Kit for rapidly capturing, releasing and detecting circulating tumor cells without damage

A technology for detection kits and tumor cells, applied in the field of release and detection kits, rapid and non-damaging capture of circulating tumor cells, can solve the problems of high cost of antibodies, short shelf life, affecting the true characteristics of CTCs, etc., to improve prognosis and survival rate , fast magnetic response ability, controllable and easy coupling modification effect

Inactive Publication Date: 2020-07-28
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has certain limitations: 1) The high cost of antibodies, poor stability, and short shelf life make immunomagnetic separation costly; 2) The process of tumor metastasis to form CTCs will undergo epithelial-mesenchymal transition (EMT) , Epithelial-mesenchymal transition leads to down-regulation or even non-expression of Ep...

Method used

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  • Kit for rapidly capturing, releasing and detecting circulating tumor cells without damage
  • Kit for rapidly capturing, releasing and detecting circulating tumor cells without damage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The enrichment and separation of CTCs in the cell suspension comprises the following steps:

[0049] (1) When HEK293 cells were cultured to 70%-80% confluence, they were digested with trypsin and then pipet and mixed to prepare a single cell suspension (the concentration of the cell suspension was 10 6 / mL, monocytes in simulated blood), take 1mL and add it to a 2mL bovine serum albumin-coated centrifuge tube;

[0050] (2) Different numbers of pre-stained cervical cancer cells (Hela) were added to the HEK293 cell suspension to simulate CTCs;

[0051] (3) After mixing the cell suspension, add 100 μL of folic acid-modified CTCs magnetic nano-capture probe solution, place it on a rotary mixer and incubate at room temperature for 15 minutes;

[0052] (4) Then the centrifuge tube was placed in a 0.6T magnetic stand, magnetically separated for 2 minutes, and the supernatant was discarded;

[0053] (5) 100 μL of PBS resuspended magnetic nanoprobe-CTCs complex containing 1% B...

Embodiment 2

[0061] The capture of CTCs in blood includes the following steps:

[0062] (1) Take 1 mL of blood and add it to a 10 mL centrifuge tube, add pre-stained cervical cancer cells (Hela) into the blood to simulate CTCs, and pipette gently to mix;

[0063] (2) Add 3 mL of erythrocyte lysate to the centrifuge tube, let the centrifuge tube stand for 10 minutes, and shake it several times during the period;

[0064] (3) After mixing the cell suspension, add 100 μL of folic acid-modified CTCs magnetic nanocapture probe, place it on a rotary mixer and incubate at room temperature for 15 minutes;

[0065] (4) After the lysis, 2000rpm, 3min, discard the supernatant, resuspend the pellet in 1mL of PBS and transfer to a 2mL BSA-coated centrifuge tube;

[0066] (5) 100 μL of folic acid-modified CTCs magnetic nanocapture probe solution was added thereto, placed on a rotary mixer and incubated at room temperature for 15 minutes;

[0067] (6) Then the centrifuge tube was placed in a 0.6T magne...

Embodiment 3

[0076] The release efficiency verification of magnetically captured CTCs includes the following steps:

[0077] (1) In this example, about 200 pre-stained Hela cells were added to 1 mL of HEK293 cell suspension, and the centrifuge tube was inverted several times to mix well, and then 100 μL of CTCs magnetic nano-capture probe solution was added, and placed in a rotating mixture above Continue to incubate at room temperature for 15 minutes;

[0078] (2) Then the centrifuge tube was placed in a 0.6T magnetic stand, magnetically separated for 2 minutes, and the supernatant was discarded;

[0079] (3) 1mL of CTCs-containing release solution to resuspend the magnetic nanoprobe-CTCs complex should be placed in a constant temperature water bath at 37°C for continuous incubation for 30 minutes;

[0080] (4) Then the centrifuge tube was placed in a 0.6T magnetic stand, magnetically separated for 2 minutes, and the supernatant was discarded;

[0081] (5) 100 μL of PBS resuspended magn...

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Abstract

The invention discloses a kit for rapidly capturing, releasing and detecting circulating tumor cells without damage, and relates to the field of biotechnology and biomedicine. The kit includes a CTCsmagnetic nano capture probe solution, an erythrocyte lysis solution, a CTCs releasing solution, a CTCs detecting solution and washing buffer; CTCs magnetic nano capture probes are the magnetic nanoparticles whose surfaces are modified with folic acid; the folic acid on the CTCs magnetic nano capture probes specifically bind to the folate receptors on the surfaces of the circulating tumor cells, sothat the capturing and separation of the circulating tumor cells can be realized at an external magnetic field; and the CTCs detecting solution can perform fluorescence labeling on the released CTCs,so that observation and counting can be realized under a fluorescence microscope. The CTCs captured and released by the kit retain very high activity, so that the kit has significant meaning on the early in vitro screening and diagnosis of cancer and the follow-up study of the CTCs; and the components in the kit can be selectively used according to specific demands, so that simple and convenientoperation can be realized, and therefore, the kit can have wide application prospects.

Description

technical field [0001] The invention relates to the fields of biotechnology and biomedicine, in particular to a rapid and non-damaging capture, release and detection kit for circulating tumor cells. Background technique [0002] Circulating tumor cells (CTCs) are important biomarkers in tumor liquid biopsy. CTCs refer to the general term for tumor cells that have shed from the primary tumor or metastases into the peripheral blood. CTCs appear earlier than solid tumors visible on imaging, and can be used as markers for early diagnosis of tumors. In addition, the number and phenotype of CTCs are closely related to tumor progression, metastasis and prognosis. Clinical detection and counting of CTCs can be used for dynamic monitoring of tumor occurrence and development, evaluation of tumor treatment effects, and guidance for the formulation and implementation of individualized tumor treatment plans. However, the content of CTCs is extremely rare, and its detection first needs...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12M1/42
CPCC12M47/04C12N5/0693C12N2509/00
Inventor 赵玉芬李福来刘艳王敏凝蔡华欢
Owner XIAMEN UNIV
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