WO2012139517A1 - 微流控装置及其用途 - Google Patents

微流控装置及其用途 Download PDF

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Publication number
WO2012139517A1
WO2012139517A1 PCT/CN2012/073958 CN2012073958W WO2012139517A1 WO 2012139517 A1 WO2012139517 A1 WO 2012139517A1 CN 2012073958 W CN2012073958 W CN 2012073958W WO 2012139517 A1 WO2012139517 A1 WO 2012139517A1
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Prior art keywords
cells
sequencing
cell
microfluidic
biological sample
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PCT/CN2012/073958
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English (en)
French (fr)
Inventor
王琳琳
刘笔锋
吴逵
侯勇
陈璞
董迎松
宋卢挺
徐迅
Original Assignee
深圳华大基因科技有限公司
深圳华大基因研究院
华中科技大学
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Application filed by 深圳华大基因科技有限公司, 深圳华大基因研究院, 华中科技大学 filed Critical 深圳华大基因科技有限公司
Priority to CN201280016505.6A priority Critical patent/CN103620016B/zh
Publication of WO2012139517A1 publication Critical patent/WO2012139517A1/zh

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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
    • 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
    • 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
    • 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
    • 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
    • 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

Definitions

  • the present invention relates to a microfluidic device and its use, and in particular to a microfluidic device, a method of determining whether a biological sample has genetic variation, and a system for determining whether a biological sample has genetic variation.
  • Microfluidic chip technology originated from the field of analytical chemistry. It integrates basic operation units such as preparation, reaction, separation and detection of samples involved in biological, medical and chemical fields into several square centimeter chips through micro-machining technology. A network formed by microchannels with controlled fluids throughout the system to replace the various functions of conventional analytical laboratories. Microfluidic technology has shown great advantages in cell separation, nucleic acid extraction, purification, and PCR amplification.
  • the present invention aims to solve at least one of the technical problems existing in the prior art.
  • the invention proposes a microfluidic device.
  • the microfluidic device has a microfluidic channel adapted to separate single cells.
  • the use of a microfluidic device in accordance with an embodiment of the present invention can effectively separate single cells from a biological sample.
  • the invention provides a method for determining whether a biological sample has genetic variation, characterized in that it comprises the steps of: separating a cell sample from a biological sample using the microfluidic device described above; At least a portion of the genetic material contained in the isolated cell sample is amplified to obtain an amplification product; the amplification product is sequenced to obtain a sequencing result; and the biological sample is determined based on the sequencing result Whether it has genetic variation.
  • the invention provides a system for determining whether a biological sample has genetic variation, characterized by comprising: a microfluidic device as described above, said microfluidic device for separating from a biological sample a cell sample; an amplification device coupled to the microfluidic device and adapted to amplify at least a portion of the genetic material contained in the isolated cell sample to obtain an amplification product; sequencing a device, the sequencing device being coupled to the amplification device and adapted to sequence the amplification product to obtain a sequencing result; and an analysis device coupled to the sequencing device and adapted to be based on The sequencing result determines whether the biological sample has genetic variation.
  • the aforementioned method can be effectively implemented, and the single cell can be efficiently separated by the microfluidic device according to the embodiment of the present invention, so that it can be based on the pair
  • the genetic material of the cell such as a whole genome, is sequenced, and it is effectively determined whether the biological sample has genetic variation by analyzing the sequencing result to obtain whether a single cell genetic material such as an entire genome has an abnormality.
  • the present invention is directed to a low-abundance cell acquisition model for low-abundance cell acquisition, and a low-abundance cell screening model based on high-throughput sequencing is established, and the parameters of the model are adjusted by bioinformatics simulation to demonstrate the The detection domain, sensitivity, accuracy, and reproducibility of the model in screening for low-abundance cells.
  • the present invention first establishes an automated microfluidic cell sorting model, and then uses amplification techniques and next generation sequencing techniques to generate a certain number of specific sequence data for effective screening of different variability of low abundance cells.
  • the present invention demonstrates a method model for automated screening of low abundance cell variability information, the experimental procedure of which is: (a) the purpose of allowing the sample to enter the microfluidic device, separating and enriching the sample through multiple microchannels or microchambers. Single cell; (b) adding a lysis reagent to a microchannel or microchamber on a microfluidic chip or a single cell obtained by a thermal cleavage step (a) under light, using a cleavage product DNA or RA as a template for expansion (c) structuring the amplification product obtained in step (b) and sequencing on a high-throughput sequencing platform including, but not limited to, Illumina/Solexa, ABI Solid, and Roche 454; (d) processing the sequencing data obtained in step (c).
  • microfluidic device for automatically screening for low abundance cell variability
  • the microfluidic device comprising at least one inlet and outlet interconnected by microchannels or microchambers,
  • the flow control chip, the drive system and the detection system are composed.
  • the microfluidic chip of the microfluidic device comprises a cell separation and enrichment unit and a cell manipulation unit.
  • the cell separation and enrichment unit is provided with a structure or an obstacle
  • the cell manipulation unit is provided with a liquid flow and a gas flow control.
  • the microfluidic chip of the microfluidic device further comprises a DNA and/or R A extraction and amplification unit.
  • the method for separating and enriching low-abundance cells according to the microfluidic device comprises flowing a sample through a microchannel or a micro-chamber of the microfluidic chip, using a microchannel Or magnetic beads in the micro-chamber capture target cells.
  • the method of lysing cells according to the microfluidic device comprises flowing a sample through a microchannel or a microchamber of the microfluidic chip, using magnetic beads captured in a microchannel or a microchamber The target cells, as well as the lysing reagent or the magnetic beads to generate heat under the light, lyse the cells.
  • the method for amplifying a target DNA or RA from a cell according to the microfluidic device comprises flowing a sample through a microchannel or a microchamber of the microfluidic chip, using a microchannel or The magnetic beads in the micro-chamber capture the target cells, add the lysis reagent or blast the cells under the light to generate heat, lyse the cells, extract the target DNA or RA, and amplify with the target DNA or RA as a template; Genomic amplification.
  • the present invention establishes a technical model of low-abundance cell screening based on microfluidic technology and high-throughput sequencing, and demonstrates the detection domain, sensitivity, accuracy, and feasibility of the model. Reproducibility enables high-throughput automated screening of genetic information from low-abundance cells in complex samples, enabling high-throughput automated labor in the laboratory, providing the basis for medical diagnostic research in genetics.
  • the present invention designs a microfluidic chip having a microchannel or a micro chamber, which integrates the functions of separating, enriching and culturing a small amount of cells and extracting and amplifying DNA or RA in a cell, thereby reducing The cost of the experimental device is easy to promote.
  • FIG. 1 to 6 show schematic views of the structure of a microfluidic device according to an embodiment of the present invention. Detailed description of the invention
  • first and second are used for descriptive purposes only, and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, features defining “first”, “second” may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, “multiple” means two or more unless otherwise stated.
  • low-abundance cells single cells
  • micro-cells are used interchangeably herein to refer to cells containing a desired type in a complex sample, but in small amounts, generally only One to several.
  • the invention proposes a microfluidic device.
  • the microfluidic device has a microfluidic channel adapted to separate single cells.
  • the use of a microfluidic device in accordance with an embodiment of the present invention can effectively separate single cells from a biological sample.
  • the form of the microfluidic channel suitable for separating single cells in the microfluidic device is not particularly limited.
  • one-step or multiple-step separation of cells can be designed and fabricated on the chip according to the differences in physical, chemical and biological properties of different types of cells.
  • Single cell microchannels are isolated to obtain the final target single cell. It is to be noted that a combination of various methods suitable for separating single cells can be employed, and the order is not particularly limited as long as separation of single cells can be achieved.
  • the mixed sample cells can be first introduced into the first microfluidic channel for preliminary separation to remove most of the interfering cells; and then the initially separated cells are passed to the second ⁇ : flow control channel In the middle, further enrichment of the cells of interest is performed.
  • the microfluidic channel can be based on the physical, chemical properties of the cell, such as size, shape, deformability, cell surface properties (eg, cell surface receptors, antigen, membrane permeability), intracellular properties (eg expressing a protein, specific enzyme, etc.) to achieve separation of single cells.
  • cell surface properties eg, cell surface receptors, antigen, membrane permeability
  • intracellular properties eg expressing a protein, specific enzyme, etc.
  • a plurality of microcolumns are formed on the microfluidic channel.
  • a column also referred to as an obstacle
  • it acts as a barrier to the flow of cells in the fluid direction, so that separation of single cells can be achieved based on the difference in cell size.
  • regularly arranged and spaced-sized obstacles may be constructed in the microfluidic channel (the form of the obstacle is not particularly limited and may be a cylinder, Elliptical, square, etc.), a fixed amount of lateral displacement occurs between each row of obstacles. Referring to FIG.
  • the obstacles arranged by the internal micro-fluidic chip channel are arranged in an orderly manner (for example, a plurality of ⁇ :columns are arranged in an array on the ⁇ :fluidic channel;), the cells to be separated are used as particles in the fluid.
  • the cell When entering the channel, it collides with the obstacle.
  • the cell When the cell is larger than a certain size (related to the spacing between the obstacles), the cells are offset according to a certain angle (related to the lateral displacement between each row of obstacles), less than the Cells of the size flow according to the original fluid trajectory to achieve separation of cells of different sizes (Fig. 1).
  • the obstacle spacing such as the distance between the micropillars, including the lateral spacing and the longitudinal spacing, can be adjusted depending on the size of the cells to be separated.
  • the distance between adjacent two microcolumns is 10 to 100 micrometers in the longitudinal direction, and, in the lateral direction, the distance between adjacent two microcolumns is 10 to 100 micrometers.
  • each row of microcolumns is relatively offset by 6-7 microns.
  • the obstacle array structure can be set to be single-stage or multi-stage. Depending on the environment of the cells to be separated, such as separating fixed-size cells from body fluids, a single-stage column array can be realized, and complex samples like blood can be realized.
  • the diameter of the column is increased in the direction of the fluid. Thereby, the efficiency of separating single cells can be further improved.
  • an obstacle array of a plurality of different pitch sizes can be established to further refine the separation of cells of different sizes. Cells of different sizes are collected at the exit for further isolation or for subsequent testing.
  • the diameter of the microcolumns is constant along the direction of the fluid.
  • the steady flow of the fluid has a parabolic distribution, the flow velocity in the middle of the channel is the largest, the centrifugal force is the largest, and the fluid flow rate near the channel wall is the smallest.
  • eddy currents are generated. The particles in the fluid are thus interacted by buoyancy and Dean forces. At a certain flow rate, a focused flow is formed.
  • the microfluidic channel is a curved tube.
  • the curved conduit used is at least one selected from the group consisting of an Archimedean spiral channel (as shown in Figure 4) and a Nautilus spiral channel (shown in Figure 5).
  • the cells are magnetic, mainly divided into two types, one is magnetic with itself, such as red blood cells, because red blood cells contain hemoglobin, hemoglobin is ferritin, so red blood cells have paramagnetic properties, and can be deoxidized to improve their magnetic properties;
  • the other is to connect antibodies, aptamers, etc., which can specifically capture the target cells, with magnetic microspheres, and to make specificity through antigen-antibody reaction.
  • the cells are magnetic. Magnetic cells can separate magnetic cells from non-magnetic cells by applying a magnetic field, as shown in Figure 6.
  • the microfluidic device is further provided with a magnet.
  • the arrangement position of the magnet is not particularly limited as long as the microfluidic channel can be made to be within the effective magnetic field range of the magnet, and the magnet can be placed in the microfluidic channel according to an embodiment of the present invention.
  • the separation efficiency can be further improved.
  • the microfluidic channel has a width of 0.5 mm and a length of 50 mm. Thereby, the efficiency of separating single cells can be further improved.
  • Microfluidic devices since the chip is a major component that implements device functionality, and thus also referred to herein as a microfluidic chip, can be prepared by methods known to those skilled in the art.
  • the microfluidic chip is made of silicon, glass, quartz, poly(methylmethacrylate), PMMA, polystyrene, polycarbonate, and polyethylene.
  • Silicone rubber such as poly(dimethylsiloxane), PDMS), epoxy resin, etc., such as silicon, glass, quartz, etc., using wet etching method: processing of structural channels; When fabricating a silicon wafer with a high aspect ratio and a fine structure, a deep reactive ion etching method is required for processing the microstructure; and a thermosetting polymer material chip such as PDMS or epoxy resin is fabricated. Etc., it is generally necessary to build a mold, which can be made of silicon, glass, photo-adhesive, PDMS, etc.
  • the methods for making molds generally include the techniques of light, LIGA (Lithographie galvanoformung and abformung), Xuan 1 insect, soft Xuan 1 insect, etc. 'The microstructured channels are then made by casting; while thermoplastic polymers such as polycarbonate, PMMA, etc. are generally produced by hot pressing. Ionomer materials, such as PMMA, polystyrene, etc. can be made use of laser ablation. Biological sample to determine whether the genetic variation of the method and system
  • the invention proposes a method of determining whether a biological sample has genetic variation.
  • the method may comprise the following steps:
  • the cell sample is separated from the biological sample using the microfluidic device described above, which has been described in detail above with respect to the microfluidic device and will not be described again. It should be noted that all of the features and advantages previously described with respect to microfluidic devices are applicable to methods for determining whether a biological sample has genetic variation.
  • the term "genetic material” as used herein shall be understood broadly to mean any substance contained in a cell sample that carries its genetic information, either DNA or RA, may be whole genome, or Is a transcriptome.
  • the biological sample is at least one selected from the group consisting of blood, body fluids, tissue samples, and cell cultures. Thereby, single cells can be efficiently separated from the biological sample, thereby improving the efficiency of subsequent analysis.
  • the cell sample is at least one selected from the group consisting of nucleated red blood cells, tumor cells, embryonic stem cells, and immune cells.
  • the cell sample is at least one selected from the group consisting of fetal nucleated red blood cells and circulating tumor cells.
  • amplifying at least a portion of the genetic material contained in the isolated cell sample further comprises: lysing the isolated cell sample to release the genetic material of the cell sample; The released genetic material is amplified to obtain the amplification product. Thereby, the genetic material can be efficiently amplified from the separated cells.
  • the isolated cell sample is cleaved using a lysis reagent, wherein the lysis reagent is at least one selected from the group consisting of potassium hydroxide, sodium hydroxide, SDS, and proteinase K.
  • the cell sample can be lysed by physical means, for example, by placing the cell sample in an acoustic field to lyse the cells; by heating the cells, the cells are lysed. In order for a low abundance of nucleic acids in a single cell to meet the needs of subsequent sequencing, it is necessary to perform whole genome and transcriptome extraction and amplification.
  • the genetic material is a single cell whole genome of the cell sample.
  • the genetic information of the sample can be determined efficiently by sequencing the single-cell whole genome.
  • the amplification of the released genetic material is carried out by at least one of a PCR-based amplification reaction and a thermostatic amplification reaction.
  • the amplification of the released genetic material is carried out by at least one of MDA, DOP-PCR, cDNA exponential amplification, and linear amplification of RNA based on T7 RNA polymerase.
  • sequencing the amplification product further comprises: constructing a sequencing library for the amplification product; and sequencing the sequencing library to obtain the sequencing result.
  • the amplified product can be efficiently sequenced.
  • the sequencing library is sequenced by sequencing the whole genome sequencing library using at least one selected from the group consisting of a second generation high throughput sequencing platform and a third generation high throughput sequencing platform. Thereby, the sequencing efficiency can be further improved.
  • sequencing technology There are two main types of sequencing, one is the second generation of high-throughput sequencing technology, including Illumina's Gemone Anayzer system (the Solexa sequencer, later developed into the HisSeq 2000 system), ABI's Solid system and Roche. 454 company's GS-FLX system. Second-generation sequencing technologies have their own standard processes, which are sequenced according to standard procedures. The other is third-generation sequencing technology, single-molecule sequencing technology, including Helicos's true single-molecule sequencing technology, and Pacific Biosciences' single Molecular real-time sequencing technology, and Oxford Nanopore Technologies' nanopore sequencing technology.
  • This kind of technology can directly sequence DNA, RA, microR A, protein and other molecules without the need to amplify whole-genome or transcriptome of nucleic acids in single cells, and more intuitively reflect the number of DNA and RA in a single cell. Sequence changes.
  • the method according to the present invention it is possible to efficiently separate single cells by means of a microfluidic device according to an embodiment of the present invention, thereby enabling sequencing based on genetic analysis of single cells, such as whole genome, effectively obtained by analyzing sequencing results. Whether a single cell's genetic material, such as the entire genome, is abnormal, determines whether the biological sample has genetic variation.
  • the genetic variation is at least one selected from the group consisting of a single nucleotide polymorphism, a copy number variation, a genomic structural variation, a variable splicing, a differential expression, and a transcript variation.
  • determining whether the biological sample has genetic variation based on the sequencing result further comprises: comparing the sequencing result with a reference genome; and determining whether the biological sample is based on the comparison result Has genetic variation.
  • short-sequence alignment tools such as SOAP are used to compare the sequencing data to the reference genome, and then the genomic characteristics of the target cells can be obtained based on the qualitative and quantitative parameters of the alignment results.
  • Commonly used data analysis methods for genomic DNA sequencing include: single nucleotide polymorphism (SNP) analysis, copy number variation (CNV) analysis, genomic structural variation (SV) analysis, etc.; commonly used analytical methods for transcriptome RA sequencing are: Expression (DGE) analysis, alternative splicing (AS) detection, gene fusion detection, etc.; cell development and evolution analysis for cell population sequencing data. Therefore, through the comparison and correlation analysis of the data of the separated cells, not only can the understanding of the occurrence, development and evolution of the cells, but also the research basis for the diagnosis of genetic medicine, such as existing or potential cancer, complex The disease is screened or identified to achieve early detection and early treatment of the disease.
  • SNP single nucleotide polymorphism
  • CNV copy number variation
  • SV genomic structural variation
  • DGE Expression
  • AS alternative splicing
  • gene fusion detection gene fusion detection
  • cell development and evolution analysis for cell population sequencing data. Therefore, through the comparison and correlation analysis of the data of the separated cells, not only can the understanding of the
  • the invention provides a system for determining whether a biological sample has genetic variation, characterized by comprising: a microfluidic device, an amplification device, a sequencing device, and an analysis device.
  • the microfluidic device is configured to separate the cell sample from the biological sample;
  • the amplification device is coupled to the microfluidic device and is adapted to amplify at least a portion of the genetic material contained in the separated cell sample for obtaining expansion An amplification device;
  • the sequencing device is coupled to the amplification device and is adapted to sequence the obtained amplification product to obtain a sequencing result;
  • the analysis device is coupled to the sequencing device and is adapted to determine the biological sample based on the obtained sequencing result Whether it has genetic variation.
  • the aforementioned method can be effectively implemented, and the single cell can be efficiently separated by the microfluidic device according to the embodiment of the present invention, so that it can be based on the pair
  • the genetic material of the cell such as a whole genome, is sequenced, and it is effectively determined whether the biological sample has genetic variation by analyzing the sequencing result to obtain whether a single cell genetic material such as an entire genome has an abnormality.
  • the amplification device further includes: a lysis unit, wherein the lysis unit is provided with a lysis reagent or an external energy device, The isolated cell sample is cleaved using a lysis reagent or an external energy source to release the genetic material of the cell sample; and an amplification unit, the amplification unit is coupled to the lysis unit, and is adapted to the released inheritance
  • the substance is amplified to obtain the amplification product.
  • the genetic material can be efficiently amplified.
  • a system for determining whether a biological sample has a genetic variation is at least one selected from the group consisting of potassium hydroxide, sodium hydroxide, SDS, and proteinase K, the external energy source being light, electricity, and At least one of the heat.
  • the amplification unit is adapted to perform at least one of MDA, DOP-PCR, cDNA exponential amplification, and linear amplification of RA based on T7 RA polymerase. Thereby, the single-cell whole genome can be efficiently amplified.
  • the sequencing device further comprises: a library construction unit, the library construction unit is configured to construct a sequencing library for the amplification product; and a sequencing unit, the sequencing unit and the library construction The units are connected and are adapted to sequence the sequencing library to obtain the sequencing results.
  • the amplified product can be efficiently sequenced.
  • the sequencing unit is at least one selected from the group consisting of a second generation high throughput sequencing platform and a third generation high throughput sequencing platform. Thereby, the sequencing efficiency can be further improved.
  • the analyzing device further comprises: a comparison unit configured to compare the sequencing result with a reference genome; and a variation determining unit, the variation determining unit and the ratio
  • the cells are connected and are adapted to determine whether the biological sample has a genetic variation based on the alignment result. Thereby, the efficiency of determining whether the biological sample has genetic variation can be further improved.
  • this embodiment designs microchannels for multi-step separation of cells on the chip according to the differences in physical, chemical and biological properties of different types of cells, in order to obtain the final target single cells. .
  • microfluidic chips with different structures were designed to selectively separate and enrich the cells of interest. If the size of the cells to be separated is significantly different from other cells, it can be in the channel of the microfluidic chip. Design various structures such as columns, curved channels, combs, braids, sieves, etc., allowing only cells of a certain size to pass, or to retain cells of a certain size to separate cells, or to use deterministic lateral
  • the principle of displacement designing obstacles of certain interval size in the channel, cells of different sizes collide with obstacles and enter different liquid streams to achieve the purpose of separating cells of different sizes; using the principle of fluid mechanics, according to the cytoplasm of different sizes The difference in points enables the separation of cells.
  • the curved channel is designed to control the flow and separate the cells of different sizes. If the cells can be deformed, such as red blood cells, fetal nucleated red blood cells, obstacles can be designed with different spacing, so that the obstacle spacing is smaller than the diameter of red blood cells or fetal nucleated red blood cells, deformable cells and non-deformable cells (white blood cells, tumor cells) Etc.) to separate.
  • the cells can be deformed, such as red blood cells, fetal nucleated red blood cells
  • obstacles can be designed with different spacing, so that the obstacle spacing is smaller than the diameter of red blood cells or fetal nucleated red blood cells, deformable cells and non-deformable cells (white blood cells, tumor cells) Etc.) to separate.
  • various fields such as electric fields, magnetic fields, sound waves, etc.
  • dielectrophoresis can add electric fields of different frequencies outside the chip channel.
  • different types of cells can be polarized to different degrees to produce different degrees of drift, to achieve cell separation, or to utilize
  • Electrophoresis forms an electrical conductivity gradient that drives the cell to move in a direction of low conductivity until equilibrium, with a conductivity of zero, and then uses gravity to drag the cells into the destination channel to achieve cell separation.
  • biomolecules with specific capture cells attached to the surface of the magnetic beads are combined by immunochemical reactions, and the captured cells are separated by a controllable magnetic field; or some cells have their own Magnetic, such as the paramagnetic and diamagnetic properties of red blood cells and white blood cells, achieve cell separation.
  • the separation of cells is achieved according to the nature of the cell size, density, and specific gravity.
  • cells express many specific surface molecules, according to their immunochemical properties, molecules that bind to cell surface marker molecules, such as antibodies, aptamers, etc., can be captured in the channel for specific cell capture and/or Removal of non-specific cells.
  • Immunomagnetic beads can also be sorted in the channel, and the magnetic beads are respectively connected with biomolecules that specifically capture the target cells or remove non-specific cells, thereby realizing the separation and enrichment of specific cells.
  • different manipulation methods can be selected. It can use the flow and the wide control to control the wide opening and closing and retain the cells. If the magnetic beads are used for sorting the cells in the early stage, the single cells (micro cells) can be selectively manipulated to the designated reaction area by the control of the magnetic field. An electric field can be applied to the control channel to drive individual cells (micro cells) into the reaction cell by electroosmotic flow or electrophoresis. A channel with a special structure can be designed to trap only a single cell. It is also possible to use a capillary to arrange the cells in a regular manner and drive individual cells (micro cells) to a designated reaction zone by display movement. These control channels can be integrated to achieve simultaneous manipulation of multiple single cells (micro cells).
  • the nucleic acid extraction and amplification unit may be in a micro-reaction cell of a certain regular structure, or may be through a droplet (liquid Drop)
  • a single cell (micro-cell) is wrapped to form a separate chamber as a reaction cell.
  • we can put surface-functionalized magnetic beads in the extraction unit and obtain the required trace cellular DNA molecules or RA molecules by magnetic bead sorting, and drive the extracted by controlling the magnetic beads.
  • DNA or RNA molecules exit the outlet and enter the amplification unit. If the collected nucleic acid molecules are placed in a PCR tube, a standard WGA or full transcript amplification process is performed.
  • a single cell (micro-cell) is wrapped in a droplet, and the droplet is directly wrapped with a PCR reaction solution. After cleavage, the amplification reaction is directly performed in the droplet.
  • the obtained amplification product was allowed to exit through the driving system, and the reaction liquid was collected.
  • the nucleic acid molecules are interrupted, built, and then sequenced.
  • the obtained DNA fragment can be randomly broken into small fragments, and a specific linker can be used to prepare a DNA library, and cluster amplification can be performed on the cBot, and the DNA fragment can be directly sequenced at the single end or the double end. If R A is obtained, mR A is isolated, a fragmented cDNA library is prepared, clustered on cBot, and used for sequencing by Qualcomm sequencer.
  • Illumina's Gemone Anayzer system the Solexa sequencer, later developed into the HisSeq 2000 system
  • ABI's Solid system the Solexa sequencer, later developed into the HisSeq 2000 system
  • Roche 454's GS-FLX system are the top three.
  • High-throughput sequencing technology Third-generation sequencing technology, including single-molecule sequencing technology, including Helicos's true single-molecule sequencing technology, Pacific Biosciences' single-molecule real-time sequencing technology, and nanopores from Oxford Nanopore Technologies, will be applied. Sequencing technology, etc.
  • short sequence alignment tools such as SOAP are used to compare the sequencing data to the reference genome, and the genomic features of the target cells can be obtained based on the qualitative and quantitative parameters of the alignment results.
  • Commonly used data analysis methods for genomic DNA sequencing include: single nucleotide polymorphism (SNP) analysis, copy number variation (CNV) analysis, genomic structural variation (SV) analysis, etc.; commonly used analytical methods for transcriptome RA sequencing are: Expression (DGE) analysis, alternative splicing (AS) detection, gene fusion detection, etc.; cell development and evolution analysis for cell population sequencing data.
  • SNP single nucleotide polymorphism
  • CNV copy number variation
  • SV genomic structural variation
  • DGE Expression
  • AS alternative splicing
  • gene fusion detection gene fusion detection
  • cell development and evolution analysis for cell population sequencing data.
  • Example 1 Genetic identification of fetal nucleated red blood cells
  • Separation was performed using two steps. First, separation is performed according to the difference in the size of the red blood cells, such as the chip structure in 2.1, and most of the red blood cells are removed; the recovered cells are introduced into the magnetic separation chip, and the magnetic separation of the red and white cells is performed.
  • the structure of the first chip The spacing between the microcolumns is 15 ⁇ , and the relative displacement of each microcolumn is 6.75 ⁇ .
  • a deep structure ion etch technique was used to construct a microstructure on a silicon wafer with a channel depth of 150 ⁇ , which was sealed using a glass piece.
  • Blood and buffer were passed to the first chip at 100 ⁇ 7 ⁇ using an external drive device.
  • the buffer was iDPBS (containing 1% BSA, 2 mM EDTA). Recover cells from the outlet. The recovered cells were centrifuged at 2000 rpm for 15 min. Samples were treated with 50 mM NaN0 2 for 10 min. Then pass to the Miltenyi LS Column. Rinse slowly with iDPBS and wash away unadsorbed white blood cells. The magnet is then removed and rinsed with buffer to recover magnetic cells. Cells were labeled with FITC-epsilon-globulin and individual nucleated red blood cells were picked under a fluorescence microscope and transferred to a ⁇ :well array.
  • SNP Single-nucleotide polymorphism
  • Example 2 Identification of chromosomal aneuploidy abnormalities in fetal nucleated red blood cells
  • the Nautilus chip has an inlet and 6 outlets.
  • the inlet channel has a width of 0.22 mm.
  • the curvature of the channel is gradually enlarged according to a certain ratio at the exit.
  • the channel width is 3.88 mm.
  • Chip Channel Structure The su-8 negative photoresist was masked by photolithography, and the PDMS prepolymer was mixed and cast on the mold and turned over. Sealing is performed using a glass piece.
  • the structure of the second chip used as shown in Figure 6. There are two entrances and two exits.
  • the channel has a width of 0.5mm and a length of 50mm.
  • Chip Channel Structure The su-8 negative photoresist was masked by photolithography, and the PDMS prepolymer was mixed and cast on the mold and turned over. Sealing is performed using a glass piece. An external magnet is attached to one side of the channel to form a magnetic chip.
  • the first step uses the first chip to remove most of the red blood cells; it will contain nucleated red blood cells.
  • the mixed cell sample is passed to the second chip and separated by the difference in magnetic properties of the red blood cells.
  • An externally driven microinjection pump was used to drive 10% dilute blood (diluted with 1% BSA in iDPBS buffer) into the channel at a flow rate of 300 ⁇ 7 ⁇ .
  • the cell solution was collected at the outlet.
  • the recovered cells were centrifuged at 2000 rpm for 15 min. Samples were treated with 50 mM NaN0 2 for 10 min.
  • the cells were passed from the sample inlet into the chip while the other inlet was passed into iDPBS buffer at a flow rate of 10 ⁇ 7 ⁇ .
  • the target cells are recovered at the outlet.
  • the cells were stained with FITC-CD71 antibody, and single positive cells were picked for subsequent experiments.
  • Example 3 Screening of circulating tumor cells (CTCs) in cancer patients
  • the first step is to separate according to the size of the cells, such as the chip structure in Figure 1, to remove most of the red blood cells and some of the outer cells; the second step is to capture CTCs cells with EpCAM antibodies. specifically,
  • the structure of the first chip The spacing between the microcolumns is 20 ⁇ , and the relative displacement of each microcolumn is 6 ⁇ .
  • a deep reactive ion etching technique was used to construct a ⁇ : structure on a silicon wafer with a channel depth of 150 mm, which was sealed using a glass piece.
  • CTCs were captured using a Miltenyi LS/MS Column (Miltenyi BioTech).
  • Blood and buffer were pumped to the first chip at 100 ⁇ 7 ⁇ using an external drive device.
  • the buffer was iDPBS (containing 1% BSA, 2 mM EDTA). Recover cells from the outlet. The recovered cells were centrifuged at 300 g for 10 min. The magnetic beads to which the CD326 (EpCAM) antibody was ligated were then incubated with the cells. Rinse with iDPBS. The cell and magnetic bead mixture was passed into the Miltenyi LS/MS Column. Rinse slowly with iDPBS and wash away unadsorbed white blood cells. The magnet is then removed and rinsed with buffer to recover magnetic cells. CTCs were counted.
  • Cell lysis Cells were lysed using lysate KOH or NaOH.
  • SNP single nucleotide polymorphism
  • CTCs circulating tumor cells
  • the micro-injection pump is used to flow the blood and PBS buffer into the separation and enrichment unit of the microfluidic chip in a laminar flow.
  • the cylindrical microcolumns are arranged in the channel, and the microcolumn spacing is 16 ⁇ , and the blood cells pass through The microcolumn collides, and the smaller size red blood cells are separated according to the cell size, and the larger size white blood cells enter the next separation channel.
  • the immunomagnetic beads to which the anti-EpCAM antibody molecule is attached are placed in the channel, and the immunomagnetic beads capture CTCs which specifically express EpCAM molecules on the cell surface by colliding with the leukocytes flowing in the channel. Rinse with PBS to remove non-specifically bound cells. The enzymatic hydrolysate is introduced to release the CTCs from the magnetic beads.
  • the cells are regularly arranged into the channel, and a single CTC volume CTCs are trapped in different chambers by opening and closing the gas.
  • the separated red blood cells and white blood cells are controlled, and a small number of cells enter the respective channels that allow only a single cell to pass through, and the individual cells (micro cells) are separated into separate cells by the gas sap and liquid flow control, and used as a control.
  • micro-cells Through fluid control and open-air opening, individual cells (micro-cells) are pushed out from their respective outlets and collected in PCR tubes.
  • the collected single cells (micro cells) were subjected to standard amplification according to the method of MDA.
  • the amplified products were collected, and the DNA fragments were randomly divided into small fragments, and a specific linker was used to construct a DNA fragment library, which was subjected to cluster amplification on cBot for Illumina Hiseq 2000 sequencing.
  • Blood is introduced into the micro-magnetic flow channel.
  • red blood cells including ⁇ RBCs
  • white blood cells are separated according to the principle of red blood cells and WRBCs in the blood cells, and the white blood cells are diamagnetically separated into different channels.
  • Erythrocytes (including ⁇ RBCs) enter the column channel to which the anti-CD71 antibody is ligated, and the ?RBCs are captured by the antigen-antibody immunological reaction. After fNRBCs are desorbed from the microcolumn, they are manipulated into the control channel.
  • Control ⁇ RBCs enter a channel that allows only single cells to pass. Under the microscope, a single RBC is pushed into the array collection tube by the control of the flow and the air volume.
  • the white blood cells and red blood cells that are separated from the blood are controlled to enter a channel that allows only a single cell to pass through.
  • individual white blood cells and living cells are pushed into the array collection tube and used as a control.
  • the cell RA in the array collection tube was extracted, and the transcript amplification was carried out according to the linear amplification method based on T7 RA polymerase.
  • the specific process is referred to Eberwine, J. et al. Analysis of gene expression in single live neurons. Proc. Natl. Acad Sci. USA 89, 3010-3014 (1992). and Van Gelder, RN et al. Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. USA 87, 1663-1667 (1990).
  • the amplified product cDNA was collected, constructed according to the library provided by Illumina, and the library was prepared and clustered on cBot for Illumina Hiseq 2000 sequencing. data analysis.
  • Reverse transcription Directly add reverse transcriptase and primers for reverse transcription to synthesize double-stranded DNA.
  • 7.5 cDNA Index Amplification The first round of PCR was performed and purified using the QIAquick PCR purification kit. The product purified after the first round of PCR was subjected to a second round of amplification using QIAquick PCR purification kit. Purification was carried out. The gel recovered a 0.5-3 kb fragment from the PCR product.
  • microfluidic device according to an embodiment of the present invention, a method of determining whether a biological sample has genetic variation, and a system for determining whether a biological sample has genetic variation can efficiently separate single cells, thereby determining whether the biological sample has genetic variation.

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Abstract

提供了微流控装置及其用途，具体而言，提供了微流控装置、确定生物样品是否具有遗传变异的方法和确定生物样品是否具有遗传变异的***。其中，微流控装置具有适于分离单细胞的微流控通道。

Description

微流控装置及其用途 优先权信息
本申请请求 201 1 年 4 月 13 日向中国国家知识产权局提交的、 专利申请号为 201 1 10092704.2的专利申请的优先权和权益， 并且通过参照将其全文并入此处。 技术领域
本发明涉及微流控装置及其用途， 具体而言， 涉及微流控装置、 确定生物样品是否 具有遗传变异的方法和确定生物样品是否具有遗传变异的***。 背景技术
微流控芯片技术起源于分析化学领域， 是通过微细加工技术， 将生物、 医学和化学 领域中所涉及到的样品的制备、 反应、 分离、检测等基本操作单元集成到数平方厘米芯 片上， 由微通道形成网络， 以可控流体贯穿整个***， 用以取代常规分析实验室的各种 功能的技术。 微流控技术在细胞分离、 核酸提取、 纯化、 PCR扩增等方面表现出了极 强的优越性。
然而， 目前的微流控芯片仍有待改进。 发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。
在本发明的第一方面， 本发明提出了一种微流控装置。 根据本发明的实施例， 该微流 控装置具有适于分离单细胞的微流控通道。 由此， 利用根据本发明实施例的微流控装置可 以有效地从生物样本中分离单细胞。
在本发明的第二方面， 本发明提出了一种确定生物样品是否具有遗传变异的方法， 其 特征在于， 包括下列步骤： 利用前面所述的微流控装置， 从生物样品分离细胞样本； 对所 分离的细胞样本中所包含的遗传物质的至少一部分进行扩增， 以便获得扩增产物； 对所述 扩增产物进行测序， 以便获得测序结果； 以及基于所述测序结果， 确定所述生物样品是否 具有遗传变异。 通过根据本发明实施例的方法， 能够有效地借助根据本发明实施例的微流 控装置分离单细胞， 从而可以基于对单细胞的遗传物质例如全基因组进行测序， 有效地通 过对测序结果进行分析而得到单细胞的遗传物质例如全基因组中是否存在异常， 从而确定 生物样品是否具有遗传变异。 在本发明的第三方面， 本发明提出了一种确定生物样品是否具有遗传变异的***， 其特 征在于包括： 前面所述的微流控装置， 所述微流控装置用于从生物样品分离细胞样本； 扩 增装置， 所述扩增装置与所述微流控装置相连， 并且适于对所分离的细胞样本中所包含的 遗传物质的至少一部分进行扩增， 以便获得扩增产物； 测序装置， 所述测序装置与所述扩 增装置相连， 并且适于对所述扩增产物进行测序， 以便获得测序结果； 以及分析装置， 所 述分析装置与所述测序装置相连， 并且适于基于所述测序结果， 确定所述生物样品是否具 有遗传变异。 利用根据本发明实施例的确定生物样品是否具有遗传变异的***， 可以有效 地实施前面所述的方法， 能够有效地借助根据本发明实施例的微流控装置分离单细胞， 从 而可以基于对单细胞的遗传物质例如全基因组进行测序， 有效地通过对测序结果进行分析 而得到单细胞的遗传物质例如全基因组中是否存在异常， 从而确定生物样品是否具有遗传 变异。
由此， 根据本发明的实施例， 本发明针对低丰度细胞的获取难题， 建立了基于高通量测 序的低丰度细胞筛查模型， 通过生物信息模拟调节该模型的参数， 论证了该模型在筛查低 丰度细胞方面的检测域、 灵敏度、 准确性、 和可重复性。 具体地， 本发明首先建立一个自 动化微流控细胞分选模型， 接着利用扩增技术和新一代测序技术产生一定数量的特定序列 数据对低丰度细胞不同变异进行有效筛查。 将微流控技术、 全基因组扩增技术及高通量测 序技术进行整合， 实现在微流控芯片上构建单细胞（微量细胞） 的分离、 富集、 全基因组 扩增等功能， 结合高通量测序技术， 进行自动化、 高通量单细胞的检测分析， 对样品的遗 传变异方面的筛查， 该技术模型能够应用于新的遗传学方面的研究， 比如发现新的致病机 制等。
本发明论证一种自动化筛查低丰度细胞变异信息的方法模型， 其实验流程为： （ a )使样 品进入微流控装置， 经过多个微通道或微小室分离和富集样品中的目的单细胞； （b )往微 流控芯片上的微通道或微小室中加入裂解试剂或暴于光下产生热量裂解步骤（a )所得的目 的单细胞， 以裂解产物 DNA或 R A为模板进行扩增； （c )对步骤（b )所得的扩增产物进 行建库，在高通量测序平台上进行测序，所述高通量测序平台包括但不限于 Illumina/Solexa、 ABI Solid和 Roche 454; ( d )对步骤（ c )获得的测序数据进行处理。
本发明另一方面提供了一种应用于自动化筛查低丰度细胞变异信息的微流控装置，所述 微流控装置包含至少一个通过微通道或微小室互相连接的进口和出口， 由微流控芯片、 驱 动***和检测***组成。
在本发明的一个优选实施例中，所述微流控装置的微流控芯片包含细胞分离富集单元和 细胞操控单元。 在本发明的一个优选实施例中， 所述细胞分离富集单元设有^：结构或^:障碍物， 所述细 胞操控单元设有液流和气阔控制。
在本发明的一个优选实施例中， 所述微流控装置的微流控芯片还包含 DNA和 /或 R A提取 和扩增单元。
在本发明的一个优选实施例中， 根据所述微流控装置对低丰度细胞的分离富集的方法， 包括使样品流经所述微流控芯片的微通道或微小室， 利用微通道或微室内的磁珠捕获目标 细胞。
在本发明的一个优选实施例中， 根据所述微流控装置裂解细胞的方法， 包括使样品流经 所述微流控芯片的微通道或微小室， 利用微通道或微室内的磁珠捕获目标细胞， 以及加入 裂解试剂或将磁珠暴于光下产生热量而裂解细胞。
在本发明的一个优选实施例中， 根据所述微流控装置从细胞扩增目标 DNA或 R A的 方法， 包括使样品流经所述微流控芯片的微通道或微小室， 利用微通道或微室内的磁珠捕 获目标细胞，加入裂解试剂或将磁珠暴于光下产生热量而裂解细胞，提取目标 DNA或 R A, 以及以目标 DNA或 R A为模板进行扩增； 所述扩增包括全基因组扩增。
由此， 根据本发明的实施例， 本发明建立了基于微流控技术和高通量测序的低丰度细胞 筛查的技术模型， 并论证了该模型的检测域、 灵敏度、 准确性和可重复性， 可实现对复杂 样品中的低丰度细胞的遗传信息进行高通量自动化的筛查， 实现实验室高通量自动化， 为 基因学方面的医疗诊断研究提供基础。 根据本发明的实施例， 本发明设计了具有微通道或 微小室的微流控芯片， 集分离、 富集微量细胞和对细胞中 DNA或 R A进行提取和扩增的 功能于一张芯片， 降低实验装置设备成本， 易于推广。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得 明显， 或通过本发明的实践了解到。 附图说明
本发明的上述和 /或附加的方面和优点从结合下面附图对实施例的描述中将变得明 显和容易理解， 其中：
图 1-图 6显示了根据本发明实施例的微流控装置的结构示意图。 发明详细描述
下面详细描述本发明的实施例， 所述实施例的示例在附图中示出， 其中自始至终相 图描述的实施例是示例性的， 仅用于解释本发明， 而不能理解为对本发明的限制。 在本发明的描述中， 需要理解的是， 术语 "中心"、 "上"、 "下"、 "前" 、 "后" 、 "左" 、 "右" 、 "竖直" 、 "水平" 、 "顶" 、 "底" "内" 、 "外" 等指示的方位 或位置关系为基于附图所示的方位或位置关系， 仅是为了便于描述本发明和简化描述， 而不是指示或暗示所指的装置或元件必须具有特定的方位、 以特定的方位构造和操作， 因此不能理解为对本发明的限制。
需要说明的是， 术语 "第一" 、 "第二" 仅用于描述目的， 而不能理解为指示或暗 示相对重要性或者隐含指明所指示的技术特征的数量。 由此， 限定有 "第一"、 "第二" 的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中， 除非另有说明， "多个" 的含义是两个或两个以上。
术语 "低丰度细胞"、 "单 （个） 细胞" 和 "微量细胞" 在文中可互换使用， 都是指在 一个复杂样品中包含有所要类型的细胞， 但其含量很小， 一般只有一个到几个。
微流控装置
在本发明的第一方面， 本发明提出了一种微流控装置。 根据本发明的实施例， 该微流 控装置具有适于分离单细胞的微流控通道。 由此， 利用根据本发明实施例的微流控装置可 以有效地从生物样本中分离单细胞。
根据本发明的实施例， 微流控装置中适于分离单细胞的微流控通道的形式并不受特别 限制。 针对用于分离单细胞的样品的复杂性和目的细胞的稀少性， 可以根据不同类型细胞 在物理、 化学及生物学特性上的差异在芯片上设计和制作对细胞进行一步分离单细胞或多 步分离单细胞的微通道， 以便获得最终目的单细胞。 需要说明的是， 可以通过各种适于分 离单细胞的方法的组合， 并且顺序不受特别限制， 只要能够实现对单细胞的分离即可。 例 如， 可以首先将混合样品细胞通入到第一微流控通道中， 进行初步的分离， 去除大多数的 干扰细胞； 然后再将经过初步分离的细胞， 通入到第二^：流控通道中， 进行目的细胞的进 一步的富集。
根据本发明的实施例， 微流控通道可以基于细胞的物理、 化学性质， 如大小， 形状， 可否形变， 细胞表面性质 （如细胞表面受体、 抗原、 膜的通透性）， 细胞内部性质 （如表达 某种蛋白、 特异性酶等）来实现对单细胞的分离。
根据本发明的一个实施例， 所述微流控通道上形成有多个微柱。 由此， 可以通过在微 流控通道上形成 柱（也可以称为障碍物）， 作为细胞在流体方向上流动的障碍， 从而可以 基于细胞尺寸的区别， 实现对单细胞的分离。 根据本发明的实施例， 可以在微流控通道内 构建规则排列的， 并间隔固定尺寸的障碍物（障碍物的形式并不受特别限制， 可以是圆柱， 椭圆住， 方柱等）， 每行障碍物之间发生固定尺寸的横向位移。 参考图 1-3 , 通过微流控芯 片通道内部密布规则排列的障碍物（例如多个^:柱在所述^:流控通道上呈阵列分布；)， 待分 离细胞作为流体中的质点在流入通道时与障碍物发生碰撞， 当细胞大于某一尺寸 （与障碍 物之间的间距相关） 时， 细胞按照一定角度（与每行障碍物之间的横向位移相关）发生偏 移， 小于该尺寸的细胞则按照原流体轨迹流动， 以此实现不同尺寸的细胞的分离 (；如图 1)。 可以根据待分离细胞的大小来调整障碍物间距， 如微柱之间的距离， 包括横向间距和纵向 间距。 根据本发明的实施例在纵向上， 相邻两个微柱之间的距离为 10-100微米， 并且， 在 横向上， 相邻两个微柱之间的距离为 10-100微米。 由此， 可以进一步提高分离单细胞的效 率。 根据本发明的一个实施例， 每行微柱相对偏移 6-7微米。 由此， 可以进一步提高分离单 细胞的效率。 障碍物阵列结构可以设置成单级或多级， 根据待分离细胞环境的不同， 如从 体液中分离固定大小的细胞， 一般单級^:柱阵列就可以实现， 而像血液之类的复杂样品， 里面还有多种尺寸的细胞， 可以用多级微柱阵列进行精细的分离。 根据本发明的一个实施 例， 沿流体方向， 所述^：柱的直径增大。 由此， 可以进一步提高分离单细胞的效率。 进一 步， 参考图 2, 可以建立多级不同间距尺寸的障碍物阵列， 将不同尺寸的细胞的分离进一步 细化。 在出口处收集不同尺寸的细胞， 进行下一步分离或者直接用于后续的检测实验。 根 据本发明的一个实施例， 沿流体方向， 微柱的直径不变。
另外， 根据本发明的实施例， 还可以利用不同尺寸的细胞在弯曲通道中产生的惯性力 的不同， 实现在 ^：流控通道中分离细胞。 在弯曲通道中， 稳流流动的流体其流速呈现抛物 线状分布， 在通道中间的流速最大， 受到的离心力最大， 靠近通道壁的流体流速最小。 为 了保持流体的质量守恒， 会产生涡流。 这样流体中的颗粒受到了浮力和迪恩力的相互作用。 在一定的流速条件下， 形成聚焦流动。 尺寸越大的颗粒的聚焦流越靠近通道的内侧壁， 尺 寸越小的颗粒的聚焦流越远离通道的内侧壁根据本发明的一个实施例， 所述微流控通道为 弯曲管道。 由此， 通过釆用弯曲管道， 可以通过产生惯性力， 来实现对细胞的有效分离。 才艮据本发明的优选实施例， 所釆用的弯曲管道为选自阿基米德螺线通道（如图 4 所示）和 鹦鹉螺线通道（如图 5 所示） 的至少一种。 由此， 可以通过使不同尺寸的细胞通入到通道 中， 在弯曲通道中形成聚焦流， 并在不同的出口留出， 实现大小细胞不同的分离， 从而可 以进一步提高分离单细胞的效率。
另外， 根据本发明的实施例， 还可以根据细胞带有磁性进行分离。 细胞带有磁性， 主要分为两种， 一种是自身带有磁性， 如红细胞， 由于红细胞含有血红蛋白， 血红蛋白是 含铁蛋白， 因此红细胞具有顺磁性， 并可以通过脱氧化处理， 提高其磁性； 另一种是将可 以特异性捕获目的细胞的抗体、 适配体等和磁性微球连接， 通过抗原抗体反应， 使特异性 的细胞具有磁性。 具有磁性的细胞通过外加磁场， 可以将有磁性的细胞同无磁性的细胞进 行分离， 如图 6 所示。 根据本发明的一个实施例， 所述微流控装置进一步设置有磁体。 由 此， 可以基于细胞带有磁性， 而实现对细胞的分离。 根据本发明的实施例， 磁体的设置位 置并不受特别限制， 只要能够使得微流控通道处于磁体的有效磁场范围内即可， 根据本发 明的实施例， 可以将磁体设置在微流控通道的下方， 由此， 可以进一步提高分离效率。 根 据本发明的一个实施例， 所述微流控通道的宽度为 0.5毫米， 长度为 50毫米。 由此， 可以 进一步提高分离单细胞的效率。
微流控装置 （因芯片是实现装置功能的主要组件， 因而在本文中也称为微流控芯片）， 可以通过本领域技术人员已知的方法制备。 微流控芯片的制作材料包括硅、 玻璃、 石英、 聚曱基丙婦酸曱酯 （poly(methylmethacrylate), PMMA )、 聚苯乙烯（ polystyrene )、 聚碳酸 酉旨 （ polycarbonate ) ^ 聚乙烯 （ polyethylene ) ^ 硅橡胶 （ 如聚二曱基硅氧烷 ( poly(dimethylsiloxane), PDMS )、 环氧树脂等。 像是硅、 玻璃、 石英等， 使用湿刻蚀的方 法进行^：结构通道的加工； 当制作高宽比高， 结构较 ^：细的硅芯片时， 需要使用深反应离 子刻蚀的方法进行微结构的加工； 而制作热固型的高聚物材料芯片， 如 PDMS、 环氧树脂 等， 一般都需要构建模具， 可用硅、 玻璃、 光胶、 PDMS 等制作。 制作模具的方法一般有 光玄、 LIGA ( Lithographie galvanoformung and abformung )、 玄1 虫、 软玄1 虫等技术力口工。 '然 后利用浇注的方法制作微结构通道； 而热塑型的高聚物， 如聚碳酸酯、 PMMA等， 一般使 用热压的方法进行制作。 某些高聚物材料， 如 PMMA、 聚苯乙烯等可以使用激光烧蚀的方 法进行制作。 确定生物样品是否具有遗传变异的方法和***
在本发明的第二方面， 本发明提出了一种确定生物样品是否具有遗传变异的方法。 根 据本发明的实施例， 该方法可以包括下列步骤：
首先， 利用前面所述的微流控装置， 从生物样品分离细胞样本， 前面关于微流控装置 已经进行了详细描述， 不在赘述。 需要说明的是， 前面关于微流控装置所描述的所有特征 和优点均适用确定生物样品是否具有遗传变异的方法。
接下来， 对所分离的细胞样本中所包含的遗传物质的至少一部分进行扩增， 以便获得扩增 产物。 在本文中所使用的术语 "遗传物质" 应做广义理解， 其指的是细胞样本中所包含的 可以携带其遗传信息的任何物质， 可以为 DNA也可以为 R A, 可以是全基因组， 也可以 是转录组。 根据本发明的一个实施例， 所述生物样品为选自血液、 体液、 组织样品和细胞 培养物的至少一种。 由此， 可以有效地从生物样品分离单细胞， 从而提高后续分析的效率。 根据本发明的一个实施例， 所述细胞样本为选自有核红细胞、 肿瘤细胞、 胚胎干细胞、 免疫细胞的至少一种。 优选所述细胞样本为选自胎儿有核红细胞和循环肿瘤细胞的至少一 种。 由此， 可以有效地对特定来源的生物样本分离的单细胞进行检测。 根据本发明的一 个实施例， 对所分离的细胞样本中所包含的遗传物质的至少一部分进行扩增进一步包括： 对所分离的细胞样本进行裂解， 以便释放所述细胞样本的遗传物质； 以及对所释放的遗传 物质进行扩增， 以便获得所述扩增产物。 由此， 可以有效地从所分离的细胞扩增遗传物质。 根据本发明的一个实施例， 使用裂解试剂对所分离的细胞样本进行裂解， 其中， 所述裂解 试剂为选自氢氧化钾、 氢氧化钠、 SDS和蛋白酶 K的至少一种。 由此， 可以进一步提高扩 增遗传物质的效率。 另外， 还可以通过物理方法对细胞样本进行裂解， 例如， 将细胞样本 置于声波场中， 使细胞裂解； 通过对细胞进行加热， 使细胞裂解。 为了使单个细胞中低丰 度的核酸满足后续测序的需求， 需要对其进行全基因组和转录组的提取和扩增。 单个细胞 核酸的提取方法多样， 可以釆用商品化试剂盒提取单个细胞内的基因组或者 mR A; 也可 以根据 MDA或者转录本扩增等技术中的策略， 裂解后直接进行基因组或者转录组的扩增， 而无需进行核酸的提取； 或者釆用微流控的技术， 如固液萃取、 液液萃取等方法， 提取基 因组或者转录组； 而单细胞全基因组或者全转录组扩增的主要的方法为 MDA、 DOP-PCR、 cDNA指数扩增和基于 T7 R A聚合酶的线性扩增等技术。 由此， 根据本发明的一个实施 例， 所述遗传物质为所述细胞样本的单细胞全基因组。 由此， 可以有效地通过对单细胞全 基因组进行测序， 而确定样本的遗传信息。 根据本发明的一个实施例， 对所释放的遗传物 质进行扩增是通过基于 PCR的扩增反应和恒温扩增反应的至少一种进行的。 优选， 对所 释放的遗传物质进行扩增是通过 MDA、 DOP-PCR、 cDNA指数扩增和基于 T7 RNA聚合酶 的 RNA线性扩增的至少一种进行的。 由此， 可以进一步提高对全基因组进行扩增的效率。
在得到扩增产物之后， 对所得到的扩增产物进行测序， 以便获得测序结果。 根据本发 明的一个实施例， 对所述扩增产物进行测序进一步包括： 针对所述扩增产物， 构建测序文 库； 以及对所述测序文库进行测序， 以便获得所述测序结果。 由此， 可以有效地对扩增产 物进行测序。 根据本发明的一个实施例， 利用选自第二代高通量测序平台和第三代高通量 测序平台的至少一种对所述全基因组测序文库进行测序对所述测序文库进行测序。 由此， 可以进一步提高测序效率。 目前测序主要有两大类， 一类是第二代的高通量测序技术， 包 括 Illumina公司的 Gemone Anayzer***（即 Solexa测序仪，后又发展为 HisSeq 2000***）、 ABI公司的 Solid***以及 Roche 454公司的 GS-FLX***。 第二代测序技术均有其标准流 程，按照标准流程进行测序；另一类，则是第三代测序技术，即单分子测序技术，包括 Helicos 公司的真实单分子测序技术、 Pacific Biosciences公司的单分子实时测序技术， 以及 Oxford Nanopore Technologies 公司的纳米孔测序技术等。 该类技术可直接对 DNA、 R A、 microR A,蛋白等分子直接测序，无需对单细胞中的核酸进行全基因组或者转录组的扩增， 更直观的反应了单个细胞中 DNA、 R A的数量和序列的变化。
最后， 基于所述测序结果， 确定所述生物样品是否具有遗传变异。 通过根据本发明的 方法， 能够有效地借助根据本发明实施例的微流控装置分离单细胞， 从而可以基于对单细 胞的遗传物质例如全基因组进行测序， 有效地通过对测序结果进行分析而得到单细胞的遗 传物质例如全基因组中是否存在异常， 从而确定生物样品是否具有遗传变异。 根据本发明 的一个实施例， 所述遗传变异为选自单核苷酸多态性、 拷贝数变异、 基因组结构变异、 可 变剪接、 差异表达和转录本变异的至少一种。 根据本发明的一个实施例， 基于所述测序结 果， 确定所述生物样品是否具有遗传变异进一步包括： 将所述测序结果与参考基因组进行 比对； 以及基于比对结果， 确定所述生物样品是否具有遗传变异。 由此， 可以进一步提高 确定生物样品是否具有遗传变异的效率。 基于高通量测序技术获得的数据， 运用短序列比 对工具（如 SOAP )将测序数据比对到参考基因组上， 进而可根据比对结果的定性及定量参 数获取目的细胞的基因组特征。 基因组 DNA测序常用的数据分析手段有： 单核甘酸多态性 ( SNP )分析， 拷贝数变异 (CNV)分析， 基因组结构变异 (SV)分析等等； 转录组 R A测序 常用分析方法有： 基因差异表达（DGE )分析， 可变剪接 (AS)探测， 基因融合探测等等； 对于细胞群体测序数据还能进行细胞发育及进化分析。 由此， 通过对分离的细胞的数据进 行比对、 关联性分析， 不仅可以了解细胞的发生、 发展、 演化过程， 而且可以为基因医疗 诊断提供研究基础， 如对已有或者潜在的癌症、 复杂疾病进行筛查或者鉴定， 实现疾病的 早期发现、 早期治疗的目的。
在本发明的第三方面， 本发明提出了一种确定生物样品是否具有遗传变异的***， 其特 征在于包括： 微流控装置、 扩增装置、 测序装置和分析装置。 其中， 微流控装置用于从生 物样品分离细胞样本； 扩增装置与微流控装置相连， 并且适于对所分离的细胞样本中所包 含的遗传物质的至少一部分进行扩增， 以便获得扩增产物； 测序装置与扩增装置相连， 并 且适于对所得到的扩增产物进行测序， 以便获得测序结果； 以及分析装置与测序装置相连， 并且适于基于所得到的测序结果， 确定生物样品是否具有遗传变异。 利用根据本发明实施 例的确定生物样品是否具有遗传变异的***， 可以有效地实施前面所述的方法， 能够有效 地借助根据本发明实施例的微流控装置分离单细胞， 从而可以基于对单细胞的遗传物质例 如全基因组进行测序， 有效地通过对测序结果进行分析而得到单细胞的遗传物质例如全基 因组中是否存在异常， 从而确定生物样品是否具有遗传变异。 根据本发明的一个实施例， 扩增装置进一步包括： 裂解单元， 所述裂解单元中设置有裂解试剂或者外部能源装置， 以 便使用裂解试剂或外部能源对所分离的细胞样本进行裂解， 释放所述细胞样本的遗传物质； 以及扩增单元， 所述扩增单元与所述裂解单元相连， 并且适于对所释放的遗传物质进行扩 增， 以便获得所述扩增产物。 由此， 可以有效地对遗传物质进行扩增。 根据本发明的一个 实施例， 确定生物样品是否具有遗传变异的***所述裂解试剂为选自氢氧化钾、 氢氧化钠、 SDS和蛋白酶 K的至少一种， 所述外部能源为光、 电和热的至少一种。 才艮据本发明的一个 实施例， 所述扩增单元适于进行 MDA、 DOP-PCR、 cDNA指数扩增和基于 T7 R A聚合酶 的 R A线性扩增的至少一种。 由此， 可以有效地对单细胞全基因组进行扩增。 根据本发明 的一个实施例， 所述测序装置进一步包括： 文库构建单元， 所述文库构建单元用于针对所 述扩增产物， 构建测序文库； 以及测序单元， 所述测序单元与所述文库构建单元相连， 并 且适于对所述测序文库进行测序， 以便获得所述测序结果。 由此， 可以有效地对扩增产物 进行测序。 根据本发明的一个实施例， 所述测序单元为选自第二代高通量测序平台和第三 代高通量测序平台的至少一种。 由此， 可以进一步提高测序效率。 根据本发明的一个实施 例， 分析装置进一步包括： 比对单元， 所述比对单元用于将所述测序结果与参考基因组进 行比对； 以及变异确定单元， 所述变异确定单元与所述比对单元相连， 并且适于基于比 对结果， 确定所述生物样品是否具有遗传变异。 由此， 可以进一步提高确定生物样品是否 具有遗传变异的效率。
需要说明的是，前面针对微流控装置和用于确定生物样品是否具有遗传变异的方法的特 征和优点同样适用于该***， 不再赘述。 下面将结合实施例对本发明的方案进行解释。 本领域技术人员将会理解， 下面的实施 例仅用于说明本发明， 而不应视为限定本发明的范围。 实施例中未注明具体技术或条件的， 按照本领域内的文献所描述的技术或条件（例如参考 J.萨姆布鲁克等著， 黄培堂等译的《分 子克隆实验指南》， 第三版， 科学出版社）或者按照产品说明书进行。 所用试剂或仪器未注 明生产厂商者， 均为可以通过市购获得的常规产品， 例如可以釆购自 Illumina公司。
一般方法基于高通量测序的低丰度细胞筛查模型的建立
1.1微流控芯片的设计
1.1.1微流控芯片的单细胞分离和富集单元
由于样品的复杂性和目的细胞的稀少性， 本实施例根据不同类型细胞在物理、 化学及 生物学特性上的差异在芯片上设计对细胞进行多步分离的微通道， 为获得最终目的单细胞。
根据目的细胞的理化性质， 设计具有不同结构的微流控芯片， 进行目的细胞的选择性 分离和富集。 如果要分离的细胞的大小与其他细胞有明显区别， 可以在微流控芯片的通道 里设计各种结构如柱状、 弯曲沟道、 梳状、 堰状、 筛状等微结构， 仅允许一定尺寸大小的 细胞通过， 或者截留一定尺寸大小的细胞， 来分离细胞、 或者利用确定性横向位移的原理， 在通道内设计一定间隔尺寸的障碍物， 不同大小的细胞通过与障碍物碰撞， 进入到不同的 液流， 达到分离不同大小细胞的目的； 利用流体力学的原理， 根据不同大小细胞质点的不 同， 实现细胞的分离。 根据惯性， 设计弯曲通道， 控制液流， 实现不同大小细胞的迁移而 分离。 如果细胞可以变形， 如血红细胞， 胎儿有核红细胞， 可以设计不同间距的障碍物， 让障碍物间距小于红细胞或者是胎儿有核红细胞的直径，将可变形细胞与不可变形细胞（白 细胞、 肿瘤细胞等）进行分离。
根据细胞物理性质的不同， 可以在芯片上建立各种场， 如电场、 磁场、 声波等进行细 胞的分离。 如介电泳， 可以在芯片通道外加上不同频率的电场， 通过调节电场的空间分布， 会使不同种类的细胞受到不同程度的极化而产生不同程度的漂移， 实现细胞的分离， 或者 是利用介电泳形成电传导率梯度，驱动细胞向电导率低的方向移动，直到平衡， 电导率为 0, 再利用重力将细胞拖拽到目的通道内， 实现细胞分离。 利用磁场分离， 与 MACS类似， 在 磁珠表面连接有特异性的捕获细胞的生物分子， 通过免疫化学反应进行结合， 利用可控磁 场， 将捕获的细胞进行分离； 或者利用某些细胞自身具有的磁性， 如红细胞和白细胞的顺 磁性和反磁性实现细胞的分离。 利用声波的波节特性受力的不同， 根据细胞的大小、 密度、 比重等性质， 实现细胞的分离。
由于细胞会表达很多特异性的表面分子， 根据其免疫化学性质， 在通道内连接上可以 与细胞表面标志物分子进行结合的分子如抗体、 适配体等， 进行特异性细胞的捕获和 /或非 特异性细胞的去除。 也可以在通道内进行免疫磁珠的分选， 磁珠上相应连接有特异性捕获 目的细胞或者去除非特异性细胞的生物分子， 实现特异性细胞的分离和富集。
1.1.2微流控芯片的细胞操控单元
根据整体芯片的整合和前期细胞分离的要求， 可以选择不同的操控方式。 可以利用液 流和微阔的控制， 操控微阔的开闭， 截留细胞。 如果前期釆用磁珠进行细胞的分选， 可以 通过磁场的控制， 选择性地操控单个细胞（微量细胞）到指定的反应区域。 可以在操控通 道上加上电场， 利用电渗流或者电泳的方式将单个细胞（微量细胞）驱动进反应池中。 设 计特殊结构的通道， 可以仅截留单个细胞 敫量细胞）。 还可以利用毛细管， 使细胞进行规 则排列， 通过陈列移动， 将单个细胞（微量细胞）驱动到指定的反应区域。 这些控制通道 可以进行集成， 实现同时多个单细胞（微量细胞） 的操控。
1.1.3微流控芯片的单细胞核酸提取和扩增单元
核酸提取和扩增单元可以是位于一定规则结构的微反应池中， 也可以通过 droplet (液 滴）将单个细胞（微量细胞） 包裹， 形成一个个独立的小室作为反应池。 为了提取到细胞 内的 DNA或者 R A, 我们可以在提取单元里放入表面功能化的磁珠， 通过磁珠分选， 获 得需要的微量细胞 DNA分子或 R A分子， 通过控制磁珠， 驱动提取的 DNA或者 RNA分 子流出出口， 进入扩增单元。 如将收集到的核酸分子放入 PCR管中， 进行标准的 WGA或 者全转录本扩增过程。 还可以直接利用 droplet技术， 单个细胞（微量细胞） 包裹在 droplet (液滴）中， 液滴中直接包裹如 PCR反应液， 经过裂解后， 直接在 droplet中进行扩增反应。
1.2高通量测序
获得的扩增产物， 通过驱动***留出出口， 收集反应液。 按照第二代测序技术的标准 流程， 将核酸分子打断， 建库， 然后进行测序。
针对 DNA和 R A有不同的建库方式。 如果是 DNA, 可以将获得的 DNA片段随机打 断成小片段， 加上特定接头， 制备成 DNA文库， 在 cBot上进行成簇扩增， 直接对 DNA片 段进行单末端或者双末端的测序。 如果获得的是 R A, 分离出 mR A, 制备成片段化的 cDNA文库， 在 cBot上进行成簇扩增， 再用于高通测序仪测序。
目前主要釆用三种技术进行高通量测序， Illumina公司的 Gemone Anayzer ***（即 Solexa测序仪， 后又发展为 HisSeq 2000***）、 ABI公司的 Solid***以及 Roche 454公司 的 GS-FLX ***三大高通量测序技术。 而且随着科技的发展， 将会应用第三代测序技术， 即单分子测序技术， 包括 Helicos公司的真实单分子测序技术、 Pacific Biosciences公司的单 分子实时测序技术， 以及 Oxford Nanopore Technologies公司的纳米孔测序技术等。
1.3 数据分析
基于高通量测序结果， 运用短序列比对工具（如 SOAP )将测序数据比对到参考基因组 上， 进而可根据比对结果的定性及定量参数获取目的细胞的基因组特征。
基因组 DNA测序常用的数据分析手段有： 单核甘酸多态性（ SNP )分析， 拷贝数变异 (CNV)分析， 基因组结构变异 (SV)分析等等； 转录组 R A测序常用分析方法有： 基因差异 表达（DGE )分析， 可变剪接 (AS)探测， 基因融合探测等等； 对于细胞群体测序数据还能 进行细胞发育及进化分析。
通过对分离样品进行比对、 关联性分析， 不仅可以了解细胞的发生、 发展、 演化过程， 而且可以为基因医疗诊断提供研究基础， 如对已有或者潜在的癌症、 复杂疾病进行筛查或 者鉴定， 实现疾病的早期发现、 早期治疗的目的。 实施例 1 胎儿有核红细胞的遗传性鉴定
1.1 5mL怀孕 8-14周的孕妇的肘静脉外周血， EDTA-K2抗凝， 4°C振荡保存， 24 h内 进行试验。
1.2胎儿有核红细胞的分离： 使用两步进行分离。 首先根据红白细胞尺寸的不同的进行 分离， 如 2.1中的芯片结构， 去除大多数的红细胞； 将回收的细胞通入到磁性分离芯片中， 利用红白细胞的磁性的不同进行分离。
第一芯片的结构： 微柱间的间距为 15 μηι, 每行微柱相对偏移 6.75 μηι。 使用深反应离 子刻蚀技术在硅片上构建微结构， 通道深度为 150 μηι, 使用玻璃片进行封合。
第二芯片的结构： 使用 Miltenyi LS Column(Miltenyi BioTech)，夕卜置 1.4T磁铁。
使用外部驱动设备将血液和緩冲液以 100 μΙ7ηώι的速度通入到第一个芯片， 緩冲液为 iDPBS (含有 1% BSA, 2mM EDTA)。 从出口处回收细胞。 回收的细胞经过离心 2000 rpm, 15min。 样品用 50 mM NaN02处理 10min。 然后通入到 Miltenyi LS Column中。 使用 iDPBS 緩慢冲洗， 洗去未吸附的白细胞。 然后移去磁铁， 使用緩冲液冲洗， 回收具有磁性的细胞。 使用 FITC-epsilon-球蛋白标记细胞， 在荧光显微镜下挑取单个的有核红细胞， 并将其转移 到^:孔阵列中。
1.3 细胞裂解： 使用 NaOH裂解细胞。
1.4核酸的提取和扩增： 直接使用 REPLI-g Mini/Midi kits(Qiagen)进行单细胞全基因组 扩增。
1.5测序： 全基因组扩增产物经过建库， 用 Illumina技术进行测序。
1.6数据分析： 进行单核甘酸多态性（SNP )分析， 进行单基因位点或者多基因位点突 变分析。
实施例 2: 胎儿有核红细胞的染色体非整倍体异常鉴定
2.1 取样： 同 1.1
2.2 胎儿有核红细胞的分离：
所使用的第一芯片的结构： 如图 5中的鹦鹉螺芯片， 有一个进样口和 6个出样口， 入 口通道宽度为 0.22 mm, 通道弯曲曲率按照一定比例逐渐放大，在出口处的通道宽度为 3.88 mm。 芯片通道结构 su-8 负光胶经光刻工艺制作掩膜， 再将 PDMS预聚物混合浇注在模具 上， 经过翻模而成。 使用玻璃片进行封合。
所使用的第二芯片的结构： 如图 6。 有两个入口和两个出口。 通道宽度为 0.5mm, 长度 为 50mm。 芯片通道结构 su-8负光胶经光刻工艺制作掩膜， 再将 PDMS预聚物混合浇注在 模具上， 经过翻模而成。 使用玻璃片进行封合。 在通道的一侧加上外部磁铁， 构建成磁性 芯片。
釆用两步进行分离。 第一步使用第一芯片去除大多数的红细胞； 再将含有有核红细胞 的混合细胞样品通入到第二芯片， 利用红白细胞磁性的不同进行分离。
使用外部驱动设备微量注射泵，驱动稀释浓度为 10%的血液（使用含 1%BSA的 iDPBS 緩冲液稀释）通入到通道中， 流速为 300 μΙ7ηώι。 在出口处收集细胞溶液。 回收的细胞经 过离心 2000rpm, 15min。 样品用 50 mM NaN02处理 10min。 将细胞从样品入口通入到芯片 中， 同时另一个入口通入 iDPBS緩冲液，二者流速均为 10 μΙ7ηώι。在出口处回收目的细胞。 细胞经过 FITC-CD71抗体染色， 挑取单个阳性细胞进行后续实验。
2.3 细胞裂解： 使用 KOH/DTT裂解细胞。
2.4核酸的提取和扩增： 直接釆用 DOP-PCR ( GenomePlex Single Cell Whole Genome Amplification Kit， Sigma ) 方法进行单个细胞基因组的扩增。
2.5测序： 全基因组扩增产物经过建库， 用 Illumina技术进行测序。
2.6数据分析： 进行拷贝数变异 (CNV)分析， 基因组结构变异 (SV)分析等。
实施例 3: 癌症患者循环肿瘤细胞 (CTCs)筛查
3.1 取样： 抽取肿瘤患者的肘静脉外周血 10 mL , EDTA-K2抗凝。 血样 4 °C保存， 24h 内进行检测。
3.2 CTCs的分离： 使用两步芯片进行分离。 第一步根据细胞的大小进行分离， 如图 1 中的芯片结构， 去除大多数的红细胞和部分外细胞； 第二步釆用 EpCAM抗体捕获 CTCs细 胞。 具体地，
第一芯片的结构： 微柱间的间距为 20 μηι, 每行微柱相对偏移 6 μηι。 使用深反应离子 刻蚀技术在硅片上构建^:结构， 通道深度为 150 mm, 使用玻璃片进行封合。
第二芯片的结构： 使用 Miltenyi LS/MS Column(Miltenyi BioTech) , 捕获 CTCs。
使用外部驱动设备将血液和緩冲液以 100 μΙ7ηώι的速度通入到第一个芯片， 緩冲液为 iDPBS (含有 1% BSA, 2mM EDTA)。从出口处回收细胞。回收的细胞经过离心 300g, 10 min。 然后将连接有 CD326(EpCAM)抗体的磁珠与细胞混合孵育。 使用 iDPBS冲洗。 将细胞和磁 珠混合物通入到 Miltenyi LS/MS Column中。 使用 iDPBS緩慢冲洗， 洗去未吸附的白细胞。 然后移去磁铁， 使用緩冲液冲洗， 回收具有磁性的细胞。 并对 CTCs细胞进行计数。
3.3 细胞裂解： 使用裂解液 KOH或者 NaOH裂解细胞。
3.4核酸的提取和扩增： 将收集到的微量细胞按照 MDA的方法进行全基因组扩增。 3.5 测序： 全基因组扩增产物经过建库， 用 Illumina技术进行测序。
3.6数据分析： 进行单核甘酸多态性（SNP )分析， 进行单基因位点或者多基因位点突 变分析。 通过比较分离样本与其他正常细胞、 癌症实体瘤组织、 癌症相关单细胞的遗传变 异分析， 为以后的癌症的早期发现、 早期诊断及术后治疗提供证据。 实施例 4 胎儿有核红细胞的遗传性鉴定
4.1 同 1.1
4.2 同 1.2
4.3 同 1.3
4.4核酸的提取：使用磁珠（ Invitrogen, Dynabeads )的方法直接提取单细胞中的基因组。
4.5 测序： 直接使用 Oxford Nanopore Technologies进行单分子测序。
4.6 数据分析： 进行单核甘酸多态性（SNP )分析、 拷贝数变异 (CNV)分析， 基因组结 构变异 (SV)分析等 实施例 5 样品中循环肿瘤细胞（ CTCs ) 的自动化筛查
取样： 外周血 lOmL, EDTA-K2抗凝， 4°C振荡保存， 24h内进行试验。
使用微量注射泵将血液和 PBS緩冲液以层流的方式通入微流控芯片的分离富集单元， 通道内构建有规则排列的圆柱形的微柱， 微柱间距为 16 μηι, 血细胞通过与微柱碰撞， 按照 细胞尺寸将尺寸较小的红细胞分离出去， 尺寸较大的白细胞进入到下一分离通道。 通道内 置有连接有 anti-EpCAM抗体分子的免疫磁珠， 免疫磁珠通过与通道内流入的白细胞的碰 撞， 捕获到细胞表面特异性表达 EpCAM分子的 CTCs。 PBS冲洗， 去除非特异性结合细胞。 通入酶解液， 将 CTCs从磁珠上释放。
通过流体驱动，使细胞规则排列进入通道，通过气阔的开合，将单个 CTC 敫量 CTCs ) 截留在不同的小室中。
分离出去的红细胞和白细胞经控制， 少量细胞进入到各自的仅允许单个细胞排列通过 的通道， 通过气阔和液流控制， 将单个细胞（微量细胞）分隔到独立的小室， 作为对照备 用。
通过流体控制和气阔开合， 推动单个细胞（微量细胞)从各自的出口流出， 分别收集在 PCR管中。 收集到的单个细胞（微量细胞）按照 MDA的方法进行标准扩增。
收集扩增产物， 将 DNA片段随机打成小片段， 加上特定接头建立 DNA片段文库， 在 cBot上进行成簇扩增， 用于 Illumina Hiseq 2000测序。
数据分析， 基于高通量测序结果探测细胞基因组单核甘酸多态性及染色体结构变化， 寻找潜在的癌症驱动因素及候选位点。 建立癌细胞发育树， 追溯癌细胞进化发育过程。 通 过比较分离样本与其他正常细胞、 癌症实体瘤组织、 癌症相关单细胞的遗传变异分析， 为 以后的癌症的早期发现、 早期诊断及术后治疗提供证据。 实施例 6 胎儿有核红细胞的遗传性鉴定
取样： 5mL怀孕 14周的孕妇的外周血， EDTA-K2抗凝， 4°C振荡保存， 24h内进行试 验。
将血液通入到微磁流通道中， 在外加磁场的作用下， 根据血细胞中红细胞、 WRBCs具 有顺磁性， 白细胞反磁性的原理， 将红细胞（包括 ^RBCs )和白细胞分离， 分别进入不同 的通道。
红细胞 (包括 ^RBCs )进入到连接有 anti-CD71抗体的 柱通道内， 通过抗原抗体免 疫反应， 捕获到 ^RBCs。 将 fNRBCs从微柱上解吸附后， 操控其进入到控制通道中。
控制 ^ RBCs进入仅允许单细胞通过的通道。 在显 镜下， 通过液流和气阔的控制， 推 动单个 RBC依次进入到阵列收集管中。
控制血液中分离出来的白细胞和红细胞部分进入仅允许单个细胞通过的通道， 通过控 制， 将单个的白细胞、 活细胞推动进入阵列收集管中， 作为对照使用。
提取阵列收集管中的细胞 R A, 按照基于 T7 R A聚合酶的线性扩增方法进行转录本 扩增 , 具体过程参照 Eberwine, J. et al. Analysis of gene expression in single live neurons. Proc. Natl. Acad. Sci.USA89， 3010-3014 (1992).和 Van Gelder, R.N. et al. Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. USA87, 1663-1667 (1990). 进行。
收集扩增产物 cDNA, 根据 Illumina提供的文库构建文档， 制备文库， 在 cBot上进行 成簇扩增， 用于 Illumina Hiseq 2000测序。 数据分析。
基于高通量测序数据分析胎儿转录组特征， 为医疗分子诊断研究提供基础。
实施例 7 循环肿瘤细胞表达水平的研究
7.1 同 3.1
7.2 同 3.2
获得单个循环肿瘤细胞后， 后续 R A提取扩增过程及所用引物试剂等参照 Tang F, Barbacioru C, Nordman E, et al. RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nature Protocols. 2010;5(3):516-535.进行。
7.3 细胞裂解： 使用 NP40 裂解细胞。
7.4反转录： 直接加入反转录酶和引物进行反转录， 合成双链 DNA。
7.5 cDNA指数扩增： 进行第一轮 PCR反应， 使用 QIAquick PCR purification kit进行纯 化； 在将第一轮 PCR后纯化的产物进行第二轮扩增， 使用 QIAquick PCR purification kit 进行纯化。 凝胶回收 PCR产物中的 0.5-3 kb的片段。
7.6测序： cDNA扩增产物经过建库， 用 Illumina技术进行测序。
7.7数据分析： 将测序所得 reads比对到参考转录本， 根据比对所得信息进行转录本结构研 究， 转录本变异研究， 基因表达水平研究， 非编码区功能研究等。 工业实用性
根据本发明的实施例的微流控装置、 确定生物样品是否具有遗传变异的方法和确定生 物样品是否具有遗传变异的***， 能够有效地分离单细胞， 从而确定生物样品是否具有遗 传变异。
尽管本发明的具体实施方式已经得到详细的描述， 本领域技术人员将会理解。 根据已 经公开的所有教导， 可以对那些细节进行各种修改和替换， 这些改变均在本发明的保护范 围之内。 本发明的全部范围由所附权利要求及其任何等同物给出。
在本说明书的描述中， 参考术语 "一个实施例"、 "一些实施例"、 "示意性实施例"、 "示 例"、 "具体示例"、 或 "一些示例" 等的描述意指结合该实施例或示例描述的具体特征、 结 构、 材料或者特点包含于本发明的至少一个实施例或示例中。 在本说明书中， 对上述术语 的示意性表述不一定指的是相同的实施例或示例。 而且， 描述的具体特征、 结构、 材料或 者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。

Claims

权利要求书
1、 一种微流控装置， 其特征在于， 所述微流控装置具有适于分离单细胞的微流控通 道。
2、 根据权利要求 1所述的装置， 其特征在于， 所述微流控通道上形成有多个微柱。
3、 根据权利要求 2所述的装置， 其特征在于， 所述多个微柱在所述微流控通道上呈阵 列分布。
4、 根据权利要求 3所述的装置， 其特征在于， 在纵向上， 相邻两个微柱之间的距离为 10-100微米， 并且， 在横向上， 相邻两个微柱之间的距离为 10-100微米， 每行微柱相对偏 移 6-7微米。
5、 根据权利要求 2所述的装置， 其特征在于， 沿流体方向， 所述微柱的直径增大。
6、 根据权利要求 1所述的装置， 其特征在于， 所述微流控通道为弯曲管道， 所述弯曲 管道选自阿基米德螺线通道和鹦鹉螺线通道的至少一种， 所述鹦鹉螺线通道的入口宽度为 0.22毫米， 出口通道宽度为 3.88毫米。
7、 根据权利要求 1所述的装置， 其特征在于， 所述微流控装置进一步设置有磁体， 所 述微流控通道的宽度为 0.5毫米， 长度为 50毫米。
8、 一种确定生物样品是否具有遗传变异的方法， 其特征在于， 包括下列步骤： 利用权利要求 1-7任一项所述的微流控装置， 从生物样品分离细胞样本；
对所分离的细胞样本中所包含的遗传物质的至少一部分进行扩增， 以便获得扩增产物； 对所述扩增产物进行测序， 以便获得测序结果； 以及
基于所述测序结果， 确定所述生物样品是否具有遗传变异。
9、 根据权利要求 8所述的方法， 其特征在于， 所述遗传物质为所述细胞样本的单细胞 全基因组或全转录组， 基于所述测序结果， 确定所述生物样品是否具有遗传变异进一步包 括：
将所述测序结果与参考基因组或参考转录组进行比对； 以及
基于比对结果， 确定所述生物样品是否具有遗传变异。
10、 一种确定生物样品是否具有遗传变异的***， 其特征在于包括：
权利要求 1-7任一项所述的微流控装置， 所述微流控装置用于从生物样品分离细胞样 本；
扩增装置， 所述扩增装置与所述微流控装置相连， 并且适于对所分离的细胞样本中所 包含的遗传物质的至少一部分进行扩增， 以便获得扩增产物；
测序装置， 所述测序装置与所述扩增装置相连， 并且适于对所述扩增产物进行测序， 以便获得测序结果； 以及
分析装置， 所述分析装置与所述测序装置相连， 并且适于基于所述测序结果， 确定所 述生物样品是否具有遗传变异。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106047706A (zh) * 2016-06-15 2016-10-26 西北工业大学 一种基于单细胞捕获实现细胞定位培养芯片及其使用及制备方法
CN106148160A (zh) * 2015-03-27 2016-11-23 深圳华大基因科技服务有限公司 细胞分离装置和细胞分离方法

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104991055A (zh) * 2015-06-19 2015-10-21 大连理工大学 一种血栓即时检测poct产品中的血液样本延时流动仿生操控单元
CN107262171A (zh) * 2017-07-12 2017-10-20 华讯方舟科技有限公司 一种血液分离预处理芯片及血液分离装置
CN107702973A (zh) * 2017-09-08 2018-02-16 深圳市太赫兹科技创新研究院有限公司 一种全血血浆分离***及方法
CN109536590B (zh) * 2018-11-27 2021-10-29 中国科学院上海微***与信息技术研究所 一种基于微孔阵列芯片的单细胞基因检测方法
CN111363795A (zh) * 2018-12-26 2020-07-03 清华大学 一种单细胞全基因组测序的方法
CN109746064B (zh) * 2019-01-28 2021-10-15 武汉纺织大学 一种梯度磁场微流控芯片

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6277060B1 (en) * 1998-09-12 2001-08-21 Fresenius Ag Centrifuge chamber for a cell separator having a spiral separation chamber
CN101086504A (zh) * 2006-06-06 2007-12-12 北京大学 一种微流体离心芯片及其加工方法
US7390388B2 (en) * 2004-03-25 2008-06-24 Hewlett-Packard Development Company, L.P. Method of sorting cells on a biodevice
CN101643701A (zh) * 2009-07-23 2010-02-10 清华大学 基于免疫磁性分离技术的细胞分选微流控芯片及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
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
JP2009511001A (ja) * 2005-09-15 2009-03-19 アルテミス ヘルス，インク． 細胞及びその他の粒子を磁気濃縮するためのデバイス並びに方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6277060B1 (en) * 1998-09-12 2001-08-21 Fresenius Ag Centrifuge chamber for a cell separator having a spiral separation chamber
US7390388B2 (en) * 2004-03-25 2008-06-24 Hewlett-Packard Development Company, L.P. Method of sorting cells on a biodevice
CN101086504A (zh) * 2006-06-06 2007-12-12 北京大学 一种微流体离心芯片及其加工方法
CN101643701A (zh) * 2009-07-23 2010-02-10 清华大学 基于免疫磁性分离技术的细胞分选微流控芯片及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN, RUBING ET AL.: "Single-cell PCR: Technology and Application", CHEMISTRY OF LIFE, vol. 25, no. 1, 2005, pages 46 - 48 *
FAN, J.B. ET AL.: "DNA Microarrays, Part A: Array Platforms and Wet-Bench Protocols [3] Illumina Universal Bead Arrays.", METHODS IN ENZYMOLOGY, vol. 410, 29 August 2006 (2006-08-29), pages 57 - 73 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106148160A (zh) * 2015-03-27 2016-11-23 深圳华大基因科技服务有限公司 细胞分离装置和细胞分离方法
CN106148160B (zh) * 2015-03-27 2018-04-20 深圳华大基因科技服务有限公司 细胞分离装置和细胞分离方法
CN106047706A (zh) * 2016-06-15 2016-10-26 西北工业大学 一种基于单细胞捕获实现细胞定位培养芯片及其使用及制备方法

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