CN106148187A - For expressing unicellular sorting and the micro-fluidic chip of polygenic locus detection of EGFR - Google Patents
For expressing unicellular sorting and the micro-fluidic chip of polygenic locus detection of EGFR Download PDFInfo
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- CN106148187A CN106148187A CN201610576642.5A CN201610576642A CN106148187A CN 106148187 A CN106148187 A CN 106148187A CN 201610576642 A CN201610576642 A CN 201610576642A CN 106148187 A CN106148187 A CN 106148187A
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- G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
Abstract
The present invention provides a kind of unicellular sorting for expressing EGFR and the micro-fluidic chip of polygenic locus detection, described micro-fluidic chip includes the four-layer structure being stacked together successively and sealing against each other, the most respectively top cover, microfluidic channel layer, the substrate with microwell array and the base plate with cavity group.The present invention also provides for the system for the detection of EGFR gene multiple mutational site individual cell level that a kind of and described chip is supporting.The micro-fluidic chip of the present invention and corresponding detecting system thereof can be quick, easy and cheap from a large amount of cells, sub-elect purpose cell, and individual cells therein is carried out EGFR polygenic locus analysis.
Description
Technical field
The present invention relates to micro fluidic chip technical field, specifically, relate to a kind of Fang Yu and express unicellular point of EGFR
Choosing and the micro-fluidic chip of polygenic locus detection.
Background technology
In recent years, the M & M of pulmonary carcinoma rises rapidly, becomes China's the first big cancer, although treatment technology has
Large increase, but the survival rate of 5 years is not obviously improved.In pulmonary carcinoma, nonsmall-cell lung cancer (NSCLC) represents the most
Property, account for about 80%.At present, the targeted therapy for EGFR becomes the focus that people study, and serves good curative effect.
EGFR (Epidermal Growth Factor Receptor) is epithelium growth factor (EGF) cell proliferation and signal conduction
Receptor.EGFR belongs to the one of ErbB receptor family, and this family includes EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her
3 (ErbB-3) and Her 4 (ErbB-4).At present it has proven convenient that EGFR and the propagation of tumor cell, angiogenesis, tumor invasion, turn
Move and apoptotic suppression is relevant.
Owing to EGFR TYR kinases is the essential condition of signal conduction, the important target the most also becoming oncotherapy divides
Son.TYR is blocked by selective enzyme inhibitor or monoclonal antibody competitive binding extracellular ligand binding site
Kinase activation, thus suppress the activation of EGFR.And current research is primarily directed to the 18th of EGFR the, 19,20 and 21 exons
Carry out.Since 2004 find epithelial growth factor receptor (EGFR) gene activation sudden change (based on Del19 or L858R) with
Since the curative effect of EGFR tyrosine kinase inhibitor (TKI) treatment nonsmall-cell lung cancer is correlated with, existing multinomial Prospective Clinical grinds
Studying carefully confirmation, the NSCLC patient of the EGFR Activating mutations positive is significantly higher than EGFR wild type NSCLC and suffers from the response rate of EGFR-TKI
Person, Progression free survival (PFS) phase and total existence (OS) phase significantly extend.Therefore, also begin in clinic based on patient EGFR
Abrupt climatic change is precisely treated.
But due to insufficient sensitivity and the restriction of sorting technology of detection method, EGFR genetic mutation is divided for a long time
Analysis can only carry out cell colony analysis.There are some researches show, the gene between different cells there is also difference especially at cancerous cell
In.And the statistical average result obtained by cell colony analysis, mask the difference between individual cells and can be subject to big
Measure the impact of non-purpose cell, make the development in a lot of field such as biology and medical science be restricted.
Traditional EGFR detection focuses mostly at protein level, as adsorbed (ELISA) with SABC (IHC) and integrated enzyme reaction
For the detection method represented.But above two method can only substantially react the expression of EGFR albumen, can not directly examine
The abrupt information of cls gene, it is impossible to directly instruct targeting medication.Therefore, the technology of large quantities of direct gene detection sudden change is also used for
In EGFR abrupt climatic change, including direct sequencing after gene amplification (DS), sudden change enrichment PCR method, amplification refractory mutation system
(ARMS) method, scorpion shape probe amplification retardance abruptly-changing system (SARM) method, colony hybridization method (CH) etc..But these technology are also the most anti-
Answer sample populations information, it is impossible to detect in individual cells level.Owing to cell is minimum, diameter is generally 5-500 μm, body
Amass as fL to nL level, constituent content few (fmol to zmol), of a great variety so that obtain handle, expand and analyze single celled
Difficulty is the biggest.In the processing procedure of unicellular sample, also easily cause loss and the loss of sample, subsequent analysis is caused
Impact, success rate is relatively low.Owing to the quantity of unicellular DNA is the most small, the amplification of DNA before it is analyzed, must be carried out.And
Based on thermal cycle whole genome amplification technology based on PCR (polymerase chain reaction), during PCR, in original samples
Time the most rare, a lot of mistake and non-specific amplification product can be introduced in sequence, make amplification quality be substantially reduced, and one
Secondary by specific primer, individual gene site can only be checked, it is impossible to same sample is carried out the amplification of many locus genes.
In current published technology, all screen without reference to highly integrated for EGFR tumor cell, identify, crack,
Amplification and polygenic locus are analyzed, thus realize the many sites EGFR genetic mutation carrying out on individual cell level to tumor cell
Detection.
Summary of the invention
It is an object of the invention to provide a kind of unicellular sorting for expressing EGFR and the miniflow of polygenic locus detection
Control chip.
It is a further object of the present invention to provide a kind of for EGFR gene multiple mutational site individual cell level detection be
System.
In order to realize the object of the invention, a kind of unicellular sorting for expressing EGFR that the present invention provides and polygenes position
The micro-fluidic chip of some detection, described micro-fluidic chip includes the four-layer structure being stacked together successively and sealing against each other, by upper
To lower be respectively top cover, microfluidic channel layer, with microtrabeculae and the substrate of microwell array and the base plate with cavity group；
Described top cover is provided with a sample inlet, a sample export and a waste liquid port；Described sample inlet with
Sample entry port connection on the U-shaped sprue of microfluidic channel layer, described sample export and the U-shaped main flow of microfluidic channel layer
Sample flow export connection on road, described waste liquid outlet connects with the waste liquid port on the branch flow passage of microfluidic channel layer；
Described microfluidic channel layer is provided with a U-shaped sprue, and one end of U-shaped sprue is provided with sample entry port, separately
One end is provided with sample flow export；Leading portion with the sprue of sample entry port side is wider, is provided with filtering area, is used for filtering sample
Product, remove bigger impurity；The stage casing of sprue is sorting district, is used for separating tumor cell；Master with sample flow export side
The back segment of runner is narrower, is provided with trapping region, is used for limiting liquid flowing and carries out unicellular capture；Be additionally provided with on sprue one with
The vertically disposed branch flow passage in sorting district, the other end of branch flow passage is provided with waste liquid port, and it connects with the waste liquid outlet on top cover,
For waste liquid outflow (as it is shown in figure 1, the single flow channel that horizontal width is different, the two ends up and down on the left side respectively as entrance and
Outlet, the right is waste liquid port)；The filtering area of described sprue, sorting district and trapping region lay respectively at the filtering area on substrate,
Above sorting district and trapping region；Sample flows on substrate, and runner is for limiting the direction of motion of fluid sample；
The described substrate with microtrabeculae and microwell array is provided with filtering area, sorting district and trapping region；Filtering area is by spacing
The micro-pillar array composition of row's shape distribution of gradual change；Sorting district is made up of the micro-pillar array that size and spacing are unified；Trapping region with
Microwell array, the position of all microtrabeculaes and micropore matches with each subregion on corresponding microfluidic channel layer, and i.e. filtering area is micro-
Post array is arranged on below the sprue filtering area of microfluidic channel layer, and the micro-pillar array in sorting district is arranged on microfluidic channel floor
Sprue sorting district below, the microwell array of trapping region is arranged on (Fig. 1) below the sprue trapping region of microfluidic channel layer,
The inside of micropore is provided with electrode, negative electrode and anode and is arranged in pairs on micropore sidewall；Each micropore has and is connected with lower floor base plate
Logical passage (i.e. intercommunicating pore)；Each subregion is connected by runner, sequentially enter under action of a fluid filtering area, sorting district and
Trapping region；
The described base plate with cavity group is provided with multiple cavity, and each cavity is made up of collecting chamber and amplification chamber, collects
Chamber is connected by the micropore that intercommunicating pore is corresponding with substrate trapping region, the cell pyrolysis liquid in separate collection difference micropore；Receive
Connect with the micro-valve that can control switch between collection chamber and amplification chamber；Amplification intracavity presets multiple displacement amplification (MDA) reagent
And/or PCR amplifing reagent, the DNA in amplifying cells lysate and the multiple mutational sites in detection EGFR gene.
The channel depth that described microfluidic channel layer is provided with is 10～200 microns, and sprue filtering area width is 1～10
Millimeter；Sprue sorting sector width is 1～10 millimeter, and sprue trapping region width is 10～5000 microns.Runner (i.e. microfluid
Passage) filtering area, the length of each subregion matches with on counterpart substrate for sorting district and the length of trapping region.Preferably, filtering area,
Sorting section length is 1000～22000 microns, a length of 1000～42000 microns of trapping region.
Described with microtrabeculae with the substrate of microwell array, the electrode in each of which micropore can control whether it leads to respectively
Electricity.
Described with microtrabeculae with the substrate of microwell array, the micro-pillar array of filtering area is by a diameter of 5～200 microns, a height of
Cylinder row's shape distribution composition of 10～200 microns, the spacing gradual change of its micro-pillar array, is 50～500 microns；The microtrabeculae in sorting district
Array is by a diameter of 5～50 microns, and the cylinder of a height of 10～200 microns row's shape distribution composition, microtrabeculae spacing is 10～200 micro-
Rice；The micropore depth of trapping region is 5～100 microns, be the length of side be the square or rectangular of 5～100 microns, or a diameter of 5
～the circle of 100 microns.Described microwell array, the passage (intercommunicating pore) a diameter of 1 that each of which micropore is connected with lower floor cavity
～10 microns.
Preferably, the filtering area of substrate, sorting section length are 1000～20000 microns, trapping region a length of 1000～
40000 microns.
The described base plate with cavity group, its cavity group variable amounts, can be with the corresponding cavity of multiple micropores, it is also possible to
The corresponding micropore of each cavity, thus collect the cell pyrolysis liquid in different micropore respectively.
Described top cover is transparent.Described top cover, microfluidic channel layer, with the substrate of microwell array and with cavity
The base plate of group is made up of insulant, or non-insulating material by wrapped with insulation and make.
Described sample comes from the cell suspension of biological tissue, blood or In vitro culture.
The described base plate with cavity group, PCR amplifing reagent comprises contained by its amplification intracavity EGFR gene the 18th, 19,
20 and/or 21 exon specific PCR amplimers.
The micro-fluidic chip of the present invention can capture individual cells respectively, and realize the qualification of the fluorescence in situ to cell, split
Solve and amplification.
Fig. 1 is the structural representation of micro-fluidic chip of the present invention.
The present invention also provides for a kind of system for the detection of EGFR gene multiple mutational site individual cell level, this system collection
Become EGFR tumor cell to screen, identified, cracked, expanded and the function such as polygenic locus analysis.Described system includes above-mentioned micro-
Fluidic chip, fluorescent probe, fluorescence microscope, image processing equipment, syringe pump, micro-valve control, constant water bath box, voltage is sent out
Raw device and PCR amplification instrument etc..
Described fluorescent probe refers to marked the antibody of fluorescence molecule or polypeptide, can be used alone a kind of fluorescent probe and comes
Realize the qualification to single albumen (such as EGFR), it is also possible to use multiple fluorescent probe to realize multiple destination protein simultaneously
Identify.
The present invention further provides a kind of unicellular sorting for expressing EGFR and the method for polygenic locus detection, to
In above-mentioned micro-fluidic chip, stream adds cell suspension, and cell is filtering area on substrate, and big impurity and cell mass are filtered, carefully
Born of the same parents enter sorting district with unicellular form, and tumor cell is sorted entrance trapping region, after all cells both falls within micropore,
In micro-fluidic chip, it is passed through the molecular probe with fluorescence, uses fluorescence microscope to observe microwell array from chip front side, right
Applying voltage in the micropore sending fluorescence, produce electric field in cell both sides, this electric field makes cell membrane rupture, cell content
Logistics goes out, and by the passage bottom micropore, flows in the collection chamber of lower floor's base plate, by the connection between collecting chamber and amplification chamber
Valve is opened, and makes cellular content mix with multiple displacement amplification (MDA) reagent, carries out cell genomic dna amplification, finally distinguish
Add the specific primer for the design of EGFR gene different loci and carry out PCR amplification, and amplified production is checked order, it is thus achieved that
Abrupt information.
As illustrated in fig. 1 and 2, in micro-fluidic chip, it is passed through cell suspension, when sample flows through filtering area, due between microtrabeculae
Away from restriction, will be limited on micro-column structure more than the impurity of microtrabeculae spacing, only permissible less than the individual cells of microtrabeculae spacing
By filtering area, it is used for removing bigger impurity in sample；When flowing through die grading district, due to hydrodynamic interaction, different big
The little cell flow direction when flowing through microtrabeculae is different, therefore, utilizes different exit positions can collect bigger cell
(tumor cell) enters subsequent captured district, and less cell (non-tumor cell) flows out from waste liquid port.Enter the swollen of trapping region
Oncocyte can fall in micropore under the effect of gravity and fluid.After all cells both falls within microwell array, then to micro-
Fluidic chip is passed through with fluorescently-labeled EGFR antibody (or polypeptide), (or polypeptide) meeting of EGFR antibody and tumor cell surface
EGFR protein binding, use fluorescence microscope to observe microwell array from chip front side, determine cell in which micropore with
EGFR albumen, thus cell is identified.According to qualification result, the electrode of specific micropore is applied voltage, in cell both sides
Producing electric field, this electric field makes membranolysis, and intracellular fluid flows out, and by the passage bottom micropore, flows into the chamber of lower floor's base plate
Internal.Then open to connect and on bottom, connect collecting chamber and micro-valve in amplification chamber, MDA reagent is mixed with intracellular DNA.By core
Sheet is put in constant water bath box, carries out MDA amplification.Finally take out, from amplification chamber, the gene outcome expanded.Utilize special setting
Four kinds of PCR primer of meter, the gene outcome after expanding MDA carries out pcr gene amplification respectively, and directly to pcr amplification product
Order-checking, the different abrupt informations of detection EGFR.
The present invention, by introducing micro-processing technology and microfluidic chip technology, develops a kind of multiplelayer microstructure chip, real
Show and carried out unicellular screening for expression EGFR tumor cells specimens, capture, identify and expand on full genome sheet, and to it
EGFR gene site is detected.Detect for the EGFR multisite mutation on individual cell level and provide a kind of new method, it is possible to
More efficient, more accurately, more high-throughout multiple EGFR site mutations to individual cell level detect.
The invention have the advantages that
(1) achieve the sorting on individual cell level, situ identification, crack and expand, particularly at nonsmall-cell lung cancer
In the area of medical diagnostics of EGFR abrupt climatic change, single celled Accurate Analysis is brought several significant advantage: first, slender
The amount of DNA of intracellular is little, is very easy to lose and loss, the lysisin situ of present invention proposition, amplification technique, can be at utmost
Reduce loss in sample liquid transfer process, the reliability of the detection being greatly improved；Secondly, compare with conventional art, greatly
Improve greatly the degree of accuracy of analysis, such that it is able to filter out few cell in a large amount of cells, and to its EGFR abrupt information
It is analyzed；Finally, all of screening, capture and cleavage method are carried out based on physical principle, and authentication step utilizes specificity
Fluorescent probe identifies the albumen of cell surface, does not all interfere with intracellular DNA, thus reduces the shadow analyzing subsequent gene
Ring.
(2) achieving the Highgrade integration of multiple experimental procedure, the present invention utilizes microflow control technique by loaded down with trivial details cell sieve
Select, capture, identify, crack and amplification step is integrated on a micro-fluidic chip, compared with original technology, greatly reduce behaviour
Make step, thus also improve experiment success rate and reliability.Meanwhile, the chip of Highgrade integration and the operation of simplification are depended on
Step, the optical identification that the present invention relates to and based on syringe pump to reaction control can pass through shooting, graphical analysis, monolithic
Automatization's means such as machine process and programming complete, and substantially increase accuracy rate, shorten experiment while improving processing speed
Time, improve sample treatment efficiency.
(3) micro-fluidic chip, corresponding detecting system and the detection method that the present invention provides, can detect EGFR simultaneously
The different loci of gene, covers most to clinical significant mutational site, applied widely, can fully meet mesh
Front Clinical detection demand.And as required, by change detection primer, other oncogenes can be detected, and this
Change it will be understood by those skilled in the art that be easily achieved and increase cost hardly a bit, significantly expanded this chip and
The application of detecting system.
Accompanying drawing explanation
Fig. 1 is the structural representation of micro-fluidic chip of the present invention.
Fig. 2 is that in the embodiment of the present invention 1, the substrate of Fig. 1 micro-fluidic chip, along the profile of AA ', illustrates in chip and filters
District, sorting district and the concrete structure of trapping region.
The process schematic that Fig. 3 is cell capture in the embodiment of the present invention 1, identifies and cracks.
In figure, 1-is respectively sample inlet (E1) and sample export (E2)；2-top cover；3-waste liquid outlet；4-U shape microfluid
Passage (sprue)；5-is with microtrabeculae and the substrate of microwell array；6-sorts microtrabeculae；7-filters microtrabeculae；8-captures microwell array；
9-cellular content collecting chamber；10-is with the base plate of cavity group；The preset chamber of 11-MDA reagent (amplification chamber)；12-controls micro-valve；
13-unicellular capture micropore；14-cell；15-intercommunicating pore；Electrode in 16-micropore；17-branch flow passage；18-filtering area；19-
Sorting district；20-trapping region；21-waste liquid port.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
In the conventional means that is well known to those skilled in the art of technological means used, raw materials used be commercial goods.
Embodiment 1 is used for expressing the unicellular sorting of EGFR and the micro-fluidic chip of polygenic locus detection and preparation side thereof
Method
The micro-fluidic chip that the present embodiment provides includes the four-layer structure being stacked together successively and sealing against each other, by up to
It is respectively down top cover, microfluidic channel layer, the substrate with microwell array and the base plate with cavity group.
1, top cover and fluid passage
When identifying cell, needing light to pass through top cover, therefore top cover needs to use transparent material；Meanwhile, top
Lid needs to make the interface that fluid flows into and out, so being required to perforate；Finally, top cover needs and fluid passage sealing-in exists
Together, and realize sealing, so to consider the compatibility with fluid passage in the selection of material.
Baseplate material can be the material being made specific standard and shape by evaporation, cutting and thermoplastic shaping.The most excellent
Choosing is relatively thin and transparent insulating polymer.
The effect of about bundle cell flowing is played in fluid passage, and its characteristic size is in millimeter rank, so needing employing to be easy to
Processing, with low cost, the material of good biocompatibility.
The contour dimension of top cover and fluid passage to match with microwell array substrate.The width of fluid passage is the most also wanted
Match with the characteristic size (length of side or diameter) of structure on substrate.
In the preferred embodiment illustrated in fig. 1, top cover uses glass material, and fluid passage uses PDMS (poly-diformazan
Radical siloxane) material, the two uses the mode of bonding to be tightly connected.In other embodiments, also may be used in top cover and fluid passage
To use same material (such as PDMS) one-shot forming, it is not necessary to manufacture respectively and seal again.
In the preferred embodiment illustrated in fig. 1, a length of 10 millimeters of microfluid filtering area passage, width is 10 millimeters；
A length of 15 millimeters of sorting district runner, width is 5 millimeters；A length of 25 millimeters of trapping region runner, a width of 4 millimeters, all runner height
Being 40 microns, this is the optimal conditions being applied to tumor cell.When needs process other cell, can be by people in the art
Member selects suitable microfluidic channel size voluntarily.
2, with the substrate of micro structure array
In the present embodiment, microwell array is used to the critical component accommodating cell and processing it.In order to adapt to
The size of cell, it is necessary to be selected to the material of compatible existing micro-processing technology.In the preferred implementation shown in Fig. 2, choosing
Selecting silicon as microtrabeculae and the material of microwell array, being because silicon can well be compatible based on chemical wet etching and the micro Process of corrosion
Technique, thus it is readily available the microwell array being consistent with cell size.Those skilled in the art can also select according to demand
Machinable material such as glass, PDMS.It should be noted that the microwell array surface finally machined needs is insulation,
Preferred implementation shown in Fig. 2 use silicon dioxide layer silicon materials surface aoxidized to form insulation to meet this
One requirement.
In the preferred implementation shown in Fig. 2, a diameter of 50 microns of the micro-pillar array of filtration zone, a height of 40 microns.
Spacing between different rows is 200 microns, the microtrabeculae spacing in same row by graded arrangement (500 microns, 200 microns,
100 microns and 50 microns).
In the preferred implementation shown in Fig. 2, the micro-pillar array in sorting district is staggered as shown in the figure, each microtrabeculae
A diameter of 15 microns, spacing is 30 microns.
In the preferred implementation shown in Fig. 2, each micropore be the length of side be the cube of 25 microns, each two micropore
Between spacing be 25 microns.Each array group is 1 millimeter long, and the entirety of microwell array is constituted by multiple groups, each array group
Spacing 1 millimeter, whole capture array length 20 millimeters, a width of 4 millimeters, have 20000 micropores.Above-mentioned is to be applied to tumor cell
Optimal conditions.When needs process other cell, suitable pore size and number can be selected voluntarily by those skilled in the art
Amount.
On the sidewall of each micropore, it is designed with electrode positioned opposite, respectively as negative electrode and anode, for cell
Cracking.Electrode can be made up of the complex of suitable conductive material or these materials.Preferred material is that bio-compatibility is good
Material, such as gold, titanium and the PDMS (polydimethylsiloxane, a kind of conventional polymer) mixed with silver ion.Multiple when using
During material, such as a kind of conductive material (such as gold) is plated on another kind of conductive material (such as copper), and outermost material is preferably made a living
The compatible good material of thing.
In micro-fluidic chip as shown in Figure 3, electrode material is chromium and gold.First sputtering a layer thickness on substrate is
The chromium of 0.1 micron, then sputter the gold that a layer thickness is 0.5 micron.Then whole substrate carries out photoetching, and (sputtering and photoetching are
Method conventional in semiconducter process) obtain the electrode shape that needs.Then erode in layer gold and layers of chrome unwanted
Part, finally gives required electrode.Certainly, the method for the processing well known in the art such as plating metal may also be used for manufacturing electricity
Pole.In embodiment as shown in Figure 3, using chromium and Jin Lai to make electrode, the bio-compatibility being because gold is fine, and chromium is
In order to increase the adhesion between gold and baseplate material.According to different demands, other conductive material, such as aluminum, copper or
Conducting polymer may serve to manufacture electrode.In micro-fluidic chip as shown in Figure 3, electrode is wide 20 microns, deep 25 microns (with
The microwell array degree of depth is identical), thickness is 0.6 micron.
In the bottom of each micropore, having a passage leading to underlying collection chamber, this passage plays stop cell to be passed through
And allow the effect that cellular content passes through, and so its characteristic size is less than the characteristic size of cell, preferred real shown in Fig. 3
Executing in mode, the cross section of this passage is square, and the length of side is 5 microns.This is to be applied to EGFR nonsmall-cell lung cancer tumor cell
Optimal conditions, both can ensure that cell did not passed through, it is also ensured that cell pyrolysis liquid is smooth and easy to be passed through.When needs, to process other thin
During born of the same parents, suitable channel size can be selected voluntarily by those skilled in the art.
3, base plate
In the present embodiment, base plate has the microcavity accommodating cell pyrolysis liquid.In preferred implementation shown in Fig. 1 and Fig. 2,
Having multiple cavity group on base plate, each cavity group is made up of collecting chamber and amplification chamber；The collection chamber of base plate and micropore battle array above
Arrange corresponding；Amplification chamber is arranged side by side with collecting chamber, and micro-valve of collecting chamber and amplification chamber controllable switch connects.
In preferred implementation shown in Fig. 1 and Fig. 3, the material of base plate is glass.And silicon, PDMS or rustless steel can also
It is used as baseboard material very easily.For when there being multiple cavity on base plate, silicon and PDMS are more easy to processing.
4, method for sealing
In the preferred embodiment illustrated in fig. 1, top cover, microfluidic channel, array base palte, the material of base plate are respectively glass
Glass, PDMS, silicon, PDMS.The method using oxygen plasma auxiliary bonding can well realize glass and PDMS, silicon and PDMS it
Between excellent sealing sealing-in；The method using anode linkage can realize the seal between silicon and glass.
In other implementation, can select according to top cover, microfluidic channel, microfluidic channel, the material of base plate
Suitably method for sealing.
5, chip corollary system
The present embodiment also provides for the system for the detection of EGFR gene multiple mutational site individual cell level.Except miniflow
Control chip outside, in addition it is also necessary to fluorescent probe (Probe), fluorescence microscope, image processing equipment, MDA amplifing reagent, syringe pump,
Voltage generator and follow-up PCR system and specific pcr amplification primer thing constitute complete system.
Fluorescent probe is used for identification of cell, in the preferred implementation shown in Fig. 2, uses and marked green fluorescence
(FITC) EGFR antibody is as fluorescent probe, for the EGFR albumen on tumor cell surface.In other embodiments,
Different antibody or polypeptide can also be selected as probe for different albumen.
Whether fluorescence microscope has fluorescence for the cell detected in microwell array, and image processing equipment is used for analysis of fluorescence
Microscope obtain image and voltage generator is sent instruction, control the electrode in specific micropore complete cell cracking.
Syringe pump is used for driving microfluid；Voltage generator is used for producing voltage cell lysis.
MDA amplification, for the whole genome amplification of cell DNA, is preset in the amplification chamber of detection chip.Cell cracks
After liquid collects collecting chamber, open the control valve of centre, both are mixed, the DNA in cell pyrolysis liquid is carried out full genome
Group amplification.
PCR system and particular detection primer, for amplification through expanding EGFR different loci then at the beginning of MDA, check order,
Detection abrupt information.In other embodiments, it is also possible to design different primers for different gene locis and carry out other
The abrupt climatic change in site.
6, the concrete manufacture method of chip
Use following two to overlap different processing technology and successfully produce described micro-fluidic chip and detecting system.For specifically
Manufacture method be to aid in skilled artisan understands that technical scheme, and be not to device of the present invention
The material of part, size and manufacture method make restriction.
Manufacture method A:
Top cover: use 4 inches of Pyrex7740 sheet glass (Corning Incorporated), according to long 4 centimetres, the overall dimensions of wide 2 centimetres
It is cut into rectangular-shaped pieces, makes a call to three holes with laser at the ad-hoc location of every piece of small pieces.
Microfluidic channel: use N-type 4 inch silicon wafer, after making the flat shape of runner by lithography, uses quasiconductor to commonly use
ICP dry etching (induction plasma etches, and i.e. uses the high energy plasma of sulfur hexafluoride and carbon tetrafluoride to etch silicon) goes out 40
The groove that micron is deep.Liquid PDMS being poured in groove, after it solidifies, the demoulding is taken out, and excision PDMS is more than part, according to long 4 lis
Rice, the overall dimensions of wide 2 centimetres is cut into rectangular-shaped pieces, obtains microfluidic channel.
With silicon microtrabeculae and the substrate of microwell array: use N-type 4 inch silicon wafer, make the flat of microtrabeculae by lithography at front side of silicon wafer
Position, face, uses the method for potassium hydroxide wet etching that non-microtrabeculae region is corroded downwards 40 microns, forms filtering area and divides
Constituency structure.Then it is used wet oxidation, obtain one layer of 0.5 micron of thick oxide layer at silicon chip surface, then recycle
The technology of dry etching, etches downwards the micropore of 25 microns in trapping region.0.1 micron of thick chromium metal is sputtered at front side of silicon wafer
Layer, sputters 0.5 micron of thick layer gold on chromium metal level, silicon chip tilts during sputtering 45 degree and rotates, to ensure metal pair
The covering of micropore sidewall.Front side of silicon wafer is carried out vacuum aided gluing and photoetching, forms the shape of required electrode, then re-use
Liquor kalii iodide corrosion layer gold, uses ammonium ceric nitrate corrosion layers of chrome, has thus obtained the electrode in micropore.Silicon chip back side is entered
Row photoetching, forms the channel position bottom silicon micropore, uses ICP this silicon chip of dry etching break-through, finally obtains complete silicon micro-
Hole array.
Base plate: use 4 inches of Pyrex7740 sheet glass, makes the position of cavity by lithography, uses Fluohydric acid. etching glass, shape
Become cavity group, add control valve and collection chamber and amplification chamber are connected.
Sealing-in: first by silicon substrate together with glass film plates anode linkage, then by long 4 centimetres, the profile chi of wide 2 centimetres
The very little rectangular-shaped pieces that is cut into, uses oxygen plasma to process the bottom surface of clear glass top cover, the two sides of microfluidic channel, silicon substrate
End face, they are bonded together successively, final obtain complete chip.
Chip system: on the basis of micro-fluid chip, uses common plastics tube to connect the fluid on pump and top cover and comes in and goes out
Mouthful, the gold electrode exposed on substrate is carried out by ultrasonic bonding the extraction of cable, connects cables to image procossing and set
On standby and voltage generator, then voltage generator and image processing equipment are linked together, and chip is placed on fluorescence shows
Under micro mirror, when needs MDA expands, chip is sealed, puts into thermostat water bath, the structure of whole system can be completed.
Manufacture method B:
Top cover and microfluidic channel: use N-type 4 inch silicon wafer, after making the flat shape of runner by lithography, use ICP to do
Method etches 40 microns of deep grooves.Liquid PDMS being poured in groove, after it solidifies, the demoulding is taken out, according to long 4 centimetres, wide 2 lis
The overall dimensions of rice is cut into rectangular-shaped pieces, uses drill bit to punch in desired location, and the top cover and the microfluid that obtain integral type lead to
Road.
Substrate with silicon microwell array: use N-type 4 inch silicon wafer, make the plan-position of microtrabeculae at front side of silicon wafer by lithography,
Non-microtrabeculae district is etched downwards 40 microns by the method using ICP dry etching, forms micro-column structure.Then CVD is used (to change
Learn vapour deposition) method, obtain one layer of 2 microns of thick oxide layer at silicon chip surface, recycling photoetching and dry etching technology,
Etch downwards in micropore district 25 microns deep.Sputter 0.1 micron of thick chromium metal level at front side of silicon wafer, electroplate on chromium metal level
3 microns of thick layer gold, to ensure the covering to micropore sidewall.Front side of silicon wafer is carried out vacuum aided gluing and photoetching, forms institute
Need the shape of electrode, then re-use liquor kalii iodide corrosion layer gold, use ammonium ceric nitrate corrosion layers of chrome, obtain the electricity in micropore
Pole.Silicon chip back side is carried out photoetching, forms the channel position bottom silicon micropore, use this silicon chip of laser ablation break-through, finally obtain
Obtain silicon microwell array completely.By 4 inch silicon wafer according to long 4 centimetres, the overall dimensions of wide 2 centimetres is cut into rectangular-shaped pieces.
Base plate: it is corresponding with chip micropore district, with multiple chambers that process technology based on glass template produces position
Mould.PDMS is poured demoulding cut growth 4 centimetres after molding, the rectangle of wide 2 centimetres, adds control valve by collection chamber
Connect with amplification chamber.
Sealing-in: with ultraviolet curing adhesive by microfluidic channel, silicon array substrate together with base plate successively seal bond,
I.e. obtain complete micro-fluidic chip.
Chip system: on the basis of micro-fluid chip, the fluid using polyfluortetraethylene pipe to connect on pump and top cover goes out
Entrance, the gold electrode exposed on substrate is carried out the extraction of cable by ultrasonic bonding, connects cables to voltage and occurs
On device, then voltage generator and image processing equipment are linked together, and chip is placed under fluorescence microscope, at needs
During MDA amplification, chip is sealed, puts into thermostat water bath, the structure of whole system can be completed.
7, concrete application process
Following methods is used to be successfully applied to unicellular by micro-fluidic chip of the present invention and corresponding detecting system
EGFR gene multidigit point detects.The concrete grammar provided is to aid in skilled artisan understands that the function of the present invention and answering
By method, and it is not that the scope of application to device of the present invention makes restriction.
Tri-kinds of cells of NCI-1975, NCI-1650, A549 are mixed in undressed blood and makes mixing sample.Dilution
10 times, it is passed through in micro-fluidic chip.In filtering area, big cell impurities and cell cluster will be filtered, it is impossible to enter next portion
Point.In sorting district, the less non-tumor cell such as the leukocyte in blood will move downwards under the fluid effect with microtrabeculae,
Flow out from waste liquid port, and the tumor cell being relatively large in diameter will be from inflow trapping region, top, through standing, treats that cell capture completes
After, in chip, it is passed through the EGFR antibody with green fluorescence the cell of capture is dyeed, use fluorescence microscope shooting
Image also processes, and not fluorescent cell is untreated；The explanation of fluoresced green is that the purpose expressing EGFR albumen swells
Oncocyte.Acknowledgement bit postpones, and is cracked the energising of corresponding cell, and lysate enters following chamber by the intercommunicating pore under micropore
In room, opening the control valve of base plate, lysate mixes with preset MDA amplifing reagent.Chip is put into the water bath with thermostatic control of 30 DEG C
Case expands 3 hours.Then take out amplified production, be divided into 4 parts, be separately added into outside the EGFR gene the 18th, 19,20 and/21 of design
The specific primer of aobvious son amplification carries out PCR amplification.Then amplified production is directly checked order, obtain abrupt information.
Although, the present invention is described in detail the most with a general description of the specific embodiments, but
On the basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (10)
1. for expressing unicellular sorting and the micro-fluidic chip of polygenic locus detection of EGFR, it is characterised in that described micro-
Fluidic chip includes the four-layer structure being stacked together successively and sealing against each other, the most respectively top cover, microfluidic channel
Layer, with microtrabeculae and the substrate of microwell array and the base plate with cavity group；
Described top cover is provided with a sample inlet, a sample export and a waste liquid port；Described sample inlet and miniflow
Sample entry port connection on the U-shaped sprue of body channel layer, on the U-shaped sprue of described sample export and microfluidic channel layer
Sample flow export connection, described waste liquid outlet connects with the waste liquid port on the branch flow passage of microfluidic channel layer；
Described microfluidic channel layer is provided with a U-shaped sprue, and one end of U-shaped sprue is provided with sample entry port, the other end
It is provided with sample flow export；Leading portion with the sprue of sample entry port side is wider, is provided with filtering area, for filtered sample,
Remove bigger impurity；The stage casing of sprue is sorting district, is used for separating tumor cell；Sprue with sample flow export side
Back segment narrower, be provided with trapping region, be used for limiting liquid flowing and carry out unicellular capture；One and sorting it is additionally provided with on sprue
The vertically disposed branch flow passage in district, the other end of branch flow passage is provided with waste liquid port, and it connects with the waste liquid outlet on top cover, is used for
The outflow of waste liquid；The filtering area of described sprue, sorting district and trapping region lay respectively at the filtering area on substrate, sorting district and
Above trapping region；
The described substrate with microtrabeculae and microwell array is provided with filtering area, sorting district and trapping region；Filtering area is by spacing gradual change
Row shape distribution micro-pillar array composition；Sorting district is made up of the micro-pillar array that size and spacing are unified；Trapping region is with micropore
Array, all of microtrabeculae and micropore be arranged on below corresponding microfluidic channel layer, and the inside of micropore is provided with electrode, negative electrode and sun
Pole is arranged in pairs on micropore sidewall；Each micropore has the passage being connected with lower floor base plate；
The described base plate with cavity group is provided with multiple cavity, and each cavity is made up of collecting chamber and amplification chamber, and collecting chamber leads to
Cross the intercommunicating pore micropore corresponding with substrate trapping region to be connected, the cell pyrolysis liquid in separate collection difference micropore；Collecting chamber
And connect with micro-valve between amplification chamber；Amplification intracavity presets multiple displacement amplification reagent and/or PCR amplifing reagent, is used for expanding
Increase the DNA in cell pyrolysis liquid and the multiple mutational sites in detection EGFR gene.
Micro-fluidic chip the most according to claim 1, it is characterised in that the passage that described microfluidic channel layer is provided with is deep
Degree is 10～200 microns, and sprue filtering area width is 1～10 millimeter；Sprue sorting sector width is 1～10 millimeter, main flow
Trapping region, road width is 10～5000 microns.
Micro-fluidic chip the most according to claim 1 and 2, it is characterised in that described with microtrabeculae with the base of microwell array
Plate, the electrode in each of which micropore can control whether it is energized respectively.
4. according to the micro-fluidic chip described in any one of claim 1-3, it is characterised in that described with microtrabeculae with microwell array
Substrate, the micro-pillar array of filtering area by a diameter of 5～200 microns, the cylinder of a height of 10～200 microns row's shape distribution composition,
The spacing gradual change of its micro-pillar array, is 50～500 microns；Sorting district micro-pillar array by a diameter of 5～50 microns, a height of 10～
Cylinder row's shape distribution composition of 200 microns, microtrabeculae spacing is 10～200 microns；The micropore depth of trapping region is 5～100 microns,
Be the length of side be the square or rectangular of 5～100 microns, or the circle of a diameter of 5～100 microns.
5. according to the micro-fluidic chip described in any one of claim 1-4, it is characterised in that the described base plate with cavity group,
Its cavity group variable amounts, can be with the corresponding cavity of multiple micropores, it is also possible to the corresponding micropore of each cavity, thus respectively
Collect the cell pyrolysis liquid in different micropore.
6. according to the micro-fluidic chip described in any one of claim 1-5, it is characterised in that described top cover is transparent；Described
Top cover, microfluidic channel layer, it is made up of insulant with the substrate of microwell array and the base plate with cavity group, or by
Wrapped with insulation non-insulating material and is made.
7. according to the micro-fluidic chip described in any one of claim 1-6, it is characterised in that described sample comes from live body group
Knit, blood or the cell suspension of In vitro culture.
8. according to the micro-fluidic chip described in any one of claim 1-7, it is characterised in that the described base plate with cavity group,
PCR amplifing reagent contained by its amplification intracavity comprises the amplification of EGFR gene the 18th, 19,20 and/or 21 exon specific PCR draw
Thing.
9. for the system of EGFR gene multiple mutational site individual cell level detection, it is characterised in that described system includes power
Profit requires the micro-fluidic chip described in any one of 1-8, fluorescent probe, fluorescence microscope, image processing equipment, syringe pump, micro-valve
Controller, constant water bath box, voltage generator and PCR amplification instrument.
System the most according to claim 9, it is characterised in that described fluorescent probe refers to marked the anti-of fluorescence molecule
Body or polypeptide, can be used alone a kind of fluorescent probe and realize the qualification to single albumen EGFR, it is also possible to use many simultaneously
Plant fluorescent probe and realize the qualification to multiple destination protein.
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