CN206052034U - For expressing the micro-fluidic chip of the unicellular sorting and polygenic locuses detection of EGFR - Google Patents
For expressing the micro-fluidic chip of the unicellular sorting and polygenic locuses detection of EGFR Download PDFInfo
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Abstract
This utility model provides a kind of micro-fluidic chip of the unicellular sorting and polygenic locuses detection for expressing EGFR, the micro-fluidic chip includes the four-layer structure for being stacked together successively and sealing against each other, from top to bottom respectively top cover, microfluidic channel layer, the substrate with microwell array and the base plate with cavity group.This utility model also provides a kind of system for EGFR gene multiple mutational site individual cell level detection supporting with the chip.Micro-fluidic chip of the present utility model and its corresponding detecting system can be quick, easy and cheap aim cell is sub-elected from a large amount of cells, and individual cells therein are carried out with EGFR polygenic locuses analyses.
Description
Technical field
This utility model is related to micro fluidic chip technical field, specifically, is related to a kind of the slender of Fang Yu expression EGFR
The micro-fluidic chip that born of the same parents sort and polygenic locuses are detected.
Background technology
In recent years, the M ＆ M of pulmonary carcinoma rises rapidly, becomes Chinese first big cancer, although treatment technology has
Large increase, but the survival rate of 5 years do not have clear improvement.In pulmonary carcinoma, nonsmall-cell lung cancer (NSCLC) is represented the most
Property, account for 80% or so.At present, the targeted therapy for EGFR becomes the focus of people's research, and serves good curative effect.
EGFR (Epidermal Growth Factor Receptor) is epithelium growth factor (EGF) cell propagation and signal transduction
Receptor.EGFR belongs to one kind of ErbB receptor family, and the family includes EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her
3 (ErbB-3) and Her4 (ErbB-4).At present it has proven convenient that the propagation of EGFR and tumor cell, angiogenesis, tumor invasion, turning
Move and apoptotic suppression is relevant.
As EGFR TYR kinases is the essential condition of signal transduction, therefore also become the important target point of oncotherapy
Son.TYR is blocked by selective enzyme inhibitor or monoclonal antibody competitive binding extracellular ligand binding site
Kinase activation, so as to suppress the activation of EGFR.And current research is primarily directed to the exon of the 18th, 19,20 and 21 of EGFR
Carry out.Since 2004 find epithelial growth factor receptor (EGFR) gene activation mutation (based on Del19 or L858R) with
Since the curative effect correlation of EGFR tyrosine kinase inhibitors (TKI) treatment nonsmall-cell lung cancer, existing multinomial Prospective Clinical grinds
Study carefully confirmation, the positive NSCLC patient of EGFR Activating mutations is significantly higher than EGFR wild types NSCLC trouble to the response rate of EGFR-TKI
Person, Progression free survival (PFS) phase and total existence (OS) phase also significantly extend.Therefore, also begin to based on patient EGFR in clinic
Abrupt climatic change is precisely treated.
But the restriction of the insufficient sensitivity and sorting technology due to detection method, for a long time EGFR genetic mutation point
Analysis can only carry out cell colony analysis.There are some researches show, the gene between different cells there is also difference especially in cancerous cell
In.And the statistical average result for obtaining is analyzed by cell colony, mask the difference between individual cells and can be subject to big
The impact of non-aim cell is measured, is restricted the development in many fields such as biology and medical science.
Traditional EGFR detections focus mostly in protein level, such as with SABC (IHC) and integrated enzyme reaction absorption (ELISA)
For the detection method for representing.But above two method can only substantially react the expression of EGFR albumen, directly can not 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 mutation is also used for
In EGFR abrupt climatic changes, including direct sequencing after gene amplification (DS), mutation enrichment PCR methods, amplification refractory mutation system
(ARMS) method, scorpion shape probe amplification retardance abruptly-changing system (SARM) method, colony hybridization method (CH) etc..But these technologies are also simply anti-
Answer sample populations information, it is impossible to detected in individual cells level.As cell is minimum, diameter is generally 5-500 μm, body
Product is fL to nL levels, and constituent content few (fmol to zmol), species are various so that obtains and manipulates, expands and analyze single celled
Difficulty is very big.Also the loss and loss of sample are easily caused in the processing procedure of unicellular sample, subsequent analysis is caused
Affect, success rate is relatively low.As the quantity of unicellular DNA is extremely small, the amplification of DNA before analyzing to which, must be carried out.And
Based on whole genome amplification technology of the thermal cycle based on PCR (polymerase chain reaction), during PCR, in original samples
When very rare, many mistakes and non-specific amplification product can be introduced in sequence, substantially reduces amplification quality, and one
It is secondary individual gene site to be checked by specific primer, it is impossible to many locus gene amplifications are carried out to same sample.
At present in published technology, without reference to highly integrated for the screening of EGFR tumor cells, identification, cracking,
What amplification and polygenic locuses were analyzed, so as to realize that many site EGFR genetic mutations on individual cell level are carried out to tumor cell
Detection.
The content of the invention
The purpose of this utility model be to provide a kind of unicellular sorting for expressing EGFR and polygenic locuses detection
Micro-fluidic chip.
In order to realize this utility model purpose, a kind of unicellular sorting for expressing EGFR that this utility model is provided and
The micro-fluidic chip of polygenic locuses detection, the micro-fluidic chip include the four layers of knot for being stacked together successively and sealing against each other
Structure, from top to bottom respectively top cover, microfluidic channel layer, substrate and the bottom with cavity group with microtrabeculae and microwell array
Plate；
The top cover is provided with a sample inlet, a sample export and a waste liquid port；The sample inlet with
The U-shaped main flow of the sample entry port connection on the U-shaped sprue of microfluidic channel layer, the sample export and microfluidic channel layer
Sample flow export connection on road, the waste liquid outlet are connected with the waste liquid port on the branch flow passage of microfluidic channel layer；
The 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；The leading portion of the sprue with sample entry port side is wider, is provided with filtering area, for filtering sample
Product, remove larger impurity；The stage casing of sprue is sorting area, for separating tumor cell；Master with sample flow export side
The back segment of runner is narrower, is provided with trapping region, carries out unicellular capture for limiting liquid flowing；Be additionally provided with sprue one with
The vertically disposed branch flow passage in sorting area, the other end of branch flow passage are provided with waste liquid port, and which is connected with the waste liquid outlet on top cover,
Outflow for waste liquid (as shown in figure 1, the different single flow channel of horizontal width, the upper and lower ends on the left side respectively as entrance and
Outlet, the right is waste liquid port)；The filtering area of the sprue, sort filtering area that area and trapping region be located on substrate respectively,
Above sorting area and trapping region；Sample flows on substrate, and runner is used for the direction of motion for limiting fluid sample；
The substrate with microtrabeculae and microwell array is provided with filtering area, sorting area and trapping region；Filtering area is by spacing
The micro-pillar array composition of the row pattern distribution of gradual change；Sorting area is made up of the unified micro-pillar array of size and spacing；Trapping region carries
The position of microwell array, all microtrabeculaes and micropore is matched with each subregion on corresponding microfluidic channel layer, 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 for sorting area is arranged on microfluidic channel floor
Sprue sorting area 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, and negative electrode and anode are arranged in pairs on microporous side wall；Each micropore is with being 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 area and
Trapping region；
The base plate with cavity group is provided with multiple cavities, and each cavity is made up of collecting chamber and amplification chamber, is collected
Chamber is connected by intercommunicating pore micropore corresponding with substrate trapping region, for the cell pyrolysis liquid in separate collection difference micropore；Receive
Use between collection chamber and amplification chamber and can be connected with the micro-valve of controlling switch；Amplification intracavity presets multiple displacement amplification (MDA) reagent
And/or PCR amplifing reagents, for the multiple mutational sites on the DNA in amplifying cells lysate and detection EGFR gene.
The channel depth that the microfluidic channel layer is provided with is 10～200 microns, and it is 1～10 that sprue filters sector width
Millimeter；Sprue sorting sector width is 1～10 millimeter, and sprue capture sector width is 10～5000 microns.Runner (i.e. microfluid
Passage) filtering area, sorting area and trapping region length and counterpart substrate on the length of each subregion match.Preferably, filtering area,
Sorting section length is 1000～22000 microns, and capture section length is 1000～42000 microns.
The substrate with microtrabeculae and microwell array, the electrode in each of which micropore can control whether which leads to respectively
Electricity.
The substrate with microtrabeculae and microwell array, the micro-pillar array of filtering area are by a diameter of 5～200 microns, a height of
10～200 microns of cylinder row pattern distribution composition, the spacing gradual change of its micro-pillar array is 50～500 microns；The microtrabeculae in sorting area
, by a diameter of 5～50 microns, a height of 10～200 microns of cylinder row pattern distribution composition, microtrabeculae spacing are micro- for 10～200 for array
Rice；The micropore depth of trapping region is 5～100 microns, is square or rectangular that the length of side is 5～100 microns, or a diameter of 5
～100 microns of circle.The 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 be 1000～20000 microns, capture section length be 1000～
40000 microns.
The base plate with cavity group, its cavity group variable amounts can be with multiple micropores one cavity of correspondence, it is also possible to
Each cavity one micropore of correspondence, so as to collect the cell pyrolysis liquid in different micropores respectively.
The top cover is transparent.The top cover, microfluidic channel layer, the substrate with microwell array and carry cavity
The base plate of group is made up of insulant, or non-insulating material by wrapped with insulation and make.
The sample comes from the cell suspension of biological tissue, blood or In vitro culture.
The base plate with cavity group, contained by its amplification intracavity in PCR amplifing reagents comprising EGFR gene the 18th, 19,
20 and/or 21 exon specific PCR amplimers.
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 this utility model micro-fluidic chip.
This utility model also provides a kind of system for the multiple mutational site individual cell level detections of EGFR gene, and this is
System is integrated with the functions such as the screening of EGFR tumor cells, identification, cracking, amplification and polygenic locuses analysis.The system includes
State micro-fluidic chip, fluorescent probe, fluorescence microscope, image processing equipment, syringe pump, micro-valve controller, constant water bath box, electricity
Pressure generator and PCR amplification instrument etc..
The fluorescent probe refers to the antibody or polypeptide that marked fluorescence molecule, can be used alone a kind of fluorescent probe
Realize the identification to single albumen (such as EGFR), it is also possible to while realizing using various fluorescent probes to various destination proteins
Identification.
Using said detecting system, it is possible to achieve the unicellular sorting of expression EGFR and the detection of polygenic locuses.Upwards
State stream plus cell suspension in micro-fluidic chip, cell filtering area on the substrate, big impurity and cell mass are filtered, cell
Sorting area is entered with unicellular form, tumor cell is sorted and enters trapping region, after all cells are both fallen within micropore, to
The molecular probe with fluorescence is passed through in micro-fluidic chip, microwell array is observed from chip front side using fluorescence microscope, for
The micropore applied voltage of fluorescence being sent, electric field being produced in cell both sides, the electric field causes cell membrane that rupture, cellular content occur
Flow out, by the passage of micropore bottom, flow in the collection chamber of lower floor's base plate, by the connection valve between collecting chamber and amplification chamber
Open, cellular content is mixed with multiple displacement amplification (MDA) reagent, carry out cell genomic dna amplification, finally add respectively
Enter the specific primer designed for EGFR gene different loci and enter performing PCR amplification, and amplified production is sequenced, dashed forward
Change information.
As illustrated in fig. 1 and 2, cell suspension is passed through in micro-fluidic chip, 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 can be with less than the individual cells of microtrabeculae spacing
By filtering area, for removing larger impurity in sample；It is when die grading area is flowed through, due to hydrodynamic interaction, different big
Flow direction of the little cell when microtrabeculae is flowed through is different, therefore, larger cell can be collected using different exit positions
(tumor cell) is into subsequent captured area, and less cell (non-tumor cell) is flowed out from waste liquid port.Into the swollen of trapping region
Oncocyte can be fallen in micropore in the presence of gravity and fluid.After all cells are both fallen within microwell array, then to micro-
It is passed through in fluidic chip with fluorescently-labeled EGFR antibody (or polypeptide), (or polypeptide) meeting of EGFR antibody and tumor cell surface
EGFR protein binding, using fluorescence microscope from chip front side observe microwell array, determine that the cell in which micropore is carried
EGFR albumen, so as to identify to cell.According to qualification result, the electrode applied voltage to specific micropore, in cell both sides
Electric field is produced, the electric field flows out membranolysises, intracellular fluid, by the passage of micropore bottom, flows into the chamber of lower floor's base plate
In vivo.Then the micro-valve for connecting collecting chamber and amplification chamber on connection bottom is opened, MDA reagents is mixed with intracellular DNA.By core
Piece is put in constant water bath box, carries out MDA amplifications.It is last that the gene outcome for having expanded is taken out from amplification chamber.Set using special
Four kinds of PCR primers of meter, carry out pcr gene amplification respectively to the gene outcome after MDA amplifications, and directly to pcr amplification product
Sequencing, detects the different abrupt informations of EGFR.
This utility model develops a kind of multiplelayer microstructure core by introducing micro-processing technology and microfluidic chip technology
Piece, realizing carries out expanding on unicellular screening, capture, identification and full genome piece for expressing EGFR tumor cells specimens, and
Its EGFR gene site is detected.A kind of new method is provided for the EGFR multisite mutations detection on individual cell level,
Can be more efficient, more accurately, more high-throughout multiple EGFR site mutations to individual cell level are detected.
This utility model has advantages below：
(1) sorting on individual cell level, situ identification, cracking are realized and is expanded, particularly in nonsmall-cell lung cancer
In the area of medical diagnostics of EGFR abrupt climatic changes, several significant advantages are brought to single celled Accurate Analysis：First, it is slender
The amount of DNA of intracellular seldom, is very easy to lose and loses, the utility model proposes lysisin situ, amplification technique, can be with maximum
Loss of the reduction of degree in sample liquid transfer process, the reliability of the detection for greatly improving；Next, and conventional art phase
Than substantially increasing the degree of accuracy of analysis, such that it is able to few cell is filtered out in a large amount of cells, and its EGFR being mutated
Information is analyzed；Finally, all of screening, capture and cleavage method are carried out based on physical principle, and authentication step is using special
Anisotropic fluorescent probe does not interfere with intracellular DNA recognizing the albumen of cell surface, subsequent gene is analyzed so as to reduce
Impact.
(2) realize the Highgrade integration of multiple experimental procedures, this 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 as to also improving experiment success rate and reliability.Meanwhile, depend on chip and the simplification of Highgrade integration
Operating procedure, optical identification and shooting, image can be passed through to the control reacted based on syringe pump that this utility model is related to
The automation means such as analysis, single-chip microcomputer process and programming are completed, and substantially increase accuracy rate, contract while processing speed is improved
Short experimental period, improves sample treatment efficiency.
(3) this utility model is provided micro-fluidic chip, corresponding detecting system and detection method, can be detected simultaneously
The different loci of EGFR gene, covers the overwhelming majority to clinical significant mutational site, applied widely, can expire completely
The current Clinical detection demand of foot.And as needed, other oncogenes can be detected by changing detection primer,
And these change it will be understood by those skilled in the art that be easily achieved and hardly increase cost, the core has been expanded significantly
The application of piece and detecting system.
Description of the drawings
Structural representations of the Fig. 1 for this utility model micro-fluidic chip.
Fig. 2 be this utility model embodiment 1 in Fig. 1 micro-fluidic chips substrate along the profile of AA ', illustrate in chip
The concrete structure of filtering area, sorting area and trapping region.
Fig. 3 is cell capture, identification and the process schematic for cracking in this utility model embodiment 1.
In figure, 1- is respectively sample inlet (E1) and sample export (E2)；2- top covers；3- waste liquid outlets；4-U shape microfluids
Passage (sprue)；5- carries the substrate of microtrabeculae and microwell array；6- sorts microtrabeculae；7- filters microtrabeculae；8- captures microwell array；
9- cellular content collecting chambers；Base plates of the 10- with cavity group；The preset chamber of 11-MDA reagents (amplification chamber)；12- controls micro-valve；
The unicellular capture micropores of 13-；14- cells；15- intercommunicating pores；Electrode in 16- micropores；17- branch flow passages；18- filtering areas；19-
Sorting area；20- trapping regions；21- waste liquid ports.
Specific embodiment
Following examples are used to illustrate this 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 are 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 micro-fluidic chip and its preparation of the unicellular sorting for expressing EGFR and polygenic locuses detection
Method
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, microfluidic channel layer, the substrate with microwell array and the base plate with cavity group.
1st, top cover and fluid passage
When identifying to cell, need light to pass through top cover, therefore top cover needs to adopt transparent material；Meanwhile, top
Lid needs to make the interface that fluid is flowed into and out, so being required to perforate；Finally, top cover needs and fluid passage sealing-in exists
Together, and realize sealing, so the compatibility with fluid passage will be considered in the selection of material.
Baseplate material can be the material that specific standard and shape are made by evaporation, cutting and thermoplastic shaping.It is especially excellent
Choosing is relatively thin and transparent insulating polymer.
Fluid passage plays a part of to constrain cell flowing, and its characteristic size is in millimeter rank, so need to adopt being easy to
Processing, with low cost, the material of good biocompatibility.
The contour dimension of top cover and fluid passage will be matched with microwell array substrate.The width of fluid passage equally also will
Match with the characteristic size (length of side or diameter) of structure on substrate.
In the preferred embodiment illustrated in fig. 1, top cover adopts glass material, and fluid passage adopts PDMS (poly- diformazans
Radical siloxane) material, the two is tightly connected by the way of bonding.In other embodiments, top cover and fluid passage also may be used
To adopt 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 are 10 millimeters；
Sorting area's runner is a length of 15 millimeters, and width is 5 millimeters；Trapping region runner is a length of 25 millimeters, a width of 4 millimeters, all runner height
40 microns are, this is the optimal conditionss for being applied to tumor cell.When needing to process other cells, can be by people in the art
Member voluntarily selects suitable microfluidic channel size.
2nd, the substrate with micro structure array
In the present embodiment, microwell array is the critical component for accommodating cell and processed to which.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
Silicon is selected as microtrabeculae and the material of microwell array, is because that silicon can be very good compatibility based on chemical wet etching and the micro Process of corrosion
Technique, so as to be 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 for finally machining needs to be insulation,
Adopt in preferred implementation shown in Fig. 2 and silicon materials surface is aoxidized to form the silicon dioxide layer of insulation to meet this
One requires.
In the preferred implementation shown in Fig. 2, the micro-pillar array of filtration zone is a diameter of 50 microns, a height of 40 microns.
Spacing between different rows is 200 microns, the microtrabeculae spacing in the 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 for sorting area 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 is the cube that the length of side is 25 microns, each two micropore
Between spacing be 25 microns.Each array group is 1 millimeter long, and the overall of microwell array is constituted by multiple groups, each array group
1 millimeter of spacing, 20 millimeters of whole capture array length, have 20000 micropores by a width of 4 millimeters.Above-mentioned is to be applied to tumor cell
Optimal conditionss.When needing to process other cells, suitable pore size and number can be voluntarily selected by those skilled in the art
Amount.
On the side wall of each micropore, electrode positioned opposite is designed with, respectively as negative electrode and anode, for cell
Cracking.Electrode can be made up of the complex of appropriate 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.When using various
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 good material of thing compatibility.
In micro-fluidic chip as shown in Figure 3, electrode material is chromium and gold.A layer thickness is sputtered first on substrate is
0.1 micron of chromium, then sputter the gold that a layer thickness is 0.5 micron.Then photoetching is carried out to whole substrate, and (sputtering and photoetching are
In semiconducter process commonly use method) obtain need electrode shape.Then erode unwanted in layer gold and layers of chrome
Part, the electrode needed for finally giving.Certainly, the method for the processing metal well known in the art such as plating may also be used for manufacture electricity
Pole.In embodiment as shown in Figure 3, electrode is made using chromium and Jin Lai, be because the bio-compatibility of gold very well, and chromium is
In order to increase the adhesion between gold and baseplate material.According to different demands, other conductive materials, 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 (and
Microwell array depth is identical), thickness is 0.6 micron.
In the bottom of each micropore, there is a passage for leading to underlying collection chamber, the passage plays stop cell and passes through
And allow the effect that cellular content passes through, so its characteristic size is less than the characteristic size of cell, in the preferred reality shown in Fig. 3
Apply in mode, the section of the passage is square, and the length of side is 5 microns.This is to be applied to EGFR nonsmall-cell lung cancer tumor cells
Optimal conditionss, both can ensure that cell did not passed through, it is also ensured that cell pyrolysis liquid is smooth to be passed through.When process is needed, other are thin
During born of the same parents, suitable channel size can be voluntarily selected by those skilled in the art.
3rd, base plate
In the present embodiment, on base plate, there is the microcavity for accommodating cell pyrolysis liquid.In preferred implementation shown in Fig. 1 and Fig. 2,
There is multiple cavities 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
Row are corresponding；Amplification chamber is arranged side by side with collecting chamber, the micro-valve connection of collecting chamber and amplification chamber controllable switch.
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
Very easily it is used as baseboard material.For when there is multiple cavities on base plate, silicon and PDMS are easier to process.
4th, 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.Using oxygen plasma aid in the method for bonding can be very good to realize glass and PDMS, silicon and PDMS it
Between excellent sealing sealing-in；The seal between silicon and glass can be realized using the method for anode linkage.
In other implementations, can according to top cover, microfluidic channel, microfluidic channel, base plate material selecting
Suitable method for sealing.
5th, chip corollary system
System for the multiple mutational site individual cell level detections of EGFR gene is also provided in the present embodiment.Except miniflow
Control chip outside, in addition it is also necessary to fluorescent probe (Probe), fluorescence microscope, image processing equipment, MDA amplifing reagents, syringe pump,
Voltage generator and follow-up PCR system and specific pcr amplification primer thing are constituting complete system.
Fluorescent probe is used for identification of cell, in the preferred implementation shown in Fig. 2, adopts and marked green fluorescence
(FITC) EGFR antibody as fluorescent probe, for the EGFR albumen on tumor cell surface.In other embodiments,
Different albumen can also be directed to different antibody or polypeptide are selected as probe.
Fluorescence microscope is used to detect whether the cell in microwell array has fluorescence that image processing equipment to be used for analysis of fluorescence
The image of microscope acquisition simultaneously sends instruction to voltage generator, and the electrode controlled in specific micropore completes cell lysis.
Syringe pump is used to drive microfluid；Voltage generator is used to produce voltage cell lysis.
MDA expands the whole genome amplification for cell DNA, is preset in the amplification chamber of detection chip.Cell lysis
After liquid collects collecting chamber, middle control valve is opened, both is mixed, the DNA in cell pyrolysis liquid is carried out into full genome
Group amplification.
PCR system and particular detection primer, for expanding amplification EGFR different locis then at the beginning of Jing MDA, are sequenced,
Detection abrupt information.In other embodiments, it is also possible to which designing different primers for different gene locis carries out other
The abrupt climatic change in site.
6th, the concrete manufacture method of chip
The micro-fluidic chip and detecting system are successfully produced using following two sets of different processing technology.For concrete
Manufacture method be to aid in skilled artisan understands that the technical solution of the utility model, and be not new to this practicality
The material of device described in type, size and manufacture method make restriction.
Manufacture method A：
Top cover：Using 4 inches of Pyrex7740 sheet glass (Corning Incorporated), according to long 4 centimetres, wide 2 centimetres of overall dimensions
Rectangular-shaped pieces are cut into, and three holes are made a call to ad-hoc location of the laser in every piece of small pieces.
Microfluidic channel：Using 4 inch silicon wafer of N-type, after the flat shape of runner is made by lithography, commonly used using quasiconductor
ICP dry etchings (induction plasma is etched, i.e., etch silicon using the high energy plasma of sulfur hexafluoride and carbon tetrafluoride) go out 40
The deep groove of micron.Liquid PDMS is poured in groove, the demoulding after its solidification is taken out, excision PDMS is more than part, according to long 4 lis
Rice, wide 2 centimetres of overall dimensions are cut into rectangular-shaped pieces, obtain final product microfluidic channel.
Substrate with silicon microtrabeculae and microwell array：Using 4 inch silicon wafer of N-type, the flat of microtrabeculae is made by lithography in front side of silicon wafer
Non- microtrabeculae region is all corroded 40 microns downwards using the method for potassium hydroxide wet etching, forms filtering area and divide by face position
Constituency structure.Then wet oxidation is adopted to which, one layer of 0.5 micron of thick oxide layer is obtained in silicon chip surface, then recycled
The technology of dry etching, etches downwards 25 microns of micropore in trapping region.0.1 micron thick of chromium metal is sputtered in front side of silicon wafer
Layer, sputters 0.5 micron of thick layer gold on chromium metal level, silicon chip is inclined 45 degree during sputtering and is rotated, to ensure metal pair
The covering of microporous side wall.Vacuum aided gluing and photoetching are carried out to front side of silicon wafer, the shape of electrode, then reuses needed for being formed
Liquor kalii iodide corrodes layer gold, corrodes layers of chrome using ammonium ceric nitrate, has thus obtained the electrode in micropore.Silicon chip back side is entered
Row photoetching, forms the channel position of silicon micropore bottom, using the ICP dry etchings break-through silicon chip, finally obtains complete silicon micro-
Hole array.
Base plate：Using 4 inches of Pyrex7740 sheet glass, the position of cavity is made by lithography, using Fluohydric acid. etching glass, shape
Into cavity group, control valve is added by collection chamber and amplification chamber connection.
Sealing-in：First by silicon substrate together with glass film plates anode linkage, then by long 4 centimetres, wide 2 centimetres of profile chi
It is very little to be cut into rectangular-shaped pieces, the bottom surface of clear glass top cover, the two sides of microfluidic channel, silicon substrate are processed using oxygen plasma
Top surface, they are bonded together successively, complete chip is finally obtained.
Chip system：On the basis of micro-fluid chip, the fluid connected using common plastics tube on pump and top cover is come in and gone out
Mouthful, the extraction of cable is carried out on the gold electrode exposed on substrate by ultrasonic bonding, image procossing is connected cables to and is set
On standby and voltage generator, then voltage generator and image processing equipment are linked together, and chip is placed on into fluorescence and shown
Under micro mirror, when needing MDA to expand, chip is sealed, be put in thermostat water bath, you can complete the structure of whole system.
Manufacture method B：
Top cover and microfluidic channel：It is using 4 inch silicon wafer of N-type, after the flat shape of runner is made by lithography, dry using ICP
Method etches 40 microns of deep grooves.Liquid PDMS is poured in groove, the demoulding after its solidification is taken out, according to long 4 centimetres, wide 2 lis
The overall dimensions of rice are cut into rectangular-shaped pieces, are punched in desired location using drill bit, and the top cover and microfluid for obtaining integral type leads to
Road.
Substrate with silicon microwell array：Using 4 inch silicon wafer of N-type, the plan-position of microtrabeculae is made by lithography in front side of silicon wafer,
Non- microtrabeculae area is all etched into 40 microns downwards using the method for ICP dry etchings, micro-column structure is formed.Then (changed using CVD
Learn vapour deposition) method, obtain one layer of 2 microns of thick oxide layer in silicon chip surface, recycle photoetching and dry etching technology,
In micropore area, 25 microns of etching is deep downwards.0.1 micron thick of chromium metal level is sputtered in front side of silicon wafer, is electroplated on chromium metal level
3 microns of thick layer gold, to ensure the covering to microporous side wall.Vacuum aided gluing and photoetching are carried out to front side of silicon wafer, institute is formed
The shape of electrode is needed, liquor kalii iodide corrosion layer gold is then reused, is corroded layers of chrome using ammonium ceric nitrate, obtain the electricity in micropore
Pole.Photoetching is carried out to silicon chip back side, the channel position of silicon micropore bottom is formed, using the laser ablation break-through silicon chip, is finally obtained
Obtain silicon microwell array completely.By 4 inch silicon wafers according to growing 4 centimetres, wide 2 centimetres of overall dimensions are cut into rectangular-shaped pieces.
Base plate：Process technology making out position based on glass template is corresponding with chip micropore area, with multiple chambers
Mould.PDMS is poured into 4 centimetres of demoulding cut growth after molding, wide 2 centimetres of rectangle adds control valve by collection chamber
With amplification chamber connection.
Sealing-in：With ultraviolet curing adhesive by microfluidic channel, silicon array substrate together with base plate successively seal bond,
Complete micro-fluidic chip is obtained.
Chip system：On the basis of micro-fluid chip, connect the fluid on pump and top cover using polyfluortetraethylene pipe and go out
Entrance, carries out the extraction of cable on substrate by ultrasonic bonding on the gold electrode for exposing, connect cables to voltage generation
On device, then voltage generator and image processing equipment are linked together, and chip is placed under fluorescence microscope, needed
When MDA is expanded, chip is sealed, is put in thermostat water bath, you can complete 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
The many site primers of born of the same parents' EGFR gene.The concrete grammar of offer is to aid in skilled artisan understands that work(of the present utility model
Energy and application process, and be not that the scope of application to device described in the utility model 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 into next portion
Point.To move downwards in the presence of fluid with microtrabeculae in less non-tumor cells such as sorting area, the leukocyte in blood,
Flow out from waste liquid port, and the tumor cell being relatively large in diameter will flow into trapping region from top, through standing, treat that cell capture is completed
Afterwards, the EGFR antibody with green fluorescence is passed through into chip to dye the cell for capturing, shot using fluorescence microscope
Image is simultaneously processed, and not fluorescent cell is untreated；The explanation of fluoresced green is that the purpose for expressing EGFR albumen swells
Oncocyte.Acknowledgement bit is postponed, and corresponding cell is powered and is cracked, and lysate enters following chamber by the intercommunicating pore under micropore
In room, the control valve of base plate is opened, and lysate is mixed with preset MDA amplifing reagents.Chip is put into into 30 DEG C of water bath with thermostatic control
Case is expanded 3 hours.Amplified production is then taken out, is 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 enters performing PCR amplification.Then amplified production is directly sequenced, is obtained abrupt information.
Although above having made detailed description with a general description of the specific embodiments to this utility model,
But on the basis of this 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 this utility model spirit, belong to this utility model
Claimed scope.
Claims (32)
1. it is used for expressing the micro-fluidic chip of the unicellular sorting of EGFR and polygenic locuses detection, it is characterised in that described micro-
Fluidic chip includes the four-layer structure for being stacked together successively and sealing against each other, from top to bottom respectively top cover, microfluidic channel
Layer, substrate and the base plate with cavity group with microtrabeculae and microwell array；
The top cover is provided with a sample inlet, a sample export and a waste liquid port；The sample inlet and miniflow
On the U-shaped sprue of the sample entry port connection on the U-shaped sprue of body channel layer, the sample export and microfluidic channel layer
The connection of sample flow export, the waste liquid outlet connected with the waste liquid port on the branch flow passage of microfluidic channel layer；
The 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；The leading portion of the sprue with sample entry port side is wider, is provided with filtering area, for filtered sample,
Remove larger impurity；The stage casing of sprue is sorting area, for separating tumor cell；Sprue with sample flow export side
Back segment it is narrower, be provided with trapping region, for limit liquid flowing carry out unicellular capture；One is additionally provided with sprue with sorting
The vertically disposed branch flow passage in area, the other end of branch flow passage are provided with waste liquid port, and which is connected with the waste liquid outlet on top cover, is used for
The outflow of waste liquid；The filtering area of the sprue, sort filtering area, sorting area that area and trapping region be located on substrate respectively and
Above trapping region；
The substrate with microtrabeculae and microwell array is provided with filtering area, sorting area and trapping region；Filtering area is by spacing gradual change
Row pattern distribution micro-pillar array composition；Sorting area is made up of the unified micro-pillar array of size and spacing；Trapping region carries micropore
Array, all of microtrabeculae and micropore are 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 microporous side wall；Each micropore is with the passage being connected with lower floor base plate；
The base plate with cavity group is provided with multiple cavities, and each cavity is made up of collecting chamber and amplification chamber, and collecting chamber leads to
Cross intercommunicating pore micropore corresponding with substrate trapping region to be connected, for the cell pyrolysis liquid in separate collection difference micropore；Collecting chamber
Connected with micro-valve and amplification chamber between；Amplification intracavity presets multiple displacement amplification reagent and/or PCR amplifing reagents, for expanding
Increase the multiple mutational sites on the DNA and detection EGFR gene in cell pyrolysis liquid.
2. micro-fluidic chip according to claim 1, it is characterised in that the passage depth that the microfluidic channel layer is provided with
Spend for 10～200 microns, it is 1～10 millimeter that sprue filters sector width；Sprue sorting sector width is 1～10 millimeter, main flow
Road capture sector width is 10～5000 microns.
3. micro-fluidic chip according to claim 2, it is characterised in that the filtration of sprue on the microfluidic channel layer
Area, sorting section length are 1000～22000 microns, and capture section length is 1000～42000 microns.
4. the micro-fluidic chip according to any one of claim 1-3, it is characterised in that described with microtrabeculae and microwell array
Substrate, the electrode in each of which micropore can control whether which is powered respectively.
5. the micro-fluidic chip according to any one of claim 1-3, it is characterised in that described with microtrabeculae and microwell array
Substrate, the micro-pillar array of filtering area by a diameter of 5～200 microns, a height of 10～200 microns of cylinder row pattern distribution composition,
The spacing gradual change of its micro-pillar array, is 50～500 microns；The micro-pillar array in sorting area by a diameter of 5～50 microns, a height of 10～
200 microns of cylinder row pattern distribution composition, microtrabeculae spacing is 10～200 microns；The micropore depth of trapping region is 5～100 microns,
It is square or rectangular that the length of side is 5～100 microns, or a diameter of 5～100 microns of circle.
6. micro-fluidic chip according to claim 4, it is characterised in that the substrate with microtrabeculae and microwell array,
By a diameter of 5～200 microns, a height of 10～200 microns of cylinder row pattern distribution is constituted the micro-pillar array of filtering area, its microtrabeculae battle array
The spacing gradual change of row, is 50～500 microns；The micro-pillar array in sorting area is by a diameter of 5～50 microns, a height of 10～200 microns
Cylinder row pattern distribution composition, microtrabeculae spacing be 10～200 microns；The micropore depth of trapping region is 5～100 microns, is the length of side
For 5～100 microns of square or rectangular, or a diameter of 5～100 microns of circle.
7. the micro-fluidic chip according to claim 1,2,3 or 6, it is characterised in that described with microtrabeculae and microwell array
Substrate on filtering area, sorting section length be 1000～20000 microns, capture section length be 1000～40000 microns.
8. micro-fluidic chip according to claim 4, it is characterised in that on the substrate with microtrabeculae and microwell array
Filtering area, sorting section length are 1000～20000 microns, and capture section length is 1000～40000 microns.
9. micro-fluidic chip according to claim 5, it is characterised in that on the substrate with microtrabeculae and microwell array
Filtering area, sorting section length are 1000～20000 microns, and capture section length is 1000～40000 microns.
10. the micro-fluidic chip according to claim 1,2,3,6,8 or 9, it is characterised in that the bottom with cavity group
Plate, its cavity group variable amounts can be with multiple micropores one cavity of correspondence, it is also possible to each cavity one micropore of correspondence, so as to
The cell pyrolysis liquid in different micropores is collected respectively.
11. micro-fluidic chips according to claim 4, it is characterised in that the base plate with cavity group, its cavity group
Variable amounts, can be with multiple micropores one cavity of correspondence, it is also possible to each cavity one micropore of correspondence, so as to collect difference respectively
Cell pyrolysis liquid in micropore.
12. micro-fluidic chips according to claim 5, it is characterised in that the base plate with cavity group, its cavity group
Variable amounts, can be with multiple micropores one cavity of correspondence, it is also possible to each cavity one micropore of correspondence, so as to collect difference respectively
Cell pyrolysis liquid in micropore.
13. micro-fluidic chips according to claim 7, it is characterised in that the base plate with cavity group, its cavity group
Variable amounts, can be with multiple micropores one cavity of correspondence, it is also possible to each cavity one micropore of correspondence, so as to collect difference respectively
Cell pyrolysis liquid in micropore.
14. micro-fluidic chips according to claim 1,2,3,6,8,9,11,12 or 13, it is characterised in that the top cover
It is transparent；The top cover, microfluidic channel layer, the substrate with microwell array and the base plate with cavity group are by insulation material
Material is made, or non-insulating material by wrapped with insulation and make.
15. micro-fluidic chips according to claim 4, it is characterised in that the top cover is transparent；It is the top cover, micro-
Fluid channel layer, the substrate with microwell array and the base plate with cavity group are made up of insulant, or by insulation material
Material is wrapped in non-insulating material and makes.
16. micro-fluidic chips according to claim 5, it is characterised in that the top cover is transparent；It is the top cover, micro-
Fluid channel layer, the substrate with microwell array and the base plate with cavity group are made up of insulant, or by insulation material
Material is wrapped in non-insulating material and makes.
17. micro-fluidic chips according to claim 7, it is characterised in that the top cover is transparent；It is the top cover, micro-
Fluid channel layer, the substrate with microwell array and the base plate with cavity group are made up of insulant, or by insulation material
Material is wrapped in non-insulating material and makes.
18. micro-fluidic chips according to claim 10, it is characterised in that the top cover is transparent；It is the top cover, micro-
Fluid channel layer, the substrate with microwell array and the base plate with cavity group are made up of insulant, or by insulation material
Material is wrapped in non-insulating material and makes.
19. micro-fluidic chips according to claim 14, it is characterised in that the top cover is transparent；It is the top cover, micro-
Fluid channel layer, the substrate with microwell array and the base plate with cavity group are made up of insulant, or by insulation material
Material is wrapped in non-insulating material and makes.
20. micro-fluidic chips according to claim 1,2,3,6,8,9,11,12,13,15,16,17,18 or 19, which is special
Levy and be, the sample comes from the cell suspension of biological tissue, blood or In vitro culture.
21. micro-fluidic chips according to claim 4, it is characterised in that the sample comes from biological tissue, blood
Or the cell suspension of In vitro culture.
22. micro-fluidic chips according to claim 5, it is characterised in that the sample comes from biological tissue, blood
Or the cell suspension of In vitro culture.
23. micro-fluidic chips according to claim 7, it is characterised in that the sample comes from biological tissue, blood
Or the cell suspension of In vitro culture.
24. micro-fluidic chips according to claim 10, it is characterised in that the sample comes from biological tissue, blood
The cell suspension of liquid or In vitro culture.
25. micro-fluidic chips according to claim 14, it is characterised in that the sample comes from biological tissue, blood
The cell suspension of liquid or In vitro culture.
26. according to claim 1,2,3,6,8,9,11,12,13,15,16,17,18,19,21,22,23,24 or 25
Micro-fluidic chip, it is characterised in that the base plate with cavity group, which is included in PCR amplifing reagents contained by amplification intracavity
The 18th, 19,20 and/or 21 exon specific PCR amplimer of EGFR gene.
27. micro-fluidic chips according to claim 4, it is characterised in that the base plate with cavity group, its amplification chamber
The 18th, 19,20 and/or 21 exon specific PCR amplimer of EGFR gene is included in interior contained PCR amplifing reagents.
28. micro-fluidic chips according to claim 5, it is characterised in that the base plate with cavity group, its amplification chamber
The 18th, 19,20 and/or 21 exon specific PCR amplimer of EGFR gene is included in interior contained PCR amplifing reagents.
29. micro-fluidic chips according to claim 7, it is characterised in that the base plate with cavity group, its amplification chamber
The 18th, 19,20 and/or 21 exon specific PCR amplimer of EGFR gene is included in interior contained PCR amplifing reagents.
30. micro-fluidic chips according to claim 10, it is characterised in that the base plate with cavity group, its amplification
The 18th, 19,20 and/or 21 exon specific PCR amplimer of EGFR gene is included in PCR amplifing reagents contained by intracavity.
31. micro-fluidic chips according to claim 14, it is characterised in that the base plate with cavity group, its amplification
The 18th, 19,20 and/or 21 exon specific PCR amplimer of EGFR gene is included in PCR amplifing reagents contained by intracavity.
32. micro-fluidic chips according to claim 20, it is characterised in that the base plate with cavity group, its amplification
The 18th, 19,20 and/or 21 exon specific PCR amplimer of EGFR gene is included in PCR amplifing reagents contained by intracavity.
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Effective date of registration: 20200514 Address after: 100080 No. 11, north of Haidian District, Beijing, Zhongguancun Patentee after: Beijing Institute of Nanoenergy and Nanosystems Address before: 100190 No. 11, north of Haidian District, Beijing, Zhongguancun Co-patentee before: BEIJING FANJIA YUANJIE BIOTECHNOLOGY Co.,Ltd. Patentee before: Beijing Institute of Nanoenergy and Nanosystems |