CN103620016B - Micro fluidic device and application thereof - Google Patents
Micro fluidic device and application thereof Download PDFInfo
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- CN103620016B CN103620016B CN201280016505.6A CN201280016505A CN103620016B CN 103620016 B CN103620016 B CN 103620016B CN 201280016505 A CN201280016505 A CN 201280016505A CN 103620016 B CN103620016 B CN 103620016B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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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
- B01L3/502753—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 bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
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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
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
- B01L2200/0652—Sorting or classification of particles or molecules
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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
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0883—Serpentine channels
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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
- B01L2400/00—Moving or stopping fluids
- B01L2400/08—Regulating or influencing the flow resistance
- B01L2400/084—Passive control of flow resistance
- B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions
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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
- B01L3/502746—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 means for controlling flow resistance, e.g. flow controllers, baffles
Abstract
Provide micro fluidic device and application thereof, specifically, it is provided that micro fluidic device, determine whether biological sample has the method for hereditary variation and determine whether biological sample has the system of hereditary variation.Wherein, micro fluidic device has and is suitable to separate single celled microfluidic channel.
Description
Technical field
The present invention relates to micro fluidic device and application thereof, in particular to micro fluidic device, determine whether biological sample has
The method of hereditary variation and determine whether biological sample has the system of hereditary variation.
Background technology
Microfluidic chip technology originates from analytical chemistry field, is by Micrometer-Nanometer Processing Technology, by biology, medical science and chemical field
In involved sample preparation, react, separate, the basic operation unit such as detection is integrated on several square centimeter chip, by
Microchannel forms network, runs through whole system with controlled fluid, in order to replace the technology of the various functions of conventional analysis laboratory.
Microflow control technique shows extremely strong superiority at aspects such as cell separation, nucleic acid extraction, purification, PCR amplifications.
But, current micro-fluidic chip still haves much room for improvement.
Summary of the invention
It is contemplated that at least solve one of technical problem present in prior art.
In a first aspect of the present invention, the present invention proposes a kind of micro fluidic device.According to embodiments of the invention, this is micro-fluidic
Device has and is suitable to separate single celled microfluidic channel.Thus, micro fluidic device according to embodiments of the present invention is utilized to have
Effect ground separates unicellular from biological specimen.
In a second aspect of the present invention, the present invention proposes a kind of method determining whether biological sample has hereditary variation, and it is special
Levy and be, comprise the following steps: to utilize foregoing micro fluidic device, from biological sample separation cell sample；To being separated
Cell sample included in the expanding at least partially of hereditary material, in order to obtain amplified production；Described amplification is produced
Thing checks order, in order to obtain sequencing result；And based on described sequencing result, determine whether described biological sample has heredity
Variation.By method according to embodiments of the present invention, it is possible to effectively separate by micro fluidic device according to embodiments of the present invention
Unicellular, such that it is able to check order based on to single celled hereditary material such as full-length genome, effectively by sequencing result
It is analyzed and obtains whether single celled hereditary material such as full-length genome exists exception, so that it is determined that whether biological sample has
There is hereditary variation.
In a third aspect of the present invention, the present invention proposes a kind of system determining whether biological sample has hereditary variation,
It is characterized in that including: foregoing micro fluidic device, described micro fluidic device is for from biological sample separation cell sample；
Amplification device, described amplification device is connected with described micro fluidic device, and is suitable to included in the cell sample separated
Expanding at least partially of hereditary material, in order to obtain amplified production；Sequencing device, described sequencing device and described amplification
Device is connected, and is suitable to check order described amplified production, in order to obtain sequencing result；And analytical equipment, described point
Analysis apparatus is connected with described sequencing device, and is suitable to, based on described sequencing result, determine whether described biological sample has heredity
Variation.Utilize whether determination biological sample according to embodiments of the present invention has the system of hereditary variation, before can effectively implementing
Method described in face, it is possible to effectively separate unicellular by micro fluidic device according to embodiments of the present invention, such that it is able to based on
Single celled hereditary material such as full-length genome is checked order, effectively obtains unicellular by sequencing result is analyzed
Hereditary material such as full-length genome in whether there is exception, so that it is determined that whether biological sample has hereditary variation.
Thus, according to embodiments of the invention, the present invention is directed to an acquisition difficult problem for low abundance cell, establish and measure based on high pass
The low abundance cell screening model of sequence, by the parameter of bio information this model of analog regulation, demonstrates this model low rich in examination
Detecting domains, sensitivity, accuracy and repeatability in terms of degree cell.Specifically, the present invention initially sets up an automatization
Micro-current controlled cell sorting model, produces a number of particular sequence data pair followed by amplification technique and new-generation sequencing technology
Low abundance cell Different Variation carries out effective examination.Microflow control technique, whole genome amplification technology and high throughput sequencing technologies are entered
Row is integrated, it is achieved build the functions such as the separation of unicellular (few cells), enrichment, whole genome amplification on micro-fluidic chip,
In conjunction with high throughput sequencing technologies, carry out the single celled detection of automatization, high flux and analyze, to the sieve in terms of the hereditary variation of sample
Looking into, this technology model can be applied to the research in terms of new hereditism, such as finds new mechanism of causing a disease etc..
Present invention demonstrates that the method model of a kind of automatization examination low abundance cytometaplasia information, its experiment flow is: (a) makes sample
Product enter micro fluidic device, separate unicellular with the purpose in enriched sample through multiple microchannels or small room；B () is toward miniflow
Control and the microchannel on chip or small room add lytic reagent or the sudden and violent purpose producing heat cleavage step (a) gained under light
Unicellular, expand with pyrolysis product DNA or RNA for template；C the amplified production of step (b) gained is carried out by ()
Build storehouse, high-flux sequence platform check order, described high-flux sequence platform include but not limited to Illumina/Solexa,
ABI Solid and Roche454；D sequencing data that step (c) is obtained by () processes.
Another aspect of the present invention provides a kind of micro fluidic device being applied to automatization's examination low abundance cytometaplasia information, described
Micro fluidic device comprises at least one inlet and outlet interconnected by microchannel or small room, by micro-fluidic chip, driving
System and detecting system composition.
In a preferred embodiment of the invention, the micro-fluidic chip of described micro fluidic device comprise cell separation and enrichment unit and
Cell manipulation unit.
In a preferred embodiment of the invention, described cell separation and enrichment unit is provided with micro structure or micro-barrier, described carefully
Born of the same parents manipulate unit and are provided with liquid stream and Air Valve Control.
In a preferred embodiment of the invention, the micro-fluidic chip of described micro fluidic device also comprises DNA and/or RNA
Extract and amplification unit.
In a preferred embodiment of the invention, according to the described micro fluidic device method to the separation and concentration of low abundance cell,
Including making sample flow through the microchannel of described micro-fluidic chip or small room, utilize the magnetic capture target in microchannel or microchamber thin
Born of the same parents.
In a preferred embodiment of the invention, according to the method for described micro fluidic device cell lysis, flow through including making sample
The microchannel of described micro-fluidic chip or small room, utilize the magnetic capture target cell in microchannel or microchamber, and addition is split
Solve reagent or magnetic bead produced cruelly under light heat and cell lysis.
In a preferred embodiment of the invention, according to described micro fluidic device from cell amplification target dna or RNA
Method, including making sample flow through the microchannel of described micro-fluidic chip or small room, utilizes the magnetic capture in microchannel or microchamber
Target cell, adds lytic reagent or magnetic bead produces under light heat cruelly and cell lysis, extracting target dna or RNA,
And expand with target dna or RNA for template；Described amplification includes whole genome amplification.
Thus, according to embodiments of the invention, the present invention establishes the low abundance cell based on microflow control technique and high-flux sequence
The technology model of examination, and demonstrate the detecting domains of this model, sensitivity, accuracy and repeatability, can realize complicated sample
The hereditary information of the low abundance cell in product carries out high throughput automated examination, it is achieved laboratory is high throughput automated, for gene
Medical diagnosis research in terms of provides basis.According to embodiments of the invention, the present invention devises has microchannel or small room
Micro-fluidic chip, collect separation, gathering trace cell and function that DNA or RNA in cell is extracted and expand in
One chip, reduces experimental provision equipment cost, it is easy to promote.
The additional aspect of the present invention and advantage will part be given in the following description, and part will become bright from the following description
Aobvious, or recognized by the practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or the additional aspect of the present invention and advantage the accompanying drawings below description to embodiment will be apparent from from combining and
Easy to understand, wherein:
Fig. 1-Fig. 6 shows the structural representation of micro fluidic device according to embodiments of the present invention.
Detailed description of the invention
Embodiments of the invention are described below in detail, and the example of described embodiment is shown in the drawings, the most identical or
Similar label represents same or similar element or has the element of same or like function.Describe below with reference to accompanying drawing
Embodiment is exemplary, is only used for explaining the present invention, and is not considered as limiting the invention.
In describing the invention, it is to be understood that term " " center ", " on ", D score, "front", "rear", "left", "right",
The orientation of the instruction such as " vertically ", " level ", " top ", " end " " interior ", " outward " or position relationship be based on orientation shown in the drawings or
Position relationship, is for only for ease of the description present invention and simplifies description rather than instruction or imply that the device of indication or element are necessary
There is specific orientation, with specific azimuth configuration and operation, be therefore not considered as limiting the invention.
It should be noted that term " first ", " second " are only used for describing purpose, and it is not intended that instruction or imply relatively heavy
The property wanted or the implicit quantity indicating indicated technical characteristic.Thus, define " first ", the feature of " second " can be expressed
Or implicitly include one or more this feature.Further, in describing the invention, except as otherwise noted, " many
Individual " it is meant that two or more.
Term " low abundance cell ", " single (individual) cell " and " few cells " are used interchangeably, in the text all referring to multiple at one
Comprising the cell having wanted type in miscellaneous sample, but its content is the least, general only one of which is to several.
Micro fluidic device
In a first aspect of the present invention, the present invention proposes a kind of micro fluidic device.According to embodiments of the invention, this is micro-fluidic
Device has and is suitable to separate single celled microfluidic channel.Thus, micro fluidic device according to embodiments of the present invention is utilized to have
Effect ground separates unicellular from biological specimen.
According to embodiments of the invention, the form being suitable to separate single celled microfluidic channel in micro fluidic device is limited the most especially
System.For complexity and the rareness of purpose cell for separating single celled sample, can be according to dissimilar cell at thing
Difference on reason, chemistry and biological characteristics designs on chip and cell carries out a step separates the separation of unicellular or multistep with making
Single celled microchannel, in order to obtain final purpose unicellular.It should be noted that can by various be suitable to separate unicellular
The combination of method, and order is not particularly limited, as long as being capable of separating single celled.For example, it is possible to it is first
First biased sample cell is passed in the first microfluidic channel, carries out preliminary separation, remove most interference cell；So
After again by the cell through initial gross separation, be passed in the second microfluidic channel, carry out the further enrichment of purpose cell.
According to embodiments of the invention, microfluidic channel can physics based on cell, chemical property, such as size, shape, can
No deformation, cell surface character (such as cell surface receptor, antigen, the permeability of film), cell interior character is (as expressed certain
Plant albumen, enzyme-specific etc.) realize single celled separation.
According to one embodiment of present invention, described microfluidic channel is formed with multiple microtrabeculae.Thus, it is possible to by miniflow
Formation microtrabeculae (being referred to as barrier) on control passage, the obstacle flowed in the direction of flow as cell, such that it is able to base
Difference in cell size, it is achieved to single celled separation.According to embodiments of the invention, can build in microfluidic channel
Regularly arranged, and (form of barrier is not particularly restricted, and can be cylinder, oval to be spaced the barrier of fixed dimension
Live, square column etc.), often there is the lateral displacement of fixed dimension between row barrier.With reference to Fig. 1-3, by micro-fluidic chip passage
The internal barrier (the most multiple microtrabeculaes on described microfluidic channel in array distribution) gathering regularly arranged, cell to be separated
Collide with barrier when flow channel as the particle in fluid, when cell is more than a certain size (and between barrier
Spacing is correlated with) time, cell offsets according to certain angle (relevant to the lateral displacement between often row barrier), less than being somebody's turn to do
The cell of size then flows according to former fluid trajectory, realizes the separation (such as Fig. 1) of various sizes of cell with this.Can be according to treating
Point cellifugal size adjusts obstacle spacing, such as the distance between microtrabeculae, including horizontal spacing and longitudinal pitch.According to this
In the vertical, the distance between adjacent two microtrabeculaes is 10-100 micron to inventive embodiment, and, in the horizontal, adjacent
Distance between two microtrabeculaes is 10-100 micron.Single celled efficiency is separated thus, it is possible to improve further.According to this
A bright embodiment, often row microtrabeculae offsets 6-7 micron relatively.Single celled efficiency is separated thus, it is possible to improve further.
Barrier array structure can be arranged to single-stage or multistage, according to the difference of cellular environment to be separated, as fixing in separated from body fluid
The cell of size, general single-stage micro-pillar array it is achieved that and as the complex sample of blood etc, the inside also has sizes
Cell, fine separation can be carried out by multistage micro-pillar array.According to one embodiment of present invention, along flow direction, institute
The diameter stating microtrabeculae increases.Single celled efficiency is separated thus, it is possible to improve further.Further, with reference to Fig. 2, Ke Yijian
The barrier array of vertical multistage different spacing size, refines further by the separation of various sizes of cell.Collect not in exit
With the cell of size, carry out next step and separate or be directly used in follow-up test experience.According to one embodiment of present invention,
Along flow direction, the diameter of microtrabeculae is constant.
It addition, according to embodiments of the invention, it is also possible to utilize the inertia force that various sizes of cell produces in bending channel
Different, it is achieved in microfluidic channel, to separate cell.In bending channel, its flow velocity of the fluid of Stationary flow presents parabolic shape
Distribution, in the channel between flow velocity maximum, the centrifugal force being subject to is maximum, minimum near the rate of flow of fluid of conduit wall.In order to keep
The conservation of mass of fluid, can produce eddy current.So granule in fluid receives buoyancy and the interaction of Dien power.Necessarily
Flow conditions under, formed focus on flowing.The focused flow of the granule that size is the biggest is the closer to the medial wall of passage, and size is the least
The focused flow of granule further away from passage medial wall according to one embodiment of present invention, described microfluidic channel is crooked pipeline.
Thus, by using crooked pipeline, can realize cell is efficiently separated by producing inertia force.According to the present invention's
Preferred embodiment, the crooked pipeline used for selected from Archimedes spiral passage (as shown in Figure 4) and Psittacula alexandri fasciata helical passage (as
Shown in Fig. 5) at least one.Thus, it is possible to by making various sizes of cell be passed in passage, shape in bending channel
Become focused flow, and reserve in different outlets, it is achieved the separation that big minicell is different, such that it is able to it is slender to improve separation further
The efficiency of born of the same parents.
It addition, according to embodiments of the invention, it is also possible to it is magnetic according to cell band and separates.Cell band is magnetic, mainly
Being divided into two kinds, one is that self band is magnetic, and such as erythrocyte, owing to erythrocyte contains hemoglobin, hemoglobin is iron content egg
In vain, therefore erythrocyte has paramagnetism, it is possible to processed by deoxygenated, improves its magnetic；Another kind is can be with specificity
The capture antibody of purpose cell, aptamers etc. and magnetic microsphere connect, and by antigen antibody reaction, make specific cell have
Magnetic.Have magnetic cell by externally-applied magnetic field, magnetic cell can be separated with non-magnetic cell, such as figure
Shown in 6.According to one embodiment of present invention, described micro fluidic device is further provided with magnet.Thus, it is possible to based on carefully
Born of the same parents carry and are magnetic, and realize the separation to cell.According to embodiments of the invention, the position that arranges of magnet is not particularly restricted,
As long as in the range of enabling to the effective magnetic field that microfluidic channel is in magnet, according to embodiments of the invention, can be by magnetic
Body is arranged on the lower section of microfluidic channel, thus, it is possible to improve separation efficiency further.According to one embodiment of present invention,
The width of described microfluidic channel is 0.5 millimeter, a length of 50 millimeters.Thus, it is possible to it is single celled to improve separation further
Efficiency.
Micro fluidic device (because chip is the primary clustering realizing apparatus function, thus referred to herein as micro-fluidic chip),
Can be prepared from known compounds by those skilled in the known methods.The making material of micro-fluidic chip includes silicon, glass, quartz, gathers
Methyl methacrylate (poly (methylmethacrylate), PMMA), polystyrene (polystyrene), Merlon
(polycarbonate), polyethylene (polyethylene), silicone rubber (as polydimethylsiloxane (poly (dimethylsiloxane),
PDMS), epoxy resin etc..Seem silicon, glass, quartz etc., use the method for wet etching to carry out the processing of micro-structured channels；
When making, depth-width ratio is high, during the finer silicon of structure, needs the method using deep reaction ion etching to carry out adding of micro structure
Work；And make the high polymer material chip of heat curing-type, and such as PDMS, epoxy resin etc., it is typically necessary structure mould, available
Silicon, glass, optical cement, PDMS etc. make.The method making mould typically has photoetching, LIGA (Lithographie
Galvanoformung and abformung), etching, the processing of the technology such as Soft lithograph.Then the method utilizing cast makes micro-knot
Structure passage；And the high polymer of thermoplastics type, such as Merlon, PMMA etc., the method generally using hot pressing makes.Certain
A little high polymer materials, as PMMA, polystyrene etc. can use the method for laser ablation to make.
Determine whether biological sample has the method and system of hereditary variation
In a second aspect of the present invention, the present invention proposes a kind of method determining whether biological sample has hereditary variation.According to
Embodiments of the invention, the method can comprise the following steps:
First, utilize foregoing micro fluidic device, from biological sample separation cell sample, be previously with regard to micro fluidic device
Through being described in detail, do not repeating.It should be noted that be previously with regard to all features described by micro fluidic device and excellent
Point is all suitable for the method determining whether biological sample has hereditary variation.
It follows that expanding at least partially the hereditary material included in the cell sample separated, in order to obtain and expand
Volume increase thing.The term " hereditary material " used in this article should be interpreted broadly, its refer to included in cell sample can
To carry any material of its hereditary information, can be DNA can also be full-length genome for RNA, it is also possible to be to turn
Record group.According to one embodiment of present invention, described biological sample is selected from blood, body fluid, tissue sample and cell culture
At least one.Thus, it is possible to effectively separate unicellular from biological sample, thus improve the efficiency of subsequent analysis.According to
One embodiment of the present of invention, described cell sample is selected from erythroblast, tumor cell, embryonic stem cell, immunocyte
At least one.The most described cell sample is selected from least one of fetal nucleated red blood and circulating tumor cell.Thus,
Effectively can separate the biological specimen of particular source unicellular detecting.According to one embodiment of present invention, right
The amplification that carries out at least partially of the hereditary material included in cell sample separated farther includes: to the cell separated
Sample cracks, in order to discharge the hereditary material of described cell sample；And the hereditary material discharged is expanded, with
Just described amplified production is obtained.Thus, it is possible to effectively from the cell amplification hereditary material separated.According to the present invention one
Embodiment, use lytic reagent the cell sample separated is cracked, wherein, described lytic reagent be selected from potassium hydroxide,
At least one of sodium hydroxide, SDS and E.C. 3.4.21.64.Thus, it is possible to improve the efficiency of amplification hereditary material further.Separately
Outward, it is also possible to by physical method, cell sample is cracked, such as, cell sample is placed in acoustic wavefield, makes cell split
Solve；By cell is heated, cell is made to crack.In order to make low-abundance nucleic acid in individual cells meet the need of follow-up order-checking
Ask, need it is carried out full-length genome and the extraction of transcript profile and amplification.The extracting method of individual cells nucleic acid is various, can adopt
The genome in individual cells or mRNA is extracted with commercial kit；Can also be according to MDA or transcript amplification etc.
Strategy in technology, directly carries out the amplification of genome or transcript profile after cracking, and without carrying out the extraction of nucleic acid；Or adopt
By micro-fluidic technology, such as the method such as liquid-solid extraction, liquid-liquid extraction, extract genome or transcript profile；And unicellular full genome
The main method of group or full transcript profile amplification is MDA, DOP-PCR, cDNA exponential amplification and gathers based on T7RNA
The technology such as the linear amplification of synthase.Thus, according to one embodiment of present invention, described hereditary material is described cell sample
Unicellular full-length genome.Thus, it is possible to effectively by unicellular full-length genome is checked order, and determine the heredity of sample
Information.According to one embodiment of present invention, it is anti-by the amplification of PCR-based for expanding the hereditary material discharged
Should carry out with at least one of isothermal amplification reactions.Preferably, the hereditary material discharged is carried out amplification be by MDA,
At least one of DOP-PCR, cDNA exponential amplification and RNA linear amplification based on t7 rna polymerase is carried out.By
This, can improve the efficiency expanding full-length genome further.
After obtaining amplified production, obtained amplified production is checked order, in order to obtain sequencing result.According to the present invention
An embodiment, described amplified production is carried out order-checking and farther includes: for described amplified production, build sequencing library；
And described sequencing library is checked order, in order to obtain described sequencing result.Thus, it is possible to effectively amplified production is carried out
Order-checking.According to one embodiment of present invention, utilize selected from second filial generation high-flux sequence platform and third generation high-flux sequence platform
At least one is checked order in described genome sequencing library described sequencing library is checked order.Thus, it is possible to further
Improve order-checking efficiency.Order-checking at present mainly has two big classes, and a class is the high throughput sequencing technologies of the second filial generation, public including Illumina
The Gemone Anayzer system (i.e. Solexa sequenator, then develop into again HisSeq2000 system) of department, ABI company
The GS-FLX system of Solid system and Roche454 company.Second filial generation sequencing technologies all has its normal process, according to standard
Flow process checks order；Another kind of, then be third generation sequencing technologies, i.e. single-molecule sequencing technology, true including Helicos company
Real single-molecule sequencing technology, the real-time sequencing technologies of unimolecule of Pacific Biosciences company, and Oxford Nanopore
The nano-pore sequencing technologies etc. of Technologies company.Such technology can be directly to DNA, RNA, microRNA, albumen
Equimolecular direct Sequencing, it is not necessary to the nucleic acid in unicellular is carried out the amplification of full-length genome or transcript profile, more intuitively reaction
The quantity of DNA, RNA and the change of sequence in individual cells.
Finally, based on described sequencing result, determine whether described biological sample has hereditary variation.By the side according to the present invention
Method, it is possible to effectively separate unicellular by micro fluidic device according to embodiments of the present invention, such that it is able to based on to single celled
Hereditary material such as full-length genome checks order, and effectively obtains single celled hereditary material by being analyzed sequencing result
Whether full-length genome such as exist exception, so that it is determined that whether biological sample has hereditary variation.A reality according to the present invention
Executing example, described hereditary variation is selected from single nucleotide polymorphism, copy number variation, genome structure variation, alternative splicing, difference
At least one of different expression and transcript variation.According to one embodiment of present invention, based on described sequencing result, determine described
Whether biological sample has hereditary variation farther includes: compared with reference to genome by described sequencing result；And based on
Comparison result, determines whether described biological sample has hereditary variation.Thus, it is possible to improve whether determine biological sample further
There is the efficiency of hereditary variation.The data obtained based on high throughput sequencing technologies, use short sequence alignment tools (such as SOAP)
By sequencing data comparison to reference on genome, and then the base of purpose cell can be obtained according to the qualitative of comparison result and quantitative parameter
Because of stack features.The means of numerical analysis that genomic DNA order-checking is commonly used has: single nucleotide polymorphism (SNP) is analyzed, copy
Number variation (CNV) is analyzed, genome structure variation (SV) analysis etc.；Transcript profile RNA order-checking common analysis has: base
Because differential expression (DGE) is analyzed, alternative splicing (AS) detects, gene fusion detection etc.；For cell colony sequencing data
Cell development and evolutionary analysis can also be carried out.Thus, by the data of cell separated are compared, correlation analysis, no
Only it will be seen that the generation of cell, development, evolutionary process, and can be that gene medical diagnosis provides Research foundation, as to
Have or potential cancer, complex disease carries out examination or qualification, it is achieved the early discovery of disease, the purpose of early treatment.
In a third aspect of the present invention, the present invention proposes a kind of system determining whether biological sample has hereditary variation, and it is special
Levy and be to include: micro fluidic device, amplification device, sequencing device and analytical equipment.Wherein, micro fluidic device is for from biology
Sample separation cell sample；Amplification device is connected with micro fluidic device, and is suitable to included in the cell sample separated
Expanding at least partially of hereditary material, in order to obtain amplified production；Sequencing device is connected with amplification device, and is suitable to
Obtained amplified production is checked order, in order to obtain sequencing result；And analytical equipment is connected with sequencing device, and fit
In based on obtained sequencing result, determine whether biological sample has hereditary variation.Utilize determination according to embodiments of the present invention
Whether biological sample has the system of hereditary variation, can effectively implement foregoing method, it is possible to effectively by basis
The micro fluidic device of the embodiment of the present invention separates unicellular, such that it is able to enter based on to single celled hereditary material such as full-length genome
Whether row order-checking, obtain existing in single celled hereditary material such as full-length genome by being analyzed sequencing result effectively
It is abnormal, so that it is determined that whether biological sample has hereditary variation.According to one embodiment of present invention, amplification device wraps further
Include: cracking unit, described cracking unit is provided with lytic reagent or extra power device, in order to use lytic reagent or outer
The cell sample separated is cracked by portion's energy, discharges the hereditary material of described cell sample；And amplification unit, described
Amplification unit is connected with described cracking unit, and is suitable to the hereditary material to being discharged and expands, in order to obtain described amplification
Product.Thus, it is possible to effectively hereditary material is expanded.According to one embodiment of present invention, determine that biological sample is
Lytic reagent described in the no system with hereditary variation is selected from potassium hydroxide, sodium hydroxide, SDS and E.C. 3.4.21.64 at least
One, described extra power is optical, electrical and hot at least one.According to one embodiment of present invention, described amplification unit is fitted
In carrying out MDA, DOP-PCR, cDNA exponential amplification and RNA linear amplification based on t7 rna polymerase at least
A kind of.Thus, it is possible to effectively unicellular full-length genome is expanded.According to one embodiment of present invention, described order-checking
Device farther includes: library construction unit, and described library construction unit, for for described amplified production, builds sequencing library；
And order-checking unit, described order-checking unit is connected with described library construction unit, and is suitable to check order described sequencing library,
To obtain described sequencing result.Thus, it is possible to effectively amplified production is checked order.According to one embodiment of present invention,
Described order-checking unit is selected from least one of second filial generation high-flux sequence platform and third generation high-flux sequence platform.Thus, may be used
Order-checking efficiency is improved with further.According to one embodiment of present invention, analytical equipment farther includes: comparing unit, described
Comparing unit is for comparing described sequencing result with reference to genome；And variation determines that unit, described variation determine list
First and described comparing unit is connected, and is suitable to based on comparison result, determines whether described biological sample has hereditary variation.By
This, can improve the efficiency determining whether biological sample has hereditary variation further.
It should be noted that above for micro fluidic device with for determining whether biological sample has the spy of the method for hereditary variation
Advantage of seeking peace is equally applicable to this system, repeats no more.
Below in conjunction with embodiment, the solution of the present invention is explained.It will be understood to those of skill in the art that the following examples
It is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.In embodiment, unreceipted concrete technology or condition, press
(such as write with reference to J. Pehanorm Brooker etc., " the molecule that yellow training hall etc. is translated according to the technology described by the document in this area or condition
Cloning experimentation guide ", the third edition, Science Press) or carry out according to product description.Agents useful for same or instrument are unreceipted
Production firm person, be can by city available from conventional products, such as can purchase from Illumina company.
The foundation of conventional method low abundance cell screening model based on high-flux sequence
The design of 1.1 micro-fluidic chips
1.1.1 the unicellular separation of micro-fluidic chip and enrichment unit
Due to complexity and the rareness of purpose cell of sample, the present embodiment and is given birth at physics, chemistry according to dissimilar cell
Difference in thing characteristic designs the microchannel that cell carries out multistep separation on chip, unicellular for obtaining final purpose.
According to the physicochemical property of purpose cell, design has the micro-fluidic chip of different structure, and the selectivity carrying out purpose cell divides
From and enrichment.If the size of cell to be separated is clearly distinguished from other cells, can set in the passage of micro-fluidic chip
Count the micro structures such as various structure such as column, meander channel, pectination, weir shape, sieve-like, only allow the cell of certain size size to lead to
Cross, or retain the cell of certain size size, separate cell or utilize the principle of definitiveness lateral displacement, at passage
The barrier of interior design certain intervals size, different size of cell, by colliding with barrier, enters into different liquid stream, reaches
To the purpose separating different size cell；Utilize hydromechanical principle, according to the difference of different size cell particle, it is achieved thin
The separation of born of the same parents.According to inertia, design bending channel, control liquid stream, it is achieved the migration of different size cell and separate.If it is thin
Born of the same parents can deform, and such as erythrocyte, fetal nucleated red blood, can design the barrier of different spacing, make obstacle spacing little
In erythrocyte or the diameter of fetal nucleated red blood, by deformable cell and non-deformable cell (leukocyte, tumor cell
Deng) separate.
According to the difference of cell physical property, various field can be set up on chip, carry out cell such as electric field, magnetic field, sound wave etc.
Separation.Such as dielectrophoresis, the electric field of different frequency can be added outside chip channel, by regulating the spatial distribution of electric field, meeting
Different types of cell is made to be produced drift in various degree by polarization in various degree, it is achieved the separation of cell, or profit
Forming conductivity gradient with dielectrophoresis, drive cell to move to the direction that electrical conductivity is low, until balance, electrical conductivity is 0, then
Gravity is utilized to be dragged to by cell in purpose passage, it is achieved cell separation.Utilize magnetic field separation, similar with MACS, at magnetic
Bead surface connects the biomolecule having specific capture cell, is combined by immuno-chemical reaction, utilizes controllable magnetic field,
The cell of capture is separated；Or utilize the magnetic that some cell has self, as erythrocyte and leukocyte paramagnetism and
Diamagnetism realizes the separation of cell.Utilize the difference of the node characteristic stress of sound wave, according to the size of cell, density, proportion etc.
Character, it is achieved the separation of cell.
Owing to cell can express a lot of specific surface molecular, according to its immunochemistry character, connecting in passage can be with
The molecule that cell surface marker molecule is combined such as antibody, aptamers etc., carry out the capture of specific cell and/or non-spy
The removal of specific cell.Can also carry out the sorting of immunomagnetic beads in passage, on magnetic bead, corresponding connection has specificity to capture purpose
Cell or the biomolecule of removal non-specific cell, it is achieved the separation of specific cell and enrichment.
1.1.2 the cell manipulation unit of micro-fluidic chip
The requirement that integration according to overall chip separates with progenitor cells, can select different control modes.Liquid stream can be utilized
With the control of micro-valve, manipulate the opening and closing of micro-valve, entrapped cell.If early stage uses magnetic bead to carry out the sorting of cell, can pass through
The control in magnetic field, optionally manipulates individual cells (few cells) to the conversion zone specified.Can add on manipulation passage
Upper electric field, utilizes the mode of EOF or electrophoresis to be driven in reaction tank by individual cells (few cells).Design special knot
The passage of structure, can only retain individual cells (few cells).Capillary tube can also be utilized, make cell carry out regularly arranged,
Mobile by display, individual cells (few cells) is driven into the conversion zone specified.These control passage and can collect
Become, it is achieved the manipulation of the most multiple unicellular (few cells).
1.1.3 the unicellular nucleic acid extraction of micro-fluidic chip and amplification unit
Nucleic acid extraction and amplification unit can be in the micro reaction pool of certain regular texture, it is also possible to by droplet (drop)
Individual cells (few cells) is wrapped up, forms the most independent cell as reaction tank.Intracellular in order to extract
DNA or RNA, we can put into surface-functionalized magnetic bead in extraction unit, pass through magnetic bead sorting, it is thus achieved that need
Few cells DNA molecular or RNA molecule, by control magnetic bead, drive extract DNA or RNA molecule flow out
Outlet, enters amplification unit.As the nucleic acid molecules collected is put in PCR pipe, carry out the WGA of standard or entirely turn
Record this amplification procedure.Can also directly utilize droplet technology, individual cells (few cells) is wrapped in droplet (drop)
In, drop directly wraps up such as PCR reactant liquor, after cracking, in droplet, directly carries out amplified reaction.
1.2 high-flux sequence
The amplified production obtained, reserves outlet by drive system, collects reactant liquor.Normal stream according to second filial generation sequencing technologies
Journey, interrupts nucleic acid molecules, builds storehouse, then checks order.
Have for DNA and RNA and different build storehouse mode.If DNA, the DNA fragmentation of acquisition can be beaten at random
It is broken into small fragment, adds given joint, be prepared as DNA library, cBot carries out cluster amplification, directly to DNA sheet
The mono-end of Duan Jinhang or the order-checking of double end.If that obtain is RNA, isolates mRNA, be prepared as the cDNA of fragmentation
Library, carries out cluster amplification on cBot, is used further to high pass sequencer.
Three kinds of technology are currently mainly used to carry out high-flux sequence, Gemone Anayzer system (the i.e. Solexa of Illumina company
Sequenator, after develop into again HisSeq2000 system), the Solid system of ABI company and the GS-FLX of Roche454 company
The big high throughput sequencing technologies of system three.And along with the development of science and technology, it will application third generation sequencing technologies, i.e. single-molecule sequencing
Technology, true single-molecule sequencing technology, the unimolecule of Pacific Biosciences company including Helicos company check order in real time
Technology, and the nano-pore sequencing technologies etc. of Oxford Nanopore Technologies company.
1.3 data analysis
Based on high-flux sequence result, use short sequence alignment tools (such as SOAP) by sequencing data comparison to reference to genome
On, and then the genome signature of purpose cell can be obtained according to the qualitative of comparison result and quantitative parameter.
The means of numerical analysis that genomic DNA order-checking is commonly used has: single nucleotide polymorphism (SNP) is analyzed, and copy number makes a variation
(CNV) analyze, genome structure variation (SV) analysis etc.；Transcript profile RNA order-checking common analysis has: gene difference
Expressing (DGE) to analyze, alternative splicing (AS) detects, gene fusion detection etc.；Can also enter for cell colony sequencing data
Row cell development and evolutionary analysis.
By to separate sample compare, correlation analysis, be possible not only to understand the generation of cell, development, evolutionary process,
And can be that gene medical diagnosis provides Research foundation, as existing or potential cancer, complex disease are carried out examination or
Identify, it is achieved the early discovery of disease, the purpose of early treatment.
The heritability of embodiment 1 fetal nucleated red blood is identified
The ulnar vein peripheral blood of the anemia of pregnant woman in 1.15mL pregnancy 8-14 week, EDTA-K2 anticoagulant, 4 DEG C of vibrations preserve, in 24h
Test.
The separation of 1.2 fetal nucleated red bloods: use two steps to separate.First according to different the carrying out of red white corpuscle size
Separate, such as the chip structure in 2.1, remove most erythrocyte；The cell of recovery is passed in Magnetic Isolation chip,
The difference utilizing the magnetic of red white corpuscle separates.
The structure of the first chip: the spacing between microtrabeculae is 15 μm, and often row microtrabeculae offsets 6.75 μm relatively.Use deep reaction from
Sub-lithographic technique builds micro structure on silicon chip, and channel depth is 150 μm, uses sheet glass to seal.
The structure of the second chip: use Miltenyi LS Column (Miltenyi Bio Tech), external 1.4T Magnet.
Using external drive equipment that with the speed of 100 μ L/min, blood and buffer are passed into first chip, buffer is
IDPBS (containing 1%BSA, 2mM EDTA).Cell is reclaimed from exit.The cell reclaimed passes through centrifugal 2000rpm,
15min.Sample 50mM NaNO2 processes 10min.Then pass in Miltenyi LS Column.Use iDPBS
Slowly rinse, wash away unadsorbed leukocyte.Then remove Magnet, use wash buffer, reclaim and have magnetic cell.
Use FITC-epsilon-globulin labeled cell, the erythroblast that picking is single under fluorescence microscope, and be transferred into
In microwell array.
1.3 cell cracking: use NaOH cell lysis.
The extraction of 1.4 nucleic acid and amplification: directly use REPLI-g Mini/Midi kits (Qiagen) to carry out unicellular full-length genome expansion
Increase.
1.5 order-checkings: whole genome amplification product, through building storehouse, checks order by Illumina technology.
1.6 data analysiss: carry out single nucleotide polymorphism (SNP) and analyze, carry out single-gene site or polygenic locus is dashed forward
Variation is analysed.
Embodiment 2: the chromosome aneuploid of fetal nucleated red blood is abnormal to be identified
2.1 samplings: with 1.1
The separation of 2.2 fetal nucleated red bloods:
The structure of the first chip used: such as the nautilus chip in Fig. 5, have an injection port and 6 outlets, entrance
Channel width is 0.22mm, and passage bending curvature the most gradually amplifies, and the channel width in exit is 3.88
mm.Chip channel configuration su-8 bears optical cement and makes mask through photoetching process, then the mixing of PDMS prepolymer is cast on mould,
Form through overmolded.Sheet glass is used to seal.
The structure of the second chip used: such as Fig. 6.There are two entrances and two outlets.Channel width is 0.5mm, length
For 50mm.Chip channel configuration su-8 bears optical cement and makes mask through photoetching process, then the mixing of PDMS prepolymer is cast in
On mould, form through overmolded.Sheet glass is used to seal.In the side of passage plus external magnet, it is built into magnetic core
Sheet.
Two steps are used to separate.The first step uses the first chip to remove most erythrocyte；Again by containing erythroblast
Cell mixing sample is passed into the second chip, utilizes the difference of red white corpuscle magnetic to separate.
Using external drive equipment micro-injection pump, the blood driving diluted concentration to be 10% (uses the iDPBS containing 1%BSA
Buffer dilutes) it is passed in passage, flow velocity is 300 μ L/min.Cell solution is collected in exit.The cell reclaimed passes through
Centrifugal 2000rpm, 15min.Sample 50mM NaNO2 processes 10min.Cell is passed into chip from sample inlet
In, another entrance is passed through iDPBS buffer simultaneously, and the two flow velocity is 10 μ L/min.Purpose cell is reclaimed in exit.
Cell is through FITC-CD71 antibody staining, and the single positive cell of picking carries out subsequent experimental.
2.3 cell cracking: use KOH/DTT cell lysis.
The extraction of 2.4 nucleic acid and amplification: directly use DOP-PCR (GenomePlex Single Cell Whole Genome
Amplification Kit, Sigma) method carries out the amplification of individual cells genome.
2.5 order-checkings: whole genome amplification product, through building storehouse, checks order by Illumina technology.
2.6 data analysiss: carry out copy number variation (CNV) and analyze, genome structure variation (SV) analysis etc..
Embodiment 3: cancer patient's circulating tumor cell (CTCs) examination
3.1 samplings: the ulnar vein peripheral blood 10mL of extraction tumor patient, EDTA-K2 anticoagulant.Blood sample 4 DEG C preservation, 24h
Inside detect.
The separation of 3.2CTCs: use two step chips to separate.The first step separates according to the size of cell, in Fig. 1
Chip structure, remove most erythrocyte and part outer cell；Second step uses EpCAM antibody capture CTCs cell.
Specifically,
The structure of the first chip: the spacing between microtrabeculae is 20 μm, and often row microtrabeculae offsets 6 μm relatively.Use deep reactive ion is carved
Erosion technology builds micro structure on silicon chip, and channel depth is 150mm, uses sheet glass to seal.
The structure of the second chip: use Miltenyi LS/MS Column (Miltenyi BioTech), captures CTCs.
Using external drive equipment that with the speed of 100 μ L/min, blood and buffer are passed into first chip, buffer is
IDPBS (containing 1%BSA, 2mM EDTA).Cell is reclaimed from exit.The cell reclaimed is through centrifugal 300g, 10min.
Then connection have the magnetic bead of CD326 (EpCAM) antibody hatch with mixing with cells.IDPBS is used to rinse.By cell and magnetic
Pearl mixture is passed in Miltenyi LS/MS Column.Use iDPBS slowly to rinse, wash away unadsorbed leukocyte.
Then remove Magnet, use wash buffer, reclaim and have magnetic cell.And CTCs cell is counted.
3.3 cell cracking: use lysate KOH or NaOH cell lysis.
The extraction of 3.4 nucleic acid and amplification: the few cells collected is carried out whole genome amplification according to the method for MDA.
3.5 order-checkings: whole genome amplification product, through building storehouse, checks order by Illumina technology.
3.6 data analysiss: carry out single nucleotide polymorphism (SNP) and analyze, carry out single-gene site or polygenic locus is dashed forward
Variation is analysed.By comparing separating sample and other normal cells, cancer solid tumor tissue, the relevant single celled hereditary variation of cancer
Analyzing, early discovery, early diagnosis and aftertreatment for later cancer provide evidence.
The heritability of embodiment 4 fetal nucleated red blood is identified
4.1 with 1.1
4.2 with 1.2
4.3 with 1.3
The extraction of 4.4 nucleic acid: use magnetic bead (Invitrogen, Dynabeads) method extracting directly unicellular in genome.
4.5 order-checkings: directly use Oxford Nanopore Technologies to carry out single-molecule sequencing.
4.6 data analysiss: carry out single nucleotide polymorphism (SNP) analysis, copy number variation (CNV) is analyzed, and genome is tied
Structure variation (SV) analysis etc.
Automatization's examination of circulating tumor cell (CTCs) in embodiment 5 sample
Sampling: peripheral blood 10mL, EDTA-K2 anticoagulant, 4 DEG C of vibrations preserve, and test in 24h.
Use micro-injection pump that blood and PBS are passed through in the way of laminar flow the separation and concentration unit of micro-fluidic chip,
Being built with regularly arranged cylindrical microtrabeculae in passage, microtrabeculae spacing is 16 μm, hemocyte by colliding with microtrabeculae, according to
Erythrocyte less for size is separated by cell size, and larger-size leukocyte enters into next split tunnel.Passage is built-in
Have to connect and have the immunomagnetic beads of anti-EpCAM antibody molecule, immunomagnetic beads by with the collision of leukocyte that flows in passage,
Capture the CTCs of cell surface specific expressed EpCAM molecule.PBS rinses, and removes non-specific binding cell.Logical
Enter enzymolysis solution, CTCs is discharged from magnetic bead.
By fluid drives, make cell regularly arranged entrance passage, by the folding of air valve, by single CTC (trace CTCs)
It is trapped in different cells.
The erythrocyte separated and leukocyte are through controlling, and a small amount of cell enters into what respective only permission individual cells arrangement was passed through
Passage, is controlled by air valve and liquid stream, individual cells (few cells) is separated into independent cell, standby as comparison.
Controlled and air valve folding by fluid, promote individual cells (few cells) to flow out from respective outlet, be collected in respectively
In PCR pipe.The individual cells (few cells) collected carries out standard amplification according to the method for MDA.
Collect amplified production, DNA fragmentation is broken at random small fragment, adds that given joint sets up DNA fragmentation library,
Carry out cluster amplification on cBot, check order for Illumina Hiseq 2000.
Data analysis, based on high-flux sequence result detection cellular genome single nucleotide polymorphism and chromosome structure change, seeks
Look for potential cancer driving factors and candidate locus.Set up cancer cell development tree, review cancerous cell evo-devo process.By than
Compared with separating sample and other normal cells, cancer solid tumor tissue, the relevant single celled Genetic Variation Analysis of cancer, for later
The early discovery of cancer, early diagnosis and aftertreatment provide evidence.
The heritability of embodiment 6 fetal nucleated red blood is identified
The peripheral blood of sampling: the 5mL anemia of pregnant woman of conceived 14 weeks, EDTA-K2 anticoagulant, 4 DEG C of vibrations preserve, and try in 24h
Test.
Blood is passed in micro-magnetic current passage, under the effect of externally-applied magnetic field, according to erythrocyte, fNRBCs tool in hemocyte
There is paramagnetism, the diamagnetic principle of leukocyte, erythrocyte (including fNRBCs) is separated with leukocyte, respectively enters difference
Passage.
Erythrocyte (including fNRBCs) enters into and connects in the microtrabeculae passage having anti-CD71 antibody, is exempted from by antigen-antibody
Epidemic disease is reacted, and captures fNRBCs.FNRBCs after desorption, is manipulated it and enters in control passage from microtrabeculae.
Control fNRBCs entrance and only allow the unicellular passage passed through.Under the microscope, by liquid stream and the control of air valve, push away
Dynamic single fNRBC sequentially enters in array collecting pipe.
Control leukocyte and the red cell fraction entrance passage that only permission individual cells passes through separated in blood, by control
System, is forced into single leukocyte, living cells in array collecting pipe, uses as comparison.
Extract the cell RNA in array collecting pipe, carry out transcript according to linear amplification method based on t7 rna polymerase
Amplification, detailed process is with reference to Eberwine, J.et a1.Analysis of gene expression in single live neurons.Proc.
Natl.Acad.Sci.USA89,3010-3014 (1992). and Van Gelder, R.N.et a1.Amplified RNA synthesized
From limited quantities ofheterogeneous cDNA.Proc.Natl.Acad.Sci.USA87,1663-1667 (1990).
Carry out.
Collect amplified production cDNA, the library construction document provided according to Illumina, prepare library, cBot becomes
Bunch amplification, check order for Illumina Hiseq 2000.Data analysis.
Based on high-flux sequence data analysis fetus transcript profile feature, provide basis for medical treatment molecular diagnosis research.
The research of embodiment 7 circulating tumor cell expression
7.1 with 3.1
7.2 with 3.2
After obtaining single loop tumor cell, follow-up RNA extracts the reference Tang F such as amplification procedure and the primer reagent,
Barbacioru C, Nordman E, et a1.RNA-Seq analysis to capture the transcriptome landscape of a
single cell.Nature Protocols.2010；5 (3): 516-535. are carried out.
7.3 cell cracking: use NP40 cell lysis.
7.4 reverse transcriptions: be directly added into reverse transcription and primer carries out reverse transcription, synthetic dsdna.
7.5cDNA exponential amplification: carry out the reaction of first round PCR, uses QIAquick PCR purification kit to carry out pure
Change；After by first round PCR, the product of purification carries out second and takes turns amplification, uses QIAquick PCR purification kit
It is purified.Gel reclaims the fragment of the 0.5-3kb in PCR primer.
7.6 order-checkings: cDNA amplified production, through building storehouse, checks order by Illumina technology.
7.7 data analysiss: gained reads comparison of checking order, to reference to transcript, carries out transcript knot according to comparison gained information
Structure is studied, transcript variation research, and gene expression dose is studied, noncoding region functional study etc..Industrial applicibility
According to an embodiment of the invention micro fluidic device, determine whether biological sample has the method for hereditary variation and determine biology
Whether sample has the system of hereditary variation, it is possible to efficiently separate unicellular, so that it is determined that whether biological sample has heredity change
Different.
Although the detailed description of the invention of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to
Those details can be carried out various amendment and replacement by disclosed all teachings, these change all protection scope of the present invention it
In.The four corner of the present invention is given by claims and any equivalent thereof.
In the description of this specification, reference term " embodiment ", " some embodiments ", " illustrative examples ", " example ",
The description of " concrete example " or " some examples " etc. means to combine this embodiment or the specific features of example description, structure, material
Or feature is contained at least one embodiment or the example of the present invention.In this manual, the schematic table to above-mentioned term
State and be not necessarily referring to identical embodiment or example.And, the specific features of description, structure, material or feature can be
Any one or more embodiments or example combine in an appropriate manner.
Claims (5)
1. one kind separates single celled micro fluidic device from biological specimen, it is characterised in that described miniflow
Control device is for separating the erythrocyte in blood sample and/or erythroblast, and described micro fluidic device has
Have and be suitable to separate single celled microfluidic channel, described microfluidic channel is formed multiple microtrabeculae, described many
Individual microtrabeculae on described microfluidic channel in array distribution, in the vertical, the distance between adjacent two microtrabeculaes
It is 15 microns, and, in the horizontal, the distance between adjacent two microtrabeculaes is 15 microns, often goes micro-
Post offsets 6-7 micron relatively, and along flow direction, the diameter of described microtrabeculae increases.
Device the most according to claim 1, it is characterised in that described micro fluidic device sets further
Being equipped with magnet, the width of described microfluidic channel is 0.5 millimeter, a length of 50 millimeters.
3. one kind determines whether biological sample has the non-diagnostic method of hereditary variation, it is characterised in that bag
Include the following step:
Utilize the micro fluidic device described in any one of claim 1-2, from biological sample separation cell sample；
To expanding at least partially of the hereditary material included in the cell sample separated, in order to obtain
Obtain amplified production；
Described amplified production is checked order, in order to obtain sequencing result；And
Based on described sequencing result, determine whether described biological sample has hereditary variation.
Method the most according to claim 3, it is characterised in that described hereditary material is described cell
The unicellular full-length genome of sample or full transcript profile, based on described sequencing result, determine that described biological sample is
No have hereditary variation and farther include:
Described sequencing result is compared with reference to genome or reference transcript profile；And
Based on comparison result, determine whether described biological sample has hereditary variation.
5. one kind determines whether biological sample has the system of hereditary variation, it is characterised in that including:
Micro fluidic device described in any one of claim 1-2, described micro fluidic device is for from biological sample
Separate cell sample；
Amplification device, described amplification device is connected with described micro fluidic device, and is suitable to thin to separated
Expanding at least partially of hereditary material included in born of the same parents' sample, in order to obtain amplified production；
Sequencing device, described sequencing device is connected with described amplification device, and is suitable to described amplified production
Check order, in order to obtain sequencing result；And
Analytical equipment, described analytical equipment is connected with described sequencing device, and is suitable to based on described order-checking knot
Really, determine whether described biological sample has hereditary variation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109536590A (en) * | 2018-11-27 | 2019-03-29 | 中国科学院上海微系统与信息技术研究所 | A kind of unicellular gene tester based on microwell array chip |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148160B (en) * | 2015-03-27 | 2018-04-20 | 深圳华大基因科技服务有限公司 | Cell separation apparatus and cell isolation method |
| CN104991055A (en) * | 2015-06-19 | 2015-10-21 | 大连理工大学 | Blood sample time-delay flowing bionic control unit in thrombus POCT product |
| CN106047706B (en) * | 2016-06-15 | 2018-11-16 | 西北工业大学 | One kind realizing cellular localization culture chip and its use and preparation method based on unicellular capture |
| CN107262171A (en) * | 2017-07-12 | 2017-10-20 | 华讯方舟科技有限公司 | A kind of blood separation and culture chip and blood separating mechanism |
| CN107702973A (en) * | 2017-09-08 | 2018-02-16 | 深圳市太赫兹科技创新研究院有限公司 | A kind of whole blood blood plasma piece-rate system and method |
| CN111363795A (en) * | 2018-12-26 | 2020-07-03 | 清华大学 | Single cell whole genome sequencing method |
| CN109746064B (en) * | 2019-01-28 | 2021-10-15 | 武汉纺织大学 | Gradient magnetic field micro-fluidic chip |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004037374A2 (en) * | 2002-10-23 | 2004-05-06 | The Trustees Of Princeton University | Method for continuous particle separation using obstacle arrays asymmetrically aligned to fields |
| CN101086504A (en) * | 2006-06-06 | 2007-12-12 | 北京大学 | Microfluid centrifugal chip and its processing method |
| CN101305087A (en) * | 2005-09-15 | 2008-11-12 | 阿尔特弥斯康复公司 | Devices and methods for magnetic enrichment of cells and other particles |
| CN101643701A (en) * | 2009-07-23 | 2010-02-10 | 清华大学 | Cell sorter micro-fluidic chip based on immunomagnetic separation technology and application thereof in aspect of enrichment of rare cells |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19841835C2 (en) * | 1998-09-12 | 2003-05-28 | Fresenius Ag | Centrifuge chamber for a cell separator |
| US7390388B2 (en) * | 2004-03-25 | 2008-06-24 | Hewlett-Packard Development Company, L.P. | Method of sorting cells on a biodevice |
-
2012
- 2012-04-12 CN CN201280016505.6A patent/CN103620016B/en active Active
- 2012-04-12 WO PCT/CN2012/073958 patent/WO2012139517A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004037374A2 (en) * | 2002-10-23 | 2004-05-06 | The Trustees Of Princeton University | Method for continuous particle separation using obstacle arrays asymmetrically aligned to fields |
| CN101305087A (en) * | 2005-09-15 | 2008-11-12 | 阿尔特弥斯康复公司 | Devices and methods for magnetic enrichment of cells and other particles |
| CN101086504A (en) * | 2006-06-06 | 2007-12-12 | 北京大学 | Microfluid centrifugal chip and its processing method |
| CN101643701A (en) * | 2009-07-23 | 2010-02-10 | 清华大学 | Cell sorter micro-fluidic chip based on immunomagnetic separation technology and application thereof in aspect of enrichment of rare cells |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109536590A (en) * | 2018-11-27 | 2019-03-29 | 中国科学院上海微系统与信息技术研究所 | A kind of unicellular gene tester based on microwell array chip |
| CN109536590B (en) * | 2018-11-27 | 2021-10-29 | 中国科学院上海微系统与信息技术研究所 | Single cell gene detection method based on micropore array chip |
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| CN103620016A (en) | 2014-03-05 |
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