CN108535228A - A method of detaching the fetal cell that dissociates from maternal blood - Google Patents
A method of detaching the fetal cell that dissociates from maternal blood Download PDFInfo
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- CN108535228A CN108535228A CN201810289478.9A CN201810289478A CN108535228A CN 108535228 A CN108535228 A CN 108535228A CN 201810289478 A CN201810289478 A CN 201810289478A CN 108535228 A CN108535228 A CN 108535228A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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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
- B01L3/502707—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 method that the present invention relates to a kind of to detach the fetal cell that dissociates from maternal blood.Cell centrifugation and specificity affine recognition principle of this method using micro-fluidic chip realize high efficiency, the high-purity capture separation of maternal blood middle reaches isolated human fetal cell (fetal nucleated red blood, trophocyte etc.).In micro-fluidic chip, the molecular radical such as aptamer, antibody, polypeptide etc. with affine recognition reaction on the micro array structure with particular geometric arrangement mode, and modification is devised.The parameter for adjusting micro array structure can regulate and control the collision efficiency of various sizes of fetal cell and microarray, and then realize the capture enrichment of different size fetal cells.
Description
Technical field
The method that the present invention relates to a kind of to detach the fetal cell that dissociates from maternal blood.
Background technology
Recently as the relieving of two tire policies, elderly parturient women is continuously increased, and inborn defect rate is even more in rising trend.
In the tertiary prevention of inborn defect, the second level " Prenatal Screening and diagnosis " is to prevent means mostly important in inborn defect.Sheep
Membrane cavity centesis is the abnormal main means of the antenatal row of high-risk puerpera, but invasive strong, infection, miscarriage equivalent risk are higher.Based on tire
The noninvasive antenatal detection of youngster's dissociative DNA, is widely used to the screening of autosome aneuploid, but that there are false positive rates is high,
It cannot exclude the deficiencies of parent chromosome is abnormal.Fetal cell in the maternal peripheral blood that 1969 find has complete
Fetal genetic information, and sample it is convenient, it is invasive small, be most potential non-invasive prenatal diagnosis object.But fetus is thin
Born of the same parents do not use always the maximum challenge of pre-natal diagnosis really and are in peripheral blood that (1~10 fetus is thin for fetal cell content rareness
Born of the same parents/milliliter, 109A red blood cell, 106A leucocyte), background interference is strong, is difficult to realize the capture of its high specific.How from thousands of
Fetal cell is accurately isolated in complicated blood environment existing for a background cells up to ten thousand, is established efficient, quick, economy outer
All blood fetal cell enrichment methods are the bottleneck problems for realizing noninvasive, accurate pre-natal diagnosis.
The problem of fetal cell separating trap is primarily present at present include：1, capture rate is very low.Fetal cell now
The main method of acquisition is Beads enrichment method, flow cytometry and cell smear method.Beads enrichment method and flow cytometry cell
Loss Rate is very high, and 10 milliliters or more of peripheral blood is generally required for the capture of fetal cell, and highest even needs 30 milliliters,
So high sample requirement amount be difficult allow all pregnant woman all to receive, and the capture quantity of its fetal cell but still 10 with
Under, accumulation rate is too low.And cell smear method is to use for reference fetal cell in traditional erythroblast detection method human peripheral blood to carry out
Analysis, cell smear method relies primarily on eye recognition, needs a large amount of time and manpower, working time of a sample is long,
Efficiency is low, and can only identify and cannot achieve target cell separation.2, cell activity is low, these methods be directed to cell into
The post-processings such as capable fixation, it is unicellular inactive or active low, it is difficult to carry out effective single cell analysis.
Micro-fluidic chip has the characteristics that high-throughput, small, consumption is small, and in recent years, micro-fluidic chip is swollen in cycle
The fields such as oncocyte separation and concentration, unicellular sequencing are yielded unusually brilliant results, it can be achieved that the efficiently separating of circulating tumor cell in peripheral blood,
Enrichment, release, sequencing etc..It is based primarily upon two kinds of principles currently with the method that micro-fluidic chip carries out cell separation：1) it utilizes
Compatibility identification capture；2) physical separation is utilized, using the methods of micro porous filtration or hydrodynamics, ultrasonic Separation.But it is different
In the big advantage of circulating tumor cell size, fetal cell size is smaller, it is difficult to cell is detached by means such as filterings,
And part fetal cell surface marker is indefinite, it is difficult to carry out the separation and concentration of the fetal cell of specificity.
Invention content
Technical problem to be solved by the invention is to provide a kind of from maternal blood detaches the side of the fetal cell that dissociates
Method.
The purpose of the present invention is realized by the following technical solution：
A method of it detaching the fetal cell that dissociates from maternal blood, includes the following steps：
1) micro-fluidic chip for fetal cell capture is prepared；Micro-fluidic chip design contains at least one injection port
With at least one outlet；Include for the microarray arranged with particular geometric, microarray fashion between injection port and outlet
Round or triangle；The distance between microarray is 0-50 microns, preferably, can be 10/20/30/40 micron), after
One pillar is 0-50 ° compared with the deviation angle of previous pillar, preferably, can be 10/20/30/40 °；
Preferably, the whole critical dimension of micro-fluidic chip is set as Dc, by formula Dc=1.4 × G × (Δ λ/λ
)0.48Setting, wherein the level interval between pillar is set as G, and size is between 0 to 50 microns；With a line intermediate cam shape orthocenter
The distance between be set as λ, size is between 100-150 microns；With compared to first triangle of second triangle in a line
The value that offsets up of shape is set as Δ λ, and size is between 0 to 20 microns；The whole critical dimension of micro-fluidic chip is set as Dc.
2) know molecule in microarray surface modification fetal cell specificity；
3) cut off diameter of regulation and control microarray is 0-50 microns, preferably, can be 10/20/30/40 micron, flow velocity
For 0-10mL/h, preferably, can be 1/2/3/4/5/6/7/8/9mL/h so that the cell capture efficiency of micro-fluidic chip
It is optimal；
4) 2-10 milliliters of maternal bloods are subjected to gradient centrifugation separation, obtain monocyte；
5) monocyte that step 4) obtains is resuspended in phosphate buffer, is injected into micro-fluidic chip and carries out tire
The capture of youngster's cell.
In the preferred embodiments of the invention, the micro-fluidic chip overall dimensions size is to be about 1 to 5 centimetres, wide about
Between 0.5 to 2 centimetres, size design is specifically set according to cell total amount size needed to be separated.
In the preferred embodiments of the invention, microarray pillar can be cylinder, can also be triangle pillar, straight
Diameter or length of side size are between 10 to 200 microns, preferably, can be 20/40/60/80/100 micron etc..
In the preferred embodiments of the invention, the level interval wherein between pillar is set as G, size 0 to 50 microns it
Between, can be 10 microns, 20 microns, 30 microns etc.；It is set as λ with the distance between a line intermediate cam shape orthocenter, size exists
It can be 100 microns, 120 microns, 130 microns etc. between 100-150 microns；With second triangle in a line compared to
The value that offsets up of one triangle is set as Δ λ, size between 0 to 20 microns, can be 1 micron, 3.5 microns, it is 6.5 micro-
Rice, 7.5 microns etc.；The whole critical dimension of micro-fluidic chip is set as Dc, by formula Dc=1.4 × G × (Δ λ/λ)0.48
It arrives.
In the preferred embodiments of the invention, the specific recognition molecules, including antibody, aptamer or affine more
Peptide；Wherein antibody is preferably transferrins (CD71) antibody or human leucocyte antigen (HLA)-G (HLA-G) antibody；Affine polypeptide is excellent
It is selected as the affine polypeptide Y1 of transferrins (CD71)；Aptamer comes out preferably by SELEX technology screenings, to CD71 or
HLA-G has the aptamer of high-affinity.
In a preferred embodiment of the present invention, micro-fluidic used can be dimethyl silicone polymer
(PDMS), slide glass material can be glass.
In a preferred embodiment of the present invention, the mode and slide glass of micro-fluidic chip using plasma bonding used
It is packaged.
The present invention a preferred embodiment in, injection port flow velocity be 0.1mL/h-10mL/h, such as 0.1mL/h,
0.3mL/h, 0.5mL/h, 1mL/h etc..
The present invention a preferred embodiment in, fetal cell release can select use chemical bond rupture, antibody from
Microarray pillar surface falls off, and cell is caused to be released from chip.
The present invention a preferred embodiment in, fetal cell release can select using laser cutting or manipulator into
The mode of row cell picking carries out.
Using above-mentioned technical proposal, technique effect of the invention is as follows：
1) according to cell size, the size of critical dimension (Dc) is adjusted, the sky of cell is realized in conjunction with certainty lateral displacement
Between detach, increase the collision frequency of cell and microarray pillar, and then improve capture rate；
2) microarray pillar is designed with rotation angle so that three face gradient shear stress are presented around pillar, are conducive to improve
Target cell capture rate and capture purity；
3) fetal cell is captured in micro-fluidic chip in conjunction with two kinds of principles of hydrodynamics and specific recognition, both
Capture rate is improved, and reduces non-specific adsorption, which greatly reduces the demand of blood, greatly improves
The probability that fetal cell obtains between different samples；
Description of the drawings
Fig. 1 is chip overall structure vertical view.Wherein (1) is cell suspension injection port, and (2) (3) are buffer solution injection port,
(4) (5) (6) are outlet.
Fig. 2 is the arrangement of microarray pillar and parameter schematic diagram, and the level interval between pillar is set as G, with a line intermediate cam shape
The distance between orthocenter is set as λ, and Δ is set as compared to the value that offsets up of first triangle with second triangle in a line
λ。
Fig. 3 A be people B lymphocytic cancer cells (Ramos) cell in different critical value chip, it is general with the collision of microarray pillar
Rate statistical chart；Fig. 3 B are the capture rate statistical chart of different cells in the chips, and left three pillars are to be modified with antibody CD71 cores
In piece, people B lymphocytic cancer cells (Ramos), human chronic polymorpho nuclear leukemia cells (K562) and leucocyte capture rate, the right side two
A pillar is to be not decorated in the control chip of antibody, the capture rate of above-mentioned three kinds of cells；Under Fig. 3 C are different in flow rate, cell
Capture rate statistics；Fig. 3 D are the capture rate statistics of the fetal cell of different numbers in the chips.
Fig. 4 A are the distribution statistics of fetal cell in the chips, and from statistical data, most cells are in chip
First half has been captured；Fig. 4 B are the image of cell capture in chip, and white bright spot is the cell of capture.
Fig. 5 is maternal blood gradient centrifugation schematic diagram.
Fig. 6 is cellular immunofluorescence image, and fetal cell is accredited as shown in the first row and the second row, mirror shown in the third line
It is set to background adherent cell-leucocyte.
Fig. 7 is cell picking diagram, the cell captured in the first behavior chip, the second behavior correspond to the cell pass through it is micro-
The cell that picking obtains.
Specific implementation mode
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, and reference
Attached drawing, the present invention is described in more detail.It should be noted that in attached drawing or specification text, it is not painted or describes
Realization method is form known to a person of ordinary skill in the art in technical field, will not be described in further detail.
1 fetal cell of embodiment captures the preparation of micro-fluidic chip
Referring to Fig. 1, micro-fluidic chip is made, chip includes two layers of PDMS, one layer of glass-chip, by PDMS thickness from the bottom to top
Block, PDMS channel layers, slide glass are bonded to complete chip using plasma successively.Chip set there are three injection port (1), (2),
(3) and three outlets (4), (5), (6) it is, triangle microarray between injection port and outlet, wherein microarray arrangement is adopted
It is arranged with DLD design principles, as shown in Figure 1.
In the present embodiment, three injection ports are located at the left side of chip, and three outlets are located at the right side of chip, use
0.7mm punching pens prepare inlet and outlet.
In the present embodiment, chip size is designed as 1 centimetre wide, 4.5 centimetres of length.
In the present embodiment, the level interval G between pillar, is set as 32 microns, between a line intermediate cam shape orthocenter away from
From λ, 122.5 microns are set as, value Δ λ is offset up compared to first triangle with second triangle in a line, is set as
3.5 microns, then the whole critical dimension Dc of the micro-fluidic chip is 8 microns.
In the present embodiment, the modification of antibody is carried out using the method for chemical modification, after being bonded above by plasma
In chip, it is passed through 4% (3- mercaptopropyis) trimethoxy silane (MPTS) for being dissolved in ethyl alcohol, it is logical primary every 5 minutes, continue 1
Hour, with ethyl alcohol irrigation channel, it is put into 100 ° of baking ovens and heats 1 hour；Taking-up is passed through the 0.01%4- maleimides for being dissolved in ethyl alcohol
Amine butyric acid N-hydroxy-succinamide ester (GMBS), it is logical primary every 5 minutes, continue 30 minutes, first uses ultrapure water later
Channel, then rinsed with 1 × PBS, 20 μ g/ml Streptavidins are passed through, are incubated 1 hour, 20 μ g/ml lifes are passed through again with PBS flushings
CD71 the or HLA-G antibody of object element is incubated 1 hour, is rinsed with PBS, it is for use to put 4 ° of refrigerators.With this, obtain repairing containing antibody
The micro-fluidic chip of decorations.
Embodiment 2 simulates fetal cell capture characterization
It the conditional parameter and is as follows：
1) the prestained target cell of fluorescence (Ramos＆K562) or control cell (WBCs) after cell count by being resuspended
To PBS buffer solutions；
2) sample is injected by sampling system from sample holes (1), buffer solution is injected by (2), (3)；
3) PBS buffer solution is injected by injection port (1,2,3) and cleans chip；
4) cell is divided by by counting statistics with the cell number of injection, calculates capture rate；
Transferrins CD-71 antibody, capture target cell efficiency is combined to be all higher than 80% on chip, leucocyte 0.016%.
In the present embodiment, the results are shown in Figure 3.
Fig. 3 is statistics of the micro-fluidic chip to simulation fetal cell capture rate.Fig. 5 A be different critical value chip in,
The statistical chart of the collision efficiency of Ramos cells, collision efficiency is higher, can obtain better capture rate；Fig. 5 B are that cell is practical
Capture situation；Fig. 5 C are the influence different in flow rate to cell efficiency；Fig. 5 D are the capture rate of different number of cells in the chips
Investigation.
Fig. 4 A are to simulate the distribution statistics of fetal cell in the chips, and from statistical data, most cells are in core
The first half of piece has been captured；Fig. 4 B are the image of cell capture in chip, and white bright spot is the cell of capture.
The preparation of mononuclear cell suspension of the embodiment 3 containing fetal cell
Two milliliters of maternal blood is taken, PBS buffer solution is added and is diluted to 4 milliliters, is handled using percoll gradient centrifugations
Sample is stated, mononuclear cell layer, washing are obtained, resuspension obtains mononuclear cell suspension.Wherein percoll density uses 1.090, centrifugation
Power is 400g, and the time is 30 minutes.Fig. 5 is maternal blood gradient centrifugation schematic diagram.
The capture and identification of 4 fetal cell of embodiment
By the mononuclear cell suspension in embodiment 3, the micro-fluidic chip in embodiment 1 is passed through by syringe pump, wherein flowing
Speed is set as 0.3mL/h, after having led to mononuclear cell suspension, is cleaned 3 times using PBS buffer solution, removal is non-specific to the full extent
The background cells of absorption.In the present embodiment, cell fixer is configured, there is the cell of fetus by above-mentioned capture using syringe
Micro-fluidic chip in.15 minutes are stood, is cleaned with PBS；It adds chip confining liquid to be closed, after standing 30 minutes, use
PBS is cleaned.
In the present embodiment, fetal cell is identified using fluorescent antibody staining method, the antibody group becomes：1)
Erythroblast：Red fluorescence CD-45 antibody+green fluorescence GPA antibody；2) trophocyte：Red fluorescence CD-45 antibody+
Blue-fluorescence HLA-G antibody+green fluorescence CK antibody.After being passed through antibody mixed liquor into chip, it is protected from light standing 1 hour, uses PBS
After washing extra dyestuff, nuclear targeting liquid is added, redyes nucleus 10 minutes, after PBS cleanings, is placed in fluorescence microscope
Lower observation cell dyeing is as a result, combination cell morphology, analysis and record fetus number of cells.Fig. 6 be cellular immunofluorescence at
As figure, it is accredited as fetal cell shown in the first row and the second row, background adherent cell-leucocyte is accredited as shown in the third line.
5 fetal cell of embodiment discharges and amplification sequencing
The fetal cell identified in embodiment 4 is subjected to the micro- picking of capillary, obtains single fetal cell, Fig. 7 is thin
Born of the same parents' picking illustrates, and the cell captured in the first behavior chip, the second behavior corresponds to the cell that the cell is obtained by micro- picking.
Gained fetal cell is subjected to unicellular amplification, commercial reagents can be used and expanded (can be MDA or Malbac methods), is expanded
Gel electrophoresis characterizes after increasing, purifies (column purification or magnetic beads for purifying), sample presentation sequencing analysis.
Claims (7)
1. a kind of method detaching the fetal cell that dissociates from maternal blood, includes the following steps：
1) micro-fluidic chip for fetal cell capture is prepared；Micro-fluidic chip design is containing at least one injection port and extremely
A few outlet；Microarray between injection port and outlet to arrange with particular geometric；The particular geometric arrangement
For the whole critical dimension of micro-fluidic chip is set as Dc, by formula Dc=1.4 × G × (Δ λ/λ)0.48Setting, wherein column
Level interval between son is set as G, and size is between 0 to 50 microns；It is set as λ with the distance between a line intermediate cam shape orthocenter,
Its size is between 100-150 microns；It is set compared to the value that offsets up of first triangle with second triangle in a line
For Δ λ, size is between 0 to 20 microns；The whole critical dimension of micro-fluidic chip is set as Dc；
2) know molecule in microarray surface modification fetal cell specificity；
3) regulation and control microarray cut off diameter be 0-50 microns, flow velocity 0-10mL/h so that the cell capture of micro-fluidic chip
Efficiency is optimal；
4) 2-10 milliliters of maternal bloods are subjected to gradient centrifugation separation, obtain monocyte；
5) monocyte that step 4) obtains is resuspended in phosphate buffer, it is thin is injected into progress fetus in micro-fluidic chip
The capture of born of the same parents.
2. a kind of method detaching the fetal cell that dissociates from maternal blood as described in claim 1, it is characterised in that：Step
It is rapid 2) described in fetal cell specific recognition molecules, including aptamer either polypeptide or antibody.
3. a kind of method detaching the fetal cell that dissociates from maternal blood as described in claim 1, it is characterised in that：It is micro-
The arrangement of array pillar adjusts 10-40 microns of the distance between microarray, the latter column according to analyzed cell object size
Deviation angle 10-40 ° of the son compared with previous pillar.
4. the method for detaching the fetal cell that dissociates in a kind of maternal blood as described in claim 1, it is characterised in that：Micro- battle array
Controlled fracture chemical group, including photocontrol either pH controls or chemical are modified between list face and specific recognition molecules
Catalytic control, the fetal cell that controllable, fixed point release captures.
5. a kind of method detaching the fetal cell that dissociates from maternal blood as described in claim 1, it is characterised in that：It is micro-
Array pillar is cylinder or triangle pillar, and diameter or length of side size are between 10 to 200 microns.
6. special such as a kind of method detaching the fetal cell that dissociates from maternal blood described in any one of claim 1 to 5
Sign is：Further include step 6), the fetal cell of capture is released from microarray.
7. a kind of method detaching the fetal cell that dissociates from maternal blood as claimed in claim 6, it is characterised in that：Step
Rapid 6) release means include that chemical reagent, laser cutting or capillary manipulator obtain the target cell in chip.
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| CN111440696A (en) * | 2020-02-26 | 2020-07-24 | 厦门大学 | Fetal cell capture module, microfluidic chip for fetal cell capture, and methods of using same |
| 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 |
| WO2022206133A1 (en) * | 2021-03-30 | 2022-10-06 | 深圳市亚辉龙生物科技股份有限公司 | Microfluidic chip, and automatic separation and detection system and method for circulating tumor cell |
| CN112980779A (en) * | 2021-05-20 | 2021-06-18 | 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 |
| WO2022242285A1 (en) * | 2021-05-20 | 2022-11-24 | 广州凯普医药科技有限公司 | Method for separating placental trophoblast cells from exfoliated cervical cells of pregnant woman |
| US11796443B2 (en) | 2021-05-20 | 2023-10-24 | Guangzhou Hybribio Medicine Technology Ltd. | Method for isolating placental trophoblast cells from cervical exfoliated cells of pregnant woman |
| 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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