 2] Investigation on the stability of fluorescent ball in complex system

[0045] Incubate FNs with 1mL pure fetal bovine serum (FBS), and monitor the changes in fluorescence intensity for 30 minutes with a fluorometer; use dynamic light scattering to measure the particle size and monodispersity changes of FNs before and after incubation; prepare samples of FNs before and after incubation Observe under an inverted fluorescence microscope.

[0046] Such as figure 1 As shown in A, the fluorescence intensity of FNs did not change significantly within 30 minutes of incubation in pure FBS; the hydrated particle size and polydispersity coefficient (PDI) after incubation were compared with those before incubation ( figure 1 B, C), almost no significant increase, indicating that FNs can still maintain excellent monodispersity in a complex system; and the results of fluorescence microscopy once again indicate that FNs are incubated in pure FBS for 30 minutes ( figure 1 D) The ...

Embodiment 3

[0047] [Example 3] Immunofluorescence ball efficiently and specifically label target cells

[0048] Incubate the green fluorescent spheres coupled with EpCAM antibody (i.e. immuno-green fluorescent spheres, IGNs) with target tumor cells MCF-7 for 30 minutes, centrifuge at 1500 rpm for 5 minutes to remove excess fluorescent spheres, and observe under an inverted fluorescent microscope; at the same time, IGNs and leukemia Cells were incubated with Jurkat T cells, and naked GNs without antibody conjugate were incubated with MCF-7 cells as a control experiment. Similarly, the red fluorescent balls coupled with CD45 antibody (ie, immunored fluorescent balls, IRNs) reacted with target cells Jurkat T cells, while IRNs were incubated with MCF-7 and RNs labeled Jurkat T cells as a control experiment. In order to compare the labeling effect of IFNs with organic dyes, Jurkat T cells were incubated with CD45 monoclonal antibody for 30 minutes and then reacted with Cy3 labeled secondary antib...

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Abstract

The invention relates to a method for identifying circulating tumor cells.The method includes the steps that antibodies for specifically identifying the tumor cells on a magnetic nanoparticle coupling are utilized to modify antibodies for specifically identifying the tumor cells and white blood cells respectively through two fluorescent nanospheres of different colors so as to obtain three nanospheres having immune functions; meanwhile the three nanospheres having the immune functions are added to a whole blood system to be detected, and synchronous capture and marking of the circulating tumor cells are achieved, enriched cells are directly observed under a fluorescence microscope through simple magnetic separation to achieve the identification purpose.The method is simple in operation, the loss of the circulating tumor cells is small, the nanospheres having the immune functions almost have no influence on the activities of target cells, and 93% of the tumor cells remain good biological activities after being identified and can be cultured in vitro.The method is an effective method for prognosis, recurrence monitoring and individualized therapies of clinical cancer patients.

Description

Technical field [0001] The invention belongs to the field of chemical biology and medical detection, and specifically relates to a method for simultaneously capturing and identifying circulating tumor cells by using nanomaterials. Background technique [0002] In recent years, cancer has gradually become a major public safety issue, and nearly tens of millions of people die of cancer each year worldwide. The main reason for the extremely high cancer mortality is that cancer is prone to metastasis, and most cancer patients eventually die from cancer metastasis. Studies have shown that circulating tumor cells play a vital role in cancer metastasis. Circulating Tumor Cells (CTCs) are tumor cells that shed from the original tumor site and enter the peripheral blood. They will break through the inner wall of the blood vessel as the peripheral blood circulates to the appropriate location and transfer to other tissues. A large number of studies have shown that the detection of CTCs ha...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N33/543
CPCG01N33/54326G01N33/54346G01N33/582
Inventor 庞代文吴玲玲张志凌
Owner WUHAN UNIV
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