CN108535228B - Method for separating free fetal cells from peripheral blood of pregnant woman - Google Patents
Method for separating free fetal cells from peripheral blood of pregnant woman Download PDFInfo
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- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
Abstract
The invention relates to a method for separating free fetal cells from the peripheral blood of a pregnant woman. The method utilizes the cell separation effect and the specific affinity recognition principle of the microfluidic chip to realize the high-efficiency and high-purity capture and separation of free fetal cells (fetal nucleated red blood cells, trophoblast cells and the like) in the peripheral blood of the pregnant woman. In the microfluidic chip, a microarray structure with a specific geometric arrangement mode and modified molecular groups with affinity recognition effects such as aptamer, antibody, polypeptide and the like are designed. The collision efficiency of the fetal cells with different sizes and the microarray can be regulated and controlled by regulating the parameters of the microarray structure, so that the capture and enrichment of the fetal cells with different sizes are realized.
Description
Technical Field
The invention relates to a method for separating free fetal cells from the peripheral blood of a pregnant woman.
Background
With the release of the two-fetus policy in recent years, the number of elderly puerperae increases, and the birth defect rate tends to increase. Of the three-stage prevention of birth defects, the second stage of prenatal screening and diagnosis is the most important means in preventing birth defects. Amniocentesis is a main means for pre-partum teratogenesis of high-risk puerpera, but has strong invasiveness and higher risks of infection, abortion and the like. Noninvasive prenatal detection based on fetal free DNA has been widely applied to screening of autosomal aneuploidy, but has the defects of high false positive rate, incapability of eliminating chromosome abnormality of parents and the like. Fetal cells found in maternal peripheral blood in 1969 have complete fetal genetic information, are convenient to sample, are minimally invasive, and are the most potential non-invasive prenatal diagnostic subjects. But instead of the other end of the tubeThe biggest challenge of fetal cells that have not been truly used for prenatal diagnosis is the rare fetal cell content in peripheral blood (1-10 fetal cells/ml, 10 fetal cells/ml)9Red blood cells, 106Individual white blood cells), the background interference is strong, and the high specificity capture is difficult to realize. How to accurately separate fetal cells from a complex blood environment in which thousands of background cells exist is a bottleneck problem in realizing noninvasive and accurate prenatal diagnosis by establishing an efficient, rapid and economical peripheral blood fetal cell enrichment method.
The main problems existing in the prior fetal cell separation and capture include: 1. the capture efficiency is very low. The main methods for fetal cell acquisition today are magnetic bead isolation, flow cytometry and cell smear. The cell loss rate of the magnetic bead separation method and the flow cytometry is very high, more than 10 milliliters of peripheral blood is often needed for capturing fetal cells, the maximum volume is even 30 milliliters, the high sample requirement is difficult to accept by all pregnant women, the capture quantity of the fetal cells is still less than 10, and the enrichment rate is too low. The cell smear method mainly depends on human eye identification, needs a large amount of time and manpower, has overlong working time and low efficiency of one sample, and can only identify but cannot realize target cell separation. 2. These methods involve post-treatment such as immobilization of cells, and therefore, single cells are inactive or have low activity, and efficient single cell analysis is difficult.
The micro-fluidic chip has the characteristics of high throughput, small volume, low consumption and the like, and in recent years, the micro-fluidic chip has been greatly and dissimilarly used in the fields of separation and enrichment of circulating tumor cells, single cell sequencing and the like, and can realize high-efficiency separation, enrichment, release, sequencing and the like of the circulating tumor cells in peripheral blood. The current method for separating cells by utilizing a microfluidic chip is mainly based on two principles: 1) recognizing and capturing by utilizing affinity; 2) physical separation is utilized, and methods such as micro-pore filtration or hydromechanics, ultrasonic separation and the like are adopted. However, unlike the advantage of large size of circulating tumor cells, fetal cells are small in size and difficult to separate by filtration, and some of the fetal cell surface markers are unclear and difficult to separate and enrich specific fetal cells.
Disclosure of Invention
The invention aims to provide a method for separating free fetal cells from peripheral blood of a pregnant woman.
The purpose of the invention is realized by the following technical scheme:
a method of isolating free fetal cells from the peripheral blood of a pregnant woman, comprising the steps of:
1) preparing a microfluidic chip for capturing fetal cells; the micro-fluidic chip is designed to comprise at least one sample inlet and at least one sample outlet; a microarray with specific geometric arrangement is arranged between the sample inlet and the sample outlet, and the microarray pattern comprises a circle or a triangle; the distance between microarrays is 0-50 microns, preferably 10/20/30/40 microns), the offset angle of the next pillar compared to the previous pillar is 0-50 °, preferably 10/20/30/40 °;
preferably, the critical dimension of the microfluidic chip as a whole is designated as Dc and is expressed by the formula Dc ═ 1.4 xg × (Δ λ/λ)0.48Setting, wherein the horizontal spacing between the pillars is set as G, and the size of the G is between 0 and 50 micrometers; the distance between the vertical centers of the triangles in the same column is set as lambda, and the size of the lambda is between 100 and 150 microns; the upward offset of the second triangle in the same row compared to the first triangle is set as Δ λ, and the magnitude of Δ λ is between 0 and 20 μm; the overall critical dimension of the microfluidic chip is designated Dc.
2) Modifying a fetal cell-specific recognition molecule on the surface of the microarray;
3) regulating the critical diameter of the microarray to be 0-50 microns, preferably 10/20/30/40 microns, and the flow rate to be 0-10mL/h, preferably 1/2/3/4/5/6/7/8/9mL/h, so that the cell capture efficiency of the microfluidic chip is optimal;
4) carrying out gradient centrifugal separation on 2-10 ml of pregnant woman peripheral blood to obtain mononuclear cells;
5) resuspending the mononuclear cells obtained in the step 4) in a phosphate buffer solution, and injecting the mononuclear cells into a microfluidic chip for capturing fetal cells.
In a preferred embodiment of the present invention, the microfluidic chip has overall dimensions of about 1 to 5 cm in length and about 0.5 to 2 cm in width, and the size is specifically designed according to the total amount of cells to be separated.
In the preferred embodiment of the present invention, the microarray pillar may be a cylindrical pillar or a triangular pillar having a diameter or side length of 10 to 200 micrometers, preferably 20/40/60/80/100 micrometers, or the like.
In the preferred embodiment of the present invention, wherein the horizontal spacing between the pillars is set to G, the size is between 0 and 50 microns, and may be 10 microns, 20 microns, 30 microns, etc.; the distance between the vertical centers of the triangles in the same column is set as lambda, the size of the lambda is between 100 and 150 micrometers, and the lambda can be 100 micrometers, 120 micrometers, 130 micrometers and the like; the upward offset of the second triangle in the same row compared to the first triangle is set as Δ λ, which is between 0 and 20 microns, and may be 1 micron, 3.5 microns, 6.5 microns, 7.5 microns, etc.; the overall critical dimension of the microfluidic chip is designated as Dc, which is expressed by the formula Dc ═ 1.4 XG × (Δ λ/λ)0.48Thus obtaining the product.
In a preferred embodiment of the invention, the specific recognition molecule comprises an antibody, a nucleic acid aptamer or an affinity polypeptide; wherein the antibody is preferably transferrin (CD71) antibody or human leukocyte antigen-G (HLA-G) antibody; the affinity polypeptide is preferably transferrin (CD71) affinity polypeptide Y1; the aptamer is preferably one selected by the SELEX technique and having a high affinity for CD71 or HLA-G.
In a preferred embodiment of the present invention, the material of the microfluidic chip may be Polydimethylsiloxane (PDMS), and the material of the slide may be glass.
In a preferred embodiment of the invention, the microfluidic chip is packaged with the slide by plasma bonding.
In a preferred embodiment of the invention, the injection port flow rate is from 0.1mL/h to 10mL/h, e.g., 0.1mL/h, 0.3mL/h, 0.5mL/h, 1mL/h, etc.
In a preferred embodiment of the present invention, the release of the fetal cells is achieved by breaking chemical bonds, and the antibodies are detached from the surface of the microarray column, resulting in the release of the cells from the chip.
In a preferred embodiment of the present invention, the release of the fetal cells can be performed by selecting a cell picking method using laser cutting or a robot.
By adopting the technical scheme, the invention has the following technical effects:
1) according to the cell size, the critical dimension (Dc) is adjusted, the space separation of the cells is realized by combining the deterministic lateral displacement, the collision frequency of the cells and the microarray column is increased, and the capture efficiency is further improved;
2) the microarray pillar is designed with a rotation angle, so that three-side gradient shear stress is formed around the pillar, and the capture efficiency and the capture purity of target cells are improved;
3) the fetal cells are captured in the microfluidic chip by combining two principles of hydrodynamics and specific recognition, so that the capture efficiency is improved, and nonspecific adsorption is reduced;
drawings
Fig. 1 is a top view of the overall chip structure. Wherein (1) is a cell suspension sample inlet, (2) and (3) are buffer solution sample inlets, and (4) and (5) and (6) are sample outlets.
FIG. 2 is a schematic diagram showing the arrangement and parameters of the pillars in the microarray, wherein the horizontal spacing between the pillars is G, the distance between the vertical centers of the triangles in the same row is λ, and the upward deviation of the second triangle from the first triangle in the same row is Δ λ.
FIG. 3A is a statistical graph of the collision probability of human B lymphoma cells (Ramos) with microarray columns in different threshold chips; FIG. 3B is a statistical chart of the capture efficiency of different cells in a chip, the left three columns are the capture efficiency of human B lymphoma cells (Ramos), human chronic myelogenous leukemia cells (K562) and leukocytes in a chip modified with antibody CD71, and the right two columns are the capture efficiency of the above three cells in a control chip without modified antibody; FIG. 3C is a statistic of cell capture efficiency at different flow rates; FIG. 3D is a graph showing the capture efficiency statistics of different numbers of fetal cells in the chip.
FIG. 4A is a statistical distribution of fetal cells in a chip, wherein statistically, most of the cells have been captured in the first half of the chip; FIG. 4B is an image of the capture of cells in the chip, the white spots being the captured cells.
Fig. 5 is a schematic diagram of pregnant woman peripheral blood gradient centrifugation.
FIG. 6 is a photograph of an image of cellular immunofluorescence with the first and second rows identified as fetal cells and the third row identified as background adsorbed cells-leukocytes.
FIG. 7 is a diagram showing cell picking in which the first action is cells captured in the chip and the second action corresponds to cells obtained by microscopically picking the cells.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and the accompanying drawings. It is to be noted that, in the drawings or in the description, the implementation manners not shown or described are all the forms known to those skilled in the art, and will not be described in detail.
Example 1 preparation of a fetal cell Capture microfluidic chip
Referring to fig. 1, a microfluidic chip is manufactured, the chip comprises two layers of PDMS and a layer of glass chip from bottom to top, and a PDMS slab, a PDMS channel layer and slide glass are sequentially bonded into a complete chip by using plasma. The chip is provided with three sample inlets (1), (2), (3) and three sample outlets (4), (5) and (6), a triangular microarray is arranged between the sample inlets and the sample outlets, wherein the microarray is arranged by adopting a DLD design principle, as shown in figure 1.
In this embodiment, three sample inlets are located on the left side of the chip, three sample outlets are located on the right side of the chip, and a 0.7mm punching pen is used to prepare the sample inlets and the sample outlets.
In this example, the chip size is designed to be 1 cm wide and 4.5 cm long.
In this embodiment, the horizontal distance G between the pillars is set to be 32 microns, the distance λ between the vertical centers of the triangles in the same row is set to be 122.5 microns, and the upward deviation Δ λ of the second triangle in the same row from the first triangle is set to be 3.5 microns, so that the overall critical dimension Dc of the microfluidic chip is 8 microns.
In this example, the modification of the antibody was performed by a chemical modification method, in the chip after the plasma bonding, 4% (3-mercaptopropyl) trimethoxysilane (MPTS) dissolved in ethanol was introduced, and the introduction was performed once every 5 minutes for 1 hour, the channel was rinsed with ethanol, and the chip was placed in a 100 ° oven and heated for 1 hour; the solution was taken out and passed through 0.01% 4-maleimidobutyrate N-hydroxysuccinimide ester (GMBS) dissolved in ethanol once every 5 minutes for 30 minutes, after which the channel was washed with ultrapure water and then with 1 XPBS, 20. mu.g/ml streptavidin was passed through, incubated for 1 hour, washed with PBS and then with 20. mu.g/ml biotinylated CD71 or HLA-G antibody, incubated for 1 hour, washed with PBS and placed in a 4 ℃ freezer for further use. Thus, the microfluidic chip containing the antibody modification is obtained.
Example 2 simulation of fetal cell capture characterization
The condition parameters and the concrete steps are as follows:
1) fluorescent pre-stained target cells (Ramos & K562) or control cells (WBCs) were resuspended in PBS buffer solution after cell counting;
2) injecting a sample from the sample injection hole (1) through a sample injection system, and injecting a buffer solution through the sample injection holes (2) and (3);
3) injecting PBS buffer solution through the sample injection ports (1, 2 and 3) to wash the chip;
4) counting and counting the cells, dividing the counted and counted cells by the number of injected cells, and calculating the capture efficiency;
the chip is combined with a transferrin CD71 antibody, the target cell capturing efficiency is more than 80 percent, and the leukocyte capturing efficiency is 0.016 percent.
In this example, the results are shown in FIG. 3.
Fig. 3 is a statistic of the microfluidic chip on the capture efficiency of the simulated fetal cells. FIG. 3A is a statistical graph of the collision efficiency of Ramos cells in chips with different critical values, wherein the higher the collision efficiency is, the better the capture efficiency can be obtained; FIG. 3B shows the actual capture of cells; FIG. 3C is a graph of the effect of different flow rates on cell efficiency; FIG. 3D is a graph showing the efficiency of capture of different cell numbers in the chip.
FIG. 4A is a statistical analysis of the distribution of fetal cells in a chip, wherein statistically most of the cells have been captured in the first half of the chip; FIG. 4B is an image of the capture of cells in the chip, the white spots being the captured cells.
EXAMPLE 3 preparation of monocyte suspension containing fetal cells
Taking two milliliters of peripheral blood of the pregnant woman, adding PBS buffer solution to dilute the blood into 4 milliliters, adopting percoll gradient centrifugation to process the sample, obtaining a monocyte layer, washing and re-suspending the monocyte suspension. Wherein the percoll density is 1.090, the centrifugal force is 400g, and the time is 30 minutes. Fig. 5 is a schematic diagram of pregnant woman peripheral blood gradient centrifugation.
Example 4 fetal cell Capture and identification
The monocyte suspension in the embodiment 3 is introduced into the microfluidic chip in the embodiment 1 through a syringe pump, wherein the flow rate is set to be 0.3mL/h, and after the monocyte suspension is introduced, the monocyte suspension is washed for 3 times by using PBS buffer solution, so that the background cells which are not specifically adsorbed are removed to the maximum extent. In this example, a cell fixing solution was prepared and passed through the microfluidic chip on which the cells of the fetus were captured by using a syringe. Standing for 15 minutes, and washing with PBS; then adding a chip sealing solution for sealing, standing for 30 minutes, and washing with PBS.
In this example, foetal cells were identified by staining with a fluorescent antibody consisting of: 1) nucleated red blood cells: red fluorescent CD-45 antibody + green fluorescent GPA antibody; 2) trophoblast cells: red fluorescent CD-45 antibody + blue fluorescent HLA-G antibody + green fluorescent CK antibody. And (3) introducing an antibody mixed solution into the chip, standing for 1 hour in a dark place, washing redundant dye by using PBS, adding a cell nucleus staining solution, re-staining the cell nucleus for 10 minutes, washing by using PBS, observing a cell staining result under a fluorescence microscope, and analyzing and recording the number of fetal cells by combining cell morphology. FIG. 6 is a photograph of an image of cellular immunofluorescence with the first and second rows identified as fetal cells and the third row identified as background adsorbed cells-leukocytes.
Example 5 fetal cell Release and amplification sequencing
The fetal cells identified in example 4 were subjected to capillary microsampling to obtain single fetal cells, and FIG. 7 is a schematic diagram of cell picking, wherein the first action is cells captured in the chip, and the second action corresponds to cells obtained by microsampling the cells. Performing single cell amplification on the obtained fetal cells, performing amplification by using a commercial reagent (MDA or Malbac method), performing gel electrophoresis characterization after amplification, purifying (column purification or magnetic bead purification), and performing sample sequencing analysis.
Claims (6)
1. A method of isolating free fetal cells from the peripheral blood of a pregnant woman, comprising the steps of:
1) preparing a microfluidic chip for capturing fetal cells; the micro-fluidic chip is designed to comprise at least one sample inlet and at least one sample outlet; a microarray with specific geometric arrangement is arranged between the sample inlet and the sample outlet; the specific geometrical arrangement is that the overall critical dimension of the microfluidic chip is designated as Dc, which is expressed by the formula Dc ═ 1.4 XGx (Δ λ/λ)0.48Setting, wherein the horizontal spacing between the pillars is set as G, and the size of the G is between 0 and 50 micrometers; the distance between the vertical centers of the triangles in the same column is set as lambda, and the size of the lambda is between 100 and 150 microns; the upward offset of the second triangle in the same row compared to the first triangle is set as Δ λ, and the magnitude of Δ λ is between 0 and 20 μm; setting the integral critical dimension of the microfluidic chip as Dc;
2) modifying a fetal cell-specific recognition molecule on the surface of the microarray; the fetal cell specific recognition molecule is transferrin CD-71 antibody;
3) regulating the critical diameter of the microarray to be 0-50 microns and the flow rate to be 0-10mL/h, so that the cell capture efficiency of the microfluidic chip is optimal;
4) carrying out gradient centrifugal separation on 2-10 ml of pregnant woman peripheral blood to obtain mononuclear cells;
5) resuspending the mononuclear cells obtained in the step 4) in a phosphate buffer solution, and injecting the mononuclear cells into a microfluidic chip to capture fetal cells; identifying fetal cells by using a fluorescent antibody staining method, wherein the antibody comprises the following components: 1) nucleated red blood cells: red fluorescent CD-45 antibody + green fluorescent GPA antibody; 2) trophoblast cells: red fluorescent CD-45 antibody + blue fluorescent HLA-G antibody + green fluorescent CK antibody.
2. The method of claim 1, wherein the isolating the free fetal cells from the peripheral blood of the pregnant woman comprises: the microarray columns are arranged such that the distance between microarrays is adjusted to 10-40 microns, depending on the size of the cell object being analyzed, and the angle of offset of the next column compared to the previous column is 10-40 °.
3. The method of claim 1, wherein the isolated free fetal cells are isolated from the peripheral blood of a pregnant woman: controllable breaking chemical groups are modified between the surface of the microarray and the specific recognition molecules, and the controllable breaking chemical groups comprise light control, pH control or chemical catalysis control, and can release the captured fetal cells in a controllable and fixed-point manner.
4. The method of claim 1, wherein the isolating the free fetal cells from the peripheral blood of the pregnant woman comprises: the microarray pillar is a cylinder or a triangular pillar having a diameter or side length of between 10 and 200 micrometers.
5. A method of isolating free foetal cells from the peripheral blood of a pregnant woman as claimed in any one of claims 1 to 4, wherein: further comprising step 6) of releasing the captured foetal cells from the microarray.
6. The method of claim 5, wherein the isolated fetal cells are isolated from the peripheral blood of the pregnant woman by: and 6) acquiring target cells in the chip by using a releasing means comprising a chemical reagent, laser cutting or a capillary manipulator.
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| CN110093247B (en) * | 2019-05-07 | 2020-11-17 | 西安交通大学 | Micro-fluidic chip for enriching and capturing target cells of different specifications and sizes |
| CN111440696B (en) * | 2020-02-26 | 2023-02-24 | 德运康明(厦门)生物科技有限公司 | Fetal cell capture module, microfluidic chip for fetal cell capture, and methods of using same |
| CN111443197A (en) * | 2020-03-04 | 2020-07-24 | 厦门大学 | Method for analyzing phenotype of circulating tumor cells of liver cancer |
| CN113019485B (en) * | 2021-03-30 | 2023-01-03 | 深圳市亚辉龙生物科技股份有限公司 | Micro-fluidic chip, circulating tumor cell automatic separation detection system and method |
| CN112980779B (en) | 2021-05-20 | 2021-08-24 | 广州凯普医药科技有限公司 | Method for separating placenta trophoblast cells from cervical exfoliated cells of pregnant women |
| CN116024067A (en) * | 2022-12-19 | 2023-04-28 | 深圳职业技术学院 | Circulating fetal cell separation device and preparation method thereof |
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| CN104977284A (en) * | 2015-07-03 | 2015-10-14 | 石莹 | Capture and identification method for fetal nucleated red blood cells |
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| CN105063181A (en) * | 2015-07-03 | 2015-11-18 | 石莹 | Method for noninvasive antenatal diagnosis through separating fetal nucleated red blood cells from peripheral circulating blood of pregnant woman |
| CN105861297A (en) * | 2016-03-29 | 2016-08-17 | 厦门大学 | Circulating tumor cell detection chip and application thereof |
| CN105950469B (en) * | 2016-06-08 | 2018-03-06 | 牛海涛 | Cell screening chip and micro-fluidic chip joint |
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| CN104977284A (en) * | 2015-07-03 | 2015-10-14 | 石莹 | Capture and identification method for fetal nucleated red blood cells |
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Effective date of registration: 20190108 Address after: 361000 15, two building, 1888 Hongxiang Road, Xiangan District, Xiamen, Fujian. Applicant after: De Yun Ming (Xiamen) Biotechnology Co., Ltd. Address before: 361000 Siming South Road, Xiamen, Fujian Province, No. 422 Applicant before: Xiamen University |
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