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Method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip

A microfluidic chip and nucleated red blood cell technology, which is applied in the field of capturing and identifying fetal nucleated red blood cells, can solve the problems of inability to distinguish the fetal origin of nucleated red blood cells, and achieve good application prospects, low price, and simple operation

Inactive Publication Date: 2018-11-06
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In addition, since pregnant women's own nucleated red blood cells will express γ-globin, the traditional use of γ-globin antibodies with fluorescent groups cannot distinguish the fetal origin of nucleated red blood cells.

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  • Method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip
  • Method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip
  • Method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip

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Embodiment 1

[0038] like figure 1 As shown, the specific steps of the method for capturing and identifying fetal nucleated red blood cells in the microfluidic chip provided in this embodiment are:

[0039] (1) Preparation of gelatin nanoparticles:

[0040] Weigh 0.625g B-type gelatin and dissolve it in 12.5mL deionized water, heat to dissolve at 50°C. Then 12.5 mL of acetone was added at a rate of 6 mL / h at 300 rpm, and after standing for 1 min, the supernatant was discarded. Add 12.5 mL of deionized water to the precipitate, and heat again at 50°C to dissolve. Use 2mol / L NaOH solution to adjust the pH of the reactant to 10, then slowly add about 45mL of acetone to the gelatin solution, and finally add glutaraldehyde (1.5%) solution, in situ cross-linking for 2 hours, and stop cross-linking by excess glycine couplet. The gelatin nanoparticles were recovered by centrifuging the above reaction solution at 10000 rpm for 10 min. Freeze-drying is carried out by a freeze dryer to remove res...

Embodiment 2

[0050] Dilute 2mL of maternal peripheral blood with PBS at a ratio of 1:1, then place it on a 4mL lymphocyte separation medium, centrifuge at 400g for 30min, obtain a mononuclear layer containing fetal nucleated red blood cells, and make 1mL of cell suspension. Then pass through the antibody-modified microfluidic chip 10, under the action of the fluid, the background interfering cells in the suspension are recovered from the outlet port along with the fluid, while the fetal nucleated red blood cells remain in the microfluidic chip through the specific recognition of the antigen antibody 10 internal, to achieve capture. like Figure 7 As shown, after the capture is completed, the fetal nucleated red blood cells are captured on the inner wall of the microfluidic chip 10 . like Figure 8 As shown, the cells spread out on the surface of the nano-coating and firmly adsorbed on the surface of the nano-coating.

Embodiment 3

[0052] To explore the effect of the concentration of gelatin nanoparticles on the efficiency of target cell sorting, the specific operation is: use different concentrations (1.0mg / mL, 1.5mg / mL, 2.0mg / mL, 2.5mg / mL) of gelatin nanoparticles in the microfluidic A nano-coating is formed inside the control chip 10 and modified with antibodies. A 1 mL sample of nucleated erythroid TF-1 cells at a concentration of 200 / mL was passed into the microfluidic chip 10 to complete capture. Observing and counting under the Olympus IX 81 microscope, the experimental results are as follows Figure 9 As shown, when the concentration of gelatin nanoparticles was 2.0 mg / mL, the capture efficiency was 81%.

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Abstract

The invention provides a method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip. The method comprises the following steps: 1, synthesizing gelatin nanoparticles bya two-step solvent removal method and preparing into a precursor liquid, and then introducing into a microfluidic chip to form a nanometer coating on the inner wall thereof, wherein the microfluidicchip comprises a plurality of serpentine main channels and the plurality of fish bone units; 2, modifying streptavidin on the nanometer coating by a chemical modification method, then grafting a biotin-modified antibody; 3, separating a mononuclear cell layer containing fetal nucleated red blood cells from peripheral blood by density-gradient centrifugation; 4, allowing mononuclear cell suspensionto flow into the microfluidic chip at a certain flow rate for capturing; 5, introducing a certain concentration of gelatinase into the microfluidic chip that captures fetal nucleated red blood cells,releasing fetal nucleated red blood cells by degrading the nanometer coating; and 6, identifying captured fetal nucleated red blood cells by using specific antibodies.

Description

technical field [0001] The invention belongs to the field of fetal cell sorting and identification methods, and in particular relates to a method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip. Background technique [0002] Fetal cells can enter the maternal peripheral blood through the placental barrier during pregnancy. Fetal cells have a variety of characteristics: ① belong to monocytes and contain all the genetic information of the fetus; ② have specific proteins on the surface that are different from normal blood cells; ③ have a short cycle in the peripheral blood of pregnant women, and are not affected by previous diagnosis when used for diagnosis. effects of pregnancy. The special properties of fetal cells provide new ideas for non-invasive prenatal diagnosis, and many studies are devoted to the enrichment of fetal cells. Due to the extremely small number of fetal cells in the peripheral blood of pregnant women, it has always be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/00C12N5/00G01N33/569
CPCC12M23/16C12M47/04G01N33/56966
Inventor 赵兴中魏小云蔡博
Owner WUHAN UNIV
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