CN206033771U - A micro -fluidic chip that is used for single cell sorting and unicellular full genome to amplify - Google Patents

Abstract

The utility model provides a micro -fluidic chip that is used for single cell sorting and unicellular full genome to amplify, micro -fluidic chip is top cap, little valve control layer, valve block layer and runner layer from top to bottom respectively including stacking in proper order together and the four -layer structure of sealing each other, micro -fluidic chip's the external magnet in bottom. The utility model provides a with the chip is supporting is used for the system of cell screening, appraisal, single cell expansion and analysis. The utility model discloses a micro -fluidic chip and corresponding detecting system can be quick, simple and convenient and low -priced divide from a large amount of cells and elect the purpose cell to single cell to wherein carries out full genome DNA cloning and analysis.

Description

For unicellular sorting and the micro-fluidic chip of unicellular whole genome amplification

Technical field

The utility model is related to micro fluidic chip technical field, specifically, is related to one kind for unicellular sorting and list The micro-fluidic chip of cell whole genome amplification.

Background technology

Gene is the functional fragment for carrying hereditary information on DNA molecular, is the material of biotransfer hereditary information.The mankind The announcement of the fine collection of illustrative plates of genome, indicates that the development of modern medicine has stepped into genome Medical Era.Especially existing Generation medical treatment aspect, genome medical science to medical diagnosis on disease, malignant tumour, organ transplant, mental illness, angiocardiopathy, infectious disease, The material impact of the aspects such as pharmacy, Medical Ethics and gene therapy first meeting clue.It is mainly reflected in prediction risk, pre- In terms of anti-disease, scientific guidance life style and disease individual character treatment.

If cancer cell can be found in cellular level and carry out genetic analysis, carry out early treatment and personalized gene is accurate Treatment, cancer will no longer be incurable disease.But the restriction of insufficient sensitivity and sorting technology due to detection method, for a long time Since genetic analysis can only carry out cell colony analysis, therefrom obtain cell in average chemical information.Research shows, in sample Gene expression regulation between individual cells there is also difference, especially in cancer cell.And obtained by cell colony analysis Statistical average result, mask the difference between individual cells, and can be affected by a large amount of non-aim cells, make biology The development of the numerous areas such as and medical science is restricted.Therefore unicellular genetic analysis great disorders such as cancers is early diagnosed, Treatment, drug screening and cell physiological, the research of pathologic process are significant, have become one of focus of research at present.

Due to cell it is minimum, generally 5-500 μm of diameter, volume is fL to nL levels, constituent content few (fmol to zmol), Species is various, makes acquisition manipulate, expand and analyze single celled difficulty and increases.Also hold very much in the processing procedure of unicellular sample The loss and loss of sample is easily caused, subsequent analysis are impacted, success rate is relatively low.Due to unicellular DNA quantity extremely It is small, the amplification of DNA must be carried out before analyzing to which.And the full-length genome based on PCR (PCR) expands Increasing technology, in PCR courses of reaction, the difference of DNA fragmentation can affect the amplification efficiency of polymerase even result in enzyme slot or Depart from from template, it is impossible to intactly covering gene group, and can introduce many mistakes and non-specific amplifications product in sequence , there is amplification bias in thing, when original samples are very rare, substantially reduce amplification quality, and can only once pass through specific Primer pair individual gene site is checked, it is impossible to carry out many site amplifications and detection to same sample, it is impossible to carry out unicellular The analysis of full genome.And the new amplification method based on MDA (multiple displacement amplification), it is inclined that amplification is there is also under existing method Lean on and measure big, amplification poor quality phenomenon.

In prior art, still there is no integrated unicellular capture, identification, full genome amplification and the whole set process analyzed.

The content of the invention

The purpose of this utility model is to provide a kind of micro-fluidic with unicellular whole genome amplification for unicellular sorting Chip.

In order to realize the utility model purpose, one kind that the utility model is provided is used for unicellular sorting and unicellular full base Because of a group micro-fluidic chip for amplification, the micro-fluidic chip includes the four-layer structure for being stacked together successively and sealing against each other, by It is up to lower to be respectively top cover, micro-valve key-course, valve block layer and flow channel layer；The external magnet in bottom of the micro-fluidic chip；

The top cover is provided with a sample inlet, sample export, three air pump connectors and several samples Acquisition port；The sample inlet is connected with the sample holes insertion of the micro-valve key-course and valve block layer of lower floor, and with flow channel layer on The well connection of sprue；The sample export is connected with the outlet insertion of the micro-valve key-course and valve block layer of lower floor, And connect with the tap hole of sprue on flow channel layer；Each air pump connector respectively with every notch end on micro-valve key-course Corresponding air pump orifice；The each sample acquisition port is corresponding with each branch flow passage on flow channel layer respectively to collect hole connection, Through micro-valve key-course and valve block layer；I.e. each sample acquisition port corresponds respectively to each sample connector on micro-valve key-course；

The micro-valve key-course is provided with three parallel array groove structures, every groove include it is multiple be interconnected it is micro- Valve control structure unit, the end of every groove are provided with an air pump interface, and each air pump interface is located at corresponding gas on top cover respectively The lower section of pump connector；Wherein, groove is arranged on the top of corresponding micro-valve between the trapping region of flow channel layer and cracking zone, and one Bar groove is arranged on the top of corresponding micro-valve between the cracking zone of flow channel layer and amplification region, and another groove is arranged on flow channel layer The top of corresponding micro-valve between amplification region and collection hole；

The valve block layer is provided with sample holes and an outlet, and some receipts corresponding with flow channel layer collection hole Ji Kou；

The branch flow passage of some parallel arrangement that the flow channel layer is provided with a sprue and is connected with sprue； Sprue is respectively arranged at two ends with a well and a tap hole；Every branch flow passage includes trapping region, cracking zone, amplification Area and collection hole, are connected with micro-valve between each several part.Sprue is used for the flowing in and out of sample (as shown in figure 1, level Single flow channel, two ends are respectively as entrance and exit)；Branch flow passage is used for single celled capture, identification and gene magnification (as schemed Shown in 1, with multiple being connected with sprue side wall, parallel, strip structures).

The width of the micro-valve key-course fovea superior slot structure is 20～100 microns, is highly 10～100 microns；It is described micro- Valve control structure unit is 20～100 microns by height, and the length of side is 100～1000 microns of square composition.

The thickness of the valve block layer is 10～50 microns, is made up of a piece of complete film.

The sprue of the flow channel layer, which is highly 10～200 microns, and width is 50～2000 microns；Branch flow passage It it is highly 15～100 microns, wherein, trapping region is a length of 30～400 microns, a width of 30～200 microns of rectangle structure；Split Xie Qu is a length of 200～2000 microns, a width of 100～1000 microns of rectangle structure；Amplification region is a length of 400～4000 micro- Rice, a width of 200～2000 microns of rectangle structure；Collect hole a diameter of 400～1000 microns.

The micro-valve arranged on the branch flow passage of the flow channel layer, its top is away from 2～50 microns of valve block layer bottom surface.Micro-valve depth For 5～20 microns.

The top cover, micro-valve key-course, valve block layer and flow channel layer are made up of the elastomeric material of transparent, water proof, trapping.

Micro-fluidic chip of the present utility model can capture individual cells respectively, and realize that the fluorescence in situ to cell reflects Fixed, cracking and amplification.

Structural representations of the Fig. 1 for the utility model micro-fluidic chip.

The utility model also provides a kind of for cell screening, identification, unicellular amplification and the system analyzed, the system Including above-mentioned micro-fluidic chip, fluorescence probe connects specific biological probe, the fluorescence microscope of magnetic bead, image processing equipment, Syringe pump, air pump and constant water bath box etc..

Can be realized to single celled analysis using said system.Add in micro-fluidic chip and combine magnetic bead biology in advance The cell suspension sample of probe, aim cell under the influence of a magnetic field, to parallel arrangement, the miniflow with successive reaction chamber The branch flow passage motion of control chip, final single aim cell are trapped in the trapping region of each branch flow passage；By to core The individual cells of capture in the presence of liquid are sequentially entered cracking zone from trapping region and carry out cell and split by the control of micro-valve on piece Solution, carries out unicellular complete genome DNA amplification into amplification region, and gained amplified production is entered to be collected in hole, for downstream analysis.

The sample comes from the cell suspension of biological tissue, blood or in vitro culture.

The magnetic bead bioprobe is the antibody or polypeptide that marked magnetic bead, so as to carry out specificity to certain specific protein Identification.

As shown in figure 3, pre-processing to cell sample first, the specific biological probe of connection magnetic bead is added, is carried out It is common to be incubated so as to specifically bind with aim cell.Cell sample suspension, cell stream are passed through to the sprue of micro-fluidic chip During Jing microfluidic channels, acted on by external magnetic field, with the cell that combined of specific biological probe of connection magnetic bead by inflow side The branch flow passage in face, and the cell of uncombined magnetic bead can be flowed out from outlet with liquid.After sample injection is finished, then to miniflow One or more is passed through in control chip with fluorescently-labeled antibody or polypeptide, and antibody (or polypeptide) can be special with some cell surfaces Fixed antigen (or albumen) is combined, and observes the trapping region of runner from chip front side using fluorescence microscope, determines which capture To cell be aim cell, so as to identify to cell, and the runner that is located to which is marked.By controlling layer open Capture cell is flowed into cracking zone by trapping region and the connection valve (micro-valve) of cracking zone.Cell pyrolysis liquid is added by sprue, it is right Cell is cracked.Then the connection valve of cracking zone and amplification region is opened again, lysate product is passed into into amplification region, is added Multiple displacement amplification (MDA) reagent, closes all connection valves.Chip is put in constant water bath box, MDA amplifications are carried out.Finally Unicellular complete genome DNA amplified production is collected from hole is collected, subsequent gene analysis is carried out.

The utility model develops a kind of multiplelayer microstructure core by introducing micro-processing technology and microfluidic chip technology Piece, realize carries out unicellular screening, capture, identification, expands on unicellular full genome piece to cell sample.For single cell analysis Field provides a kind of new method, can be more efficient, more accurately, more high-throughout to filter out single aim cell and which is carried out Gene magnification.

The utility model has advantages below：

(1) realize to single celled sorting, situ identification and amplification in situ.In the molecule involved by the utility model In field of biology and area of medical diagnostics, several significant advantages are brought to single celled Accurate Analysis：First, it is unicellular Interior DNA content is few, be very easy to lose and lose, the utility model proposes lysisin situ, amplification technique, can be with maximum journey Loss of the reduction of degree in sample liquid transfer process.Secondly, the accuracy of analysis compared with conventional art, is substantially increased, Such that it is able to filter out few cell in a large amount of cells.Finally, traditional MDA amplification methods, cell amplification transfer ratio are high, Amplification poor quality, and MDA amplifications are carried out under small chamber, bias amount can be greatly reduced, amplification quality is improved, so as to subtract Few impact to subsequent gene analysis.

(2) realize to sample high flux, quick process.The utility model utilizes micro-processing technology, size to be much smaller than and adopt With the block form arresting structure and reaction chamber of traditional approach, multiple aim cells can be captured simultaneously, and at the place of block form Gene magnification is carried out simultaneously in reason runner, compare traditional-handwork method of operating, substantially increase the flux of process.Simultaneously as The micro-fluidic chip reaction chamber is less, and reaction is abundant, the time required to can greatling save agents useful for same and reaction.Additionally, this Utility model introduces microflow control technique, can design the fluid passage of a plurality of parallel connection as needed, while carrying out, greatly improve Processing speed.

(3) realize the Highgrade integration of multiple experimental procedures.The utility model will be loaded down with trivial details thin using microflow control technique Born of the same parents' screening, capture, identification, cracking and amplification step are integrated on a micro-fluidic chip, compared with original technology, are greatly reduced Operating procedure, so that also improve experiment success rate and reliability.Meanwhile, depend on chip and the simplification of Highgrade integration Operating procedure, shortens experimental period, improves sample treatment efficiency.

(4) chip and corresponding system of the utility model design are applied widely, high degree of automation.The utility model In involved cell processing unit, its size can be adjusted according to different demands, while its cell in parallel processes single The quantity of unit can also increase and decrease according to sample feature, to adapt to wider demand.And these change to those skilled in the art For, it is easily to realize and hardly increase cost；Optical identification and be based on syringe pump to reaction that the utility model is related to Control can by shooting, graphical analysis, single-chip microcomputer process and program etc. automation means complete, improve process speed Accuracy rate is substantially increased while spending.

Description of the drawings

Structural representations of the Fig. 1 for the utility model micro-fluidic chip.

Fig. 2 be the utility model embodiment 1 in Fig. 1 micro-fluidic chips flow channel layer along the profile of AA ', illustrate chip The cell chamber of middle trapping region, the cell chamber of cracking zone, the cell chamber of amplification region and collect the concrete knot of hole and micro-valve Structure.

Fig. 3 is cell capture, identification, cracking and the schematic flow sheet for expanding in the utility model embodiment 1, is illustrated micro- The different on off states of valve.

In figure, 1- top covers；2- air pump connectors；3- micro-valve key-courses；4- micro-valve control structure units；5- samples connect Mouthful；6- valve block layers；7- sample holes；8- flow channel layers；9- collects hole；The cell chamber of 10- amplification regions；The lumen of 11- cracking zones Room；The cell chamber of 12- trapping regions；13- micro-valves space；14- cells；15- micro-valves；16- sample collection mouths；17- sample inlets； 18- sample exports；19- collection ports；20- collects hole；21- wells；22- tap holes.

Specific embodiment

Following examples are used for illustrating the utility model, but are not limited to scope of the present utility model.If not referring in particular to Bright, the conventional meanses that technological means used is well known to those skilled in the art in embodiment are raw materials used to be commercially available business Product.

Embodiment 1 is used for the micro-fluidic chip of unicellular sorting and unicellular whole genome amplification

The micro-fluidic chip that the present embodiment is provided includes the four-layer structure for being stacked together successively and sealing against each other, by up to It is respectively down top cover, micro-valve key-course, valve block layer and flow channel layer.Magnet is equipped with outside the bottom of the micro-fluidic chip.

1st, top cover and micro-valve key-course

Top cover and micro-valve key-course：By connecting the Digital Control of air pump to which, complete to chip micro-valve on an off shape The switching of state, to reach the purpose being controlled to chip reaction.During due to identifying to cell, light is needed through control Layer, therefore key-course needs to adopt transparent material；Meanwhile, key-course needs to make interface and and the control that sample is flowed into and out The interface of air pump processed, so being required to perforate, and will realize the good closed of pipe that be in communication with the outside at opening；Additionally, The micro-valve control structure unit processed by micro-processing technology is additionally provided with the key-course, so needing using compatible existing The material of micro-processing technology；Finally, key-course needs and cap layer is sealed, and realizes sealing, so the choosing of material Select and will consider sealing-in problem.Preferred material is that those can make specific standard and shape with thermoplastic shaping, and is conveniently carried out The material of secondary operation.

The contour dimension of top cover and micro-valve key-course will be matched with flow channel layer.Key-course micro-valve control structure unit it is big Little and position will be matched with the micro-valve structure on flow channel layer.

In the preferred embodiment shown in Fig. 1 and Fig. 2, key-course adopts PDMS (dimethyl silicone polymer) material.Control The width of preparative layer interface channel is 100 microns, is highly 100 microns；The square that a height of 100 microns of micro-valve control zone, its side Length is divided into 200 microns and 300 microns two kinds according to the size of corresponding runner micro-valve.This is in the preferred embodiment The optimal conditions of the design of flow channel layer.When needing to control different size micro-valve, can voluntarily select to close by those skilled in the art Suitable control area size.

2nd, valve block layer

In the utility model, valve block layer is used for separating cell flow channel layer and micro-valve key-course, therefore the portion of material will have There is trapping water isolating, while valve block layer need to utilize the elastic characteristic of its own, aid in being controlled the micro-valve of chip, therefore The portion of material will have good elastic performance；Flow channel layer is made to be in communication with the outside additionally, valve block layer also needs to perforation.Finally, The thickness of valve block layer as far as possible thin (such as 10～50 microns), to reach the purpose for passing through cell, so the portion of material Want easy to process and have good physical property.

In the preferred embodiment shown in Fig. 1 and Fig. 2, valve block layer PDMS material is made through spin coating method, thickness For 20 microns.

3rd, flow channel layer

Cell flow channel layer is the major part of the chip, and the injection and flow direction for being mainly used in laboratory sample is limited, and is provided Microcavity room environmental needed for often step reaction.In order to adapt to the size of cell and the size of reaction chamber, it is necessary to be selected to compatibility The material of existing micro-processing technology.

In the preferred embodiment shown in Fig. 2, the sprue that two ends are connected with the external world is horizontally disposed with, which is highly 50 microns, 500 microns of width.Substantial amounts of capture and amplification unit are connected the side of sprue is parallel.Each unit Runner and chamber height are 50 microns.Arresting structure is a length of 200 microns, a width of 80 microns of rectangle structure；Cracking zone For a length of 800 microns, a width of 200 microns of rectangle structure；Amplification region is a length of 1600 microns, a width of 400 microns of rectangle Structure, finally, is connected with the external world by collecting hole.4 centimetres of the chip length of the present embodiment, it is wide 2 centimetres, it is high 0.3 centimetre, altogether side by side 80 amplification units of arrangement.Each part is connected with valve arrangement, for controlling the flowing of fluid and providing confined reaction chamber.

In the preferred embodiment, special valve arrangement is devised, its amplification sectional view is as shown in Fig. 2 in cracking chamber At the connection valve of room and amplification chamber, using secondary photoetching technique, the distance of 4 microns of etching, makes thin film separation layer certainly downwards So the structure of situation and cell flow channel layer has 4 microns of spacing.When malleation is given by air pump to film, film stress shape Become, closed with following flow channel layer completely, micro-valve is closed；Conversely, when to giving negative pressure by air pump to film, Film is close to the groove top of micro-valve key-course above, and micro-valve belongs to opening；When film is in nature situation, valve block With 4 microns of runner interlamellar spacing, valve is in half-open position, the now little material (liquid etc.) with 4 microns can by and cell is (big In 4 microns) can not pass through.Meanwhile, if introducing excessive magnetic bead when cell is expanded, impact can be produced on amplification.By 4 microns are less than in used magnetic bead, it is possible to which the materials such as the magnetic bead not combined with cell are filtered out by the valve of half-open position, Increase the accuracy and reliability of amplification.

For the design parameter of the chip runner, through optimum experimental, reach high efficiency and capture individual cells and optimal Cracking and expanding effect optimal result.

4th, method for sealing

In the preferred embodiment illustrated in fig. 1, top cover is made using glass；Micro-valve key-course, valve block layer and flow channel layer Material made using PDMS material.Using oxygen plasma aid in the method for bonding can be very good to realize PDMS and PDMS, Excellent sealing sealing-in between PDMS and glass.

In other embodiments, suitable envelope can be selected according to the material of key-course, valve block layer and flow channel layer Connect method.

5th, chip corollary system

The present embodiment also provide match with above-mentioned micro-fluidic chip for cell screening, identification, it is unicellular amplification and The system of analysis.In addition to micro-fluidic chip, in addition it is also necessary to magnetic bead antibody, fluorescence probe (Probe) is connected with the magnetic bead of probe, Fluorescence microscope, image processing equipment and pump, MDA amplifing reagents and constant water bath box etc. are constituting complete system.

The magnetic bead for being connected with probe is used for sorting cells, adds identification EpCAM (Epithelial cell in the sample Adhesion molecule epithelial cell adhesion molecules) antibody immune magnetic beads be incubated, make specific cell adherence magnetic Pearl.In the presence of external magnet, aim cell is sub-elected.

Fluorescence probe is used for identification of cell, in the preferred embodiment shown in Fig. 2, adopts and marked red fluorescence The CK-19 antibody of CD-45 antibody and Green Marker as fluorescence probe, for recognizing the CD-45 albumen of leukocyte surface, and angle Matter PROTEIN C K-19 is further identified to cell.In other embodiments, it is also possible to select different for different albumen Antibody or polypeptide as probe.

Fluorescence microscope is used for detecting whether the cell of trapping region has fluorescence that it is micro- that image processing equipment is used for analysis of fluorescence The image of mirror acquisition simultaneously sends instruction to air pump and syringe pump, carries out Automated condtrol to chip.Air pump is used for controlling micro-valve, notes Pump is penetrated for driving microfluid.

MDA amplifing reagents and constant water bath box unicellular carry out full-length genome expansion in situ on chip to what is isolated Increase.

6th, the concrete preparation method of chip

Following preparation method is to aid in skilled artisan understands that manufacture method of the present utility model, and is not Material to device described in the utility model, size and manufacture method make restriction.

Top cover：Using 4 inches of Pyrex7740 sheet glass (Corning Incorporated), according to long 4 centimetres, wide 2 centimetres of appearance and size Rectangular-shaped pieces are cut into, and multiple holes are beaten with ad-hoc location of the laser in every piece of small pieces.

Micro-valve key-course：Using 4 inch silicon wafer of N-type, required figure is transferred on 4 cun of silicons using photoetching process, (induction plasma is etched the ICP dry etchings that silicon chip after photoetching is commonly used using semiconductor, i.e., using sulfur hexafluoride and four The high energy plasma of fluorocarbons is etching silicon) perform etching processing.The convex surface chip contrary with required figure is etched as mould Tool.Afterwards the PDMS jellies for having configured are poured on the Chip mold for machining, are reacted through heat cross-linking so as to into For having resilient solid structure.The demoulding after its solidification is taken out, and excision PDMS does not have slotted part, according to long 4 centimetres, wide by 2 Centimetre appearance and size be cut into rectangular-shaped pieces, obtain final product micro-valve key-course.

Flow channel layer：Using 4 inch silicon wafer of N-type, after the flat shape of runner is made by lithography, go out 50 using ICP dry etchings The deep runner mold of micron, recycles secondary photoetching technique, etches 4 microns of deep micro-valve moulds.Pour liquid PDMS into groove In, the demoulding after its solidification is taken out, and according to growing 4 centimetres, wide 2 centimetres of appearance and size is cut into rectangular-shaped pieces, produces runner Layer.

Valve block layer：By the PDMS jellies for modulating, 4 inches of N-type silicon chip bases being applied to by spin-coating method on sol evenning machine On bottom, the PDMS film of 20 micron thickness is obtained by controlling rotating speed.Then put it into baking oven to react through heat cross-linking, From on silicon chip after the demoulding, obtain having resilient valve block layer.

The assembling of chip and bonding：By four parts of chip through ultrasonic cleaning etc. process after, using Plasma etc. from Top cover and key-course are bonded together by subprocessing surface first, then successively valve block layer and flow channel layer are bonded together, and are passed through After high-temperature baking auxiliary bonding, final complete chip is formed.

Before chip use, sterilization treatment is carried out to chip.Whole chip is soaked in 75% alcohol, while using Alcohol is filled chip internal by runner by syringe.After standing 12 hours, with PBS, the alcohol of residual is removed, is carried out Subsequent experimental.

Chip system：On the basis of micro-fluid chip, connect the stream socket of pump and top cover using common plastics tube (sample gateway), Air Valve Control hole and specimen collection well, and air pump and injection pump are connected with computer are controlled, and will Chip is placed under fluorescence microscope, when needing to carry out MDA amplifications, chip is sealed, complete by being put in thermostat water bath Into the structure of whole system.

7th, concrete application method

Micro-fluidic chip described in the utility model and corresponding detecting system are successfully applied to using following methods slender Born of the same parents operate.The concrete grammar of offer is to aid in skilled artisan understands that function of the present utility model and application process, And be not that the scope of application to device described in the utility model makes restriction.

The BALB/c mouse that body weight is 18-22g is taken, enters MCF-7 cancer cells in its oxter kind, treat that tumour growth is vertical to 1 When square centimetre, 1 milliliter of mouse tail blood is taken, dilute 5 times, after removing red blood cell and impurity, add EPCAM antibody immune magnetic beads to carry out Incubation, is passed through in micro-fluidic chip afterwards.In sample injection stage, such as Fig. 3 shown in (), by the control to external air pump, Shutoff valve (1), opens valve (2) (3).Tumour cell enters trapping region under magnetic fields, rests on valve (1) front end；Cell is caught After the completion of obtaining, the CD-45 antibody with red fluorescence and the CK-19 antibody with green fluorescence is passed through into chip, using glimmering Light microscope shooting image is simultaneously processed, and selects only green fluorescence, and the unit for not having red fluorescence is marked.Afterwards, (under the state, the material less than 4 microns is passed through with liquid, and is more than 4 microns of material, is such as followed in half-open position to make valve (2) Ring tumour cell, then being stuck in valve arrangement front end can not pass through), while opening valve (1) (3), in such as Fig. 3 shown in (), injection is thin Cellular lysate liquid, under action of a fluid, the cell in trapping region will flow into cracking zone, and be stuck in valve (2) front end, now crack Area is full of cell pyrolysis liquid；Valve (3) is opened, valve (1) (2) is simultaneously closed off, in such as Fig. 3 shown in (), chip 65 DEG C of water is put into into 10 minutes in bath, cell lysis is carried out；Hereafter, as shown in () in Fig. 3, valve (1) and valve (2) is opened, shutoff valve (3) is to core The MDA amplifing reagents that prepare of pressurization injection in piece, crack product of cell lysis in chamber can under fluid pressure, with MDA amplifing reagents enter chip amplification region together；After which is full of amplification region, valve (2) and valve (3) is completely closed, in such as Fig. 3 Shown in (), chip is put into 3 hours in 30 DEG C of water-baths carries out the amplification of gene M DA；Finally, as shown in () in Fig. 3, open Valve (3), the DNA product after amplification is collected by amplified production outlet, the downstream analysis such as DNA sequencing are carried out.

Although above having made detailed description with a general description of the specific embodiments to the utility model, But on the basis of the utility model, it can be made some modifications or improvements, this is aobvious and easy to those skilled in the art See.Therefore, the these modifications or improvements on the basis of without departing from the utility model spirit, belong to the utility model Claimed scope.

Claims (14)

1. the micro-fluidic chip of unicellular sorting and unicellular whole genome amplification is used for, it is characterised in that the micro-fluidic core Piece includes the four-layer structure for being stacked together successively and sealing against each other, from top to bottom respectively top cover, micro-valve key-course, valve block layer And flow channel layer；The external magnet in bottom of the micro-fluidic chip；

The top cover is provided with a sample inlet, sample export, three air pump connectors and several sample collections Mouthful；The sample inlet is connected with the sample holes insertion of the micro-valve key-course and valve block layer of lower floor, and with main flow on flow channel layer The well connection in road；The sample export is connected with the outlet insertion of the micro-valve key-course and valve block layer of lower floor, and with The tap hole connection of sprue on flow channel layer；Each air pump connector is corresponding to every notch end on micro-valve key-course respectively Air pump orifice；The each sample acquisition port is corresponding with each branch flow passage on flow channel layer respectively to collect hole connection, runs through Micro-valve key-course and valve block layer；

The micro-valve key-course is provided with three parallel array groove structures, and every groove includes multiple micro-valve controls being interconnected Construction unit processed, the end of every groove are provided with an air pump interface, and each air pump interface is located at corresponding air pump on top cover respectively and connects The lower section of interface；Wherein, groove is arranged on the top of corresponding micro-valve between the trapping region of flow channel layer and cracking zone, and one recessed Groove is arranged on the top of corresponding micro-valve between the cracking zone of flow channel layer and amplification region, and another groove is arranged on the amplification of flow channel layer The top of corresponding micro-valve between area and collection hole；

The valve block layer is provided with sample holes and an outlet, and some collections corresponding with flow channel layer collection hole Mouthful；

The branch flow passage of some parallel arrangement that the flow channel layer is provided with a sprue and is connected with sprue；Main flow Road is respectively arranged at two ends with a well and a tap hole；Every branch flow passage include trapping region, cracking zone, amplification region with And hole is collected, it is connected with micro-valve between each several part.

2. micro-fluidic chip according to claim 1, it is characterised in that the width of the micro-valve key-course fovea superior slot structure For 20～100 microns, it is highly 10～100 microns；The micro-valve control structure unit is 20～100 microns by height, the length of side For 100～1000 microns of square composition.

3. micro-fluidic chip according to claim 1 and 2, it is characterised in that the thickness of the valve block layer is 10～50 micro- Rice.

4. micro-fluidic chip according to claim 1 and 2, it is characterised in that the sprue of the flow channel layer, which is highly 10～200 microns, width is 50～2000 microns；The height of branch flow passage is 15～100 microns.

5. micro-fluidic chip according to claim 3, it is characterised in that the sprue of the flow channel layer, which is highly 10 ～200 microns, width is 50～2000 microns；The height of branch flow passage is 15～100 microns.

6. micro-fluidic chip according to claim 4, it is characterised in that the branch flow passage of the flow channel layer, its trapping region It is a length of 30～400 microns, a width of 30～200 microns of rectangle structure；Cracking zone is a length of 200～2000 microns, a width of 100～1000 microns of rectangle structure；Amplification region is a length of 400～4000 microns, a width of 200～2000 microns of rectangle Structure；Collect hole a diameter of 400～1000 microns.

7. micro-fluidic chip according to claim 5, it is characterised in that the branch flow passage of the flow channel layer, its trapping region It is a length of 30～400 microns, a width of 30～200 microns of rectangle structure；Cracking zone is a length of 200～2000 microns, a width of 100～1000 microns of rectangle structure；Amplification region is a length of 400～4000 microns, a width of 200～2000 microns of rectangle Structure；Collect hole a diameter of 400～1000 microns.

8. the micro-fluidic chip according to 1,2,5,6,7 any one of claim, it is characterised in that the branch of the flow channel layer The micro-valve arranged on runner, away from 2～50 microns of valve block layer bottom surface, micro-valve depth is 5～20 microns on its top.

9. micro-fluidic chip according to claim 3, it is characterised in that what is arranged on the branch flow passage of the flow channel layer is micro- Valve, away from 2～50 microns of valve block layer bottom surface, micro-valve depth is 5～20 microns on its top.

10. micro-fluidic chip according to claim 4, it is characterised in that arrange on the branch flow passage of the flow channel layer Micro-valve, away from 2～50 microns of valve block layer bottom surface, micro-valve depth is 5～20 microns on its top.

11. micro-fluidic chips according to 1,2,5,6,7,9,10 any one of claim, it is characterised in that the top cover, Micro-valve key-course, valve block layer and flow channel layer are made up of the elastomeric material of transparent, water proof, trapping.

12. micro-fluidic chips according to claim 3, it is characterised in that the top cover, micro-valve key-course, valve block layer with And flow channel layer is made up of the elastomeric material of transparent, water proof, trapping.

13. micro-fluidic chips according to claim 4, it is characterised in that the top cover, micro-valve key-course, valve block layer with And flow channel layer is made up of the elastomeric material of transparent, water proof, trapping.

14. micro-fluidic chips according to claim 8, it is characterised in that the top cover, micro-valve key-course, valve block layer with And flow channel layer is made up of the elastomeric material of transparent, water proof, trapping.