CN105255703B - Micro-fluidic chip and its observation procedure for the migration observation of big sequence cell cut - Google Patents
Micro-fluidic chip and its observation procedure for the migration observation of big sequence cell cut Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
Abstract
The invention provides a kind of micro-fluidic chip and its observation procedure for the migration observation of big sequence cell cut.The micro-fluidic chip includes:Substrate and the functional layer being fixed on above substrate;Wherein, function cell array is formed in the functional layer, the upper surface of each function cell bottom substrate part is formed with the first electrode and second electrode electrically isolated in the function cell array.Micro-fluidic chip of the present invention once can be with multigroup parallel laboratory test, and flux is high, so as to greatly reduce experimental cost, realizes big sequence cell cut migration observation experiment.
Description
Technical field
The present invention relates to biological technical field, more particularly to it is a kind of for the micro-fluidic of big sequence cell cut migration observation
Chip and its observation procedure.
Background technology
Cell migration take part in the important physiology courses such as histoorgan formation, tissue damage reparation;Cell migration function is lost
Adjust, then can cause tumor cell invasion and transfer.Some disease (ischaemic cardiovasculars can be influenceed by promoting or suppressing cell migration
Pipe disease, tumour) occurrence and development with lapsing to, therefore study cell migration there is important theory significance and practical value.
At present, traditional measure cell migration method is with cell chemotaxis cell method (Boyden Chamber) and scarification is most
It is conventional.The former is made up of upper and lower two Room, and centre is separated with miillpore filter.Chemotactic factor (CF) adds lower room, forms concentration gradient, carefully
Born of the same parents are seeded in upper chamber, are influenceed by chemotactic factor (CF) concentration gradient, and opposite side is moved to through the micropore on film.Afterwards, cell is solid
Fixed, dyeing and counting, with its transfer ability of this determination.The latter is to mark one of cut on fused cell with liquid transfer gun head, is seen
Migration situation of the cell to scored area is examined, to judge the transfer ability of cell.
Micro-fluidic chip (microflow control technique), it is the product of micro-processing technology.Due to its channel size and cells of mamma animals
Linear dimension is comparable, and multidimensional network structure and the space characteristics of cell under physiological status are close, and have been widely used for cell
Biological study.Nie, which is equal to, proposes a kind of micro-fluidic chip that cell cut is formed using laminar flow for 2007, and see reference document 1.
Cheng proposed a kind of method that direct current combination Chemical modification realizes cell migration equal to 2008, and see reference document 2.
Compared with conventional method, the advantages that there is microflow control technique size can compare, and controllability is good, and precision is high.Those researchs illustrate
The feasibility of cell migration is studied by microflow control technique.
During the present invention is realized, it is found by the applicant that existing based on micro-fluidic cut technology, flux generally compared with
It is low, it is higher for the cost of a scratch experiment, it is difficult to widespread adoption.
Bibliography 1:On-Chip cell migration assay using microfluidic channels,
Biomaterials 28(2007)4017-4022;
Bibliography 2:An automatic and quantitative on-chip cell migration assay
using self-assembled monolayers combined with real-time cellular impedance
Sensing, Lab 0n a Chip-Miniaturisation for chemistry, biology&bioengineering,
Volume 8, Number 6, June 2008, Page 837-992.
The content of the invention
(1) technical problems to be solved
In view of above-mentioned technical problem, the invention provides a kind of micro-fluidic core for the migration observation of big sequence cell cut
Piece and its observation procedure, to realize big sequence cell cut migration observation experiment.
(2) technical scheme
According to an aspect of the invention, there is provided a kind of micro-fluidic core for the migration observation of big sequence cell cut
Piece.The micro-fluidic chip includes:Substrate and the functional layer being fixed on above substrate;Wherein, it is small that function is formed on a functional
Room array, the upper surface of each function cell bottom substrate part is formed with the first electricity electrically isolated in the function cell array
Pole and second electrode.
Preferably, in micro-fluidic chip of the present invention, first electrode is cut electrode, and second electrode is powered electrode, cut
Molecule of the modification with anti-protein adsorption functional group on electrode, the small interior of function has cell suspension;With anti-protein adsorption
The molecule of functional group prevents cell from being adsorbed on cut electrode;When being between powered electrode and cut electrode plus forward voltage
When, the molecule of anti-protein adsorption departs from from cut electrode, and cell can adsorb on cut electrode.
According to a further aspect of the invention, additionally providing one kind utilizes above-mentioned micro-fluidic chip to carry out cell migration observation
Method.This method includes:Step S202:With trypsin digestion and cell, and it is resuspended with culture medium, and by the thin of suspended state
Born of the same parents are seeded to the function cell of micro-fluidic chip;Step S204:The small indoor cell of function is cultivated, until cell is in cut electrode
Local adherent growth in addition;Step S206:Apply voltage between the cut electrode and powered electrode of cut cell, and continue
Preset time, make that there is the molecule of anti-protein adsorption functional group to depart from from cut electrode;Step S208:Removal contains anti-egg
The culture medium of white binding molecule, the culture medium of the chemotactic factor (CF) needed containing experiment is replaced with, photographed to record after preset time carefully
Situation of the born of the same parents to cut electrode transfer.
(3) beneficial effect
It can be seen from the above technical proposal that the present invention be used for big sequence cell cut migration observation micro-fluidic chip and
Its observation procedure has the advantages that:
(1) 384 groups of parallel laboratory tests once can be at most realized, flux is high, so as to greatly reduce experimental cost, also,
For in theory, chip can reuse, and used chip can carry out sulphur again after over cleaning and aseptic process
Alcohol is modified, and is put into scratch experiment, this greatly reduces experimental cost once more;
(2) cut border is controlled with electrode, the molecule with anti-protein adsorption functional group is removed with alive method,
Cut is consistent between each cut cell can be ensured, and cell and matrix are not damaged in the process, therefore migration results essence
It will definitely believe.
Brief description of the drawings
Fig. 1 is the schematic diagram for the micro-fluidic chip design principle that the present invention is used for the migration observation of big sequence cell cut;
Fig. 2 is the structural representation for the micro-fluidic chip for being used for the migration observation of big sequence cell cut according to the embodiment of the present invention
Figure;
Fig. 3 is the flow chart of micro-flow control chip preparation method shown in Fig. 2;
Fig. 4 is the profile that the cut cell after each step is performed in micro-flow control chip preparation method shown in Fig. 3;
Fig. 5 is the flow chart of micro-fluidic chip observation procedure shown in Fig. 2;
Fig. 6 is that the cut cell inner cell state after each step is performed in micro-fluidic chip observation procedure shown in Fig. 5
Schematic diagram.
Embodiment
Before the present invention is introduced, its design principle is illustrated first.It is poly- second to have a kind of functional group
The thiol molecule of glycol long-chain (PEG).PEG functional groups have the characteristic of anti-protein adsorption.Therefore, if will have PEG work(
The thiol molecule of energy group is modified onto metal electrode, it is possible to prevents cell absorption in electrode surface.Also, certain
Under voltage, thiol molecule can also depart from from metal electrode, so as to which cell again can be in electrode surface Absorption Growth.
Above-mentioned design principle, microfluidic chip technology are combined by the present invention with scratch experiment, there is provided one kind can be high
Flux, can be with the micro-fluidic chip of Real Time Observation cell migration, and the observation procedure of the micro-fluidic chip is provided simultaneously.
For the object, technical solutions and advantages of the present invention are more clearly understood, below in conjunction with specific embodiment, and reference
Accompanying drawing, the present invention is described in more detail.
In one exemplary embodiment of the present invention, there is provided a kind of for the micro- of big sequence cell cut migration observation
Fluidic chip.Fig. 2 is refer to, the micro-fluidic chip that the present embodiment is used for the migration observation of big sequence cell cut includes:Substrate with
And the functional layer being fixed on above substrate.
In the present embodiment, substrate uses clear glass.And in other embodiments of the present invention, organic modeling can also be used
Material, Al2O3Piece, MgO pieces are as substrate fabrication micro-fluidic chip.
In the present embodiment, functional layer is made by dimethyl silicone polymer (polydimethylsiloxane, abbreviation PDMS),
Thickness is 5mm, and thereon formed with cut cell array, totally 16 rows 24 arrange totally 384 square cut cells.
It refer in Fig. 2 C to scheme, the cut cell is the square chamber of upward opening, its length of side 3.7mm, adjacent two strokes
The center spacing 4.5mm of trace cell, it is consistent with the plate size of standard 384.In structure, the micro-fluidic chip and conventional porous plate
Technical compatibility, existing porous plate platform is adapted to completely.
It is understood that the shape of cross section of cut cell is not limited to square, it can also be rectangle, circle
Other rules such as shape, triangle or irregular figure.Also, the quantity of cut cell is also not limited to 384 or 96,
24th, the quantity in the hole of the other standards specification porous plates such as 12,6, or other quantity as defined in designer.
As shown in C in Fig. 2, each cut cell bottom is respectively provided with two kinds of electrodes-cut electrode spaced apart from each other and power-up
Electrode.Wherein, i.e., in upper surface formed with PEG.
The cut electrode of colleague's cut cell is sequentially connected, and in the left side of micro-fluidic chip and other row cut cells
Cut electrode is electrically connected with, and forms total cut electrode.Equally, the powered electrode of colleague's cut cell is sequentially connected, and in miniflow
The powered electrode on the right side and other row cut cells of controlling chip is electrically connected with, and forms total powered electrode.
, can be by total cut electrode on the left of micro-fluidic chip and total powered electrode on right side to every by being so designed that
One cut cell applies voltage, in the case of having solution in the small interior of cut, so that it may so that the PEG on cut electrode therein
Depart from, so that cell can grow in the upper surface of cut electrode.
It is understood that the connected mode of each small indoor electrode of cut is not limited to chip design part in the present invention
Described often row is connected, can also each column be connected or other connected modes.As long as ensure the positive pole and negative pole of all cut cells
It is respectively connected with and mutually disjoint connected mode, can uses.
It refer to C in Fig. 2 to scheme, cut electrode is located at cut cell middle position, isAu electrode layers.Also,
The cut electrode is electrically connected to the cut electrode for adjacent cut cell of going together by its connection electrode in the left and right sides.Cut
Electrode diametrically 1mm circle, and the connection electrode of the left and right sides is in strip-form, its width is 300 μm.
Fig. 1 is refer to, thiol molecule forms golden sulfide linkage in the side of hydrogen sulfide linkage, and is fixed on Au electrodes, i.e. cut electricity
The upper surface of pole is Thiolation, due to having polyethylene glycol long chain (PEG) on thiol molecule, therefore, has carefully in the small interior of cut
During born of the same parents' suspension, polyethylene glycol long chain (PEG) prevents cell from being adsorbed on the Au electrodes.
However, it is necessary to explanation, mercaptan and Au this reaction are reversible, under certain voltage effect, golden sulfide linkage
Fracture, mercaptan comes off, and in the case where no polyethylene glycol long chain (PEG) is disturbed, cell in cell suspension just again can be
Gold surface adherent growth.
In the present embodiment, powered electrode is Pt electrodes, and thickness isPositioned at the front and rear sides of cut electrode, and with
Cut electrode separates 400 μm.Also, both sides extend powered electrode to the left and right, the powered electrode electricity with other cut cells of going together
Property connection.
It should be noted that in addition to Pt electrodes, powered electrode can also use Au electrodes, silver electrode or copper electrode etc.
Metal electrode, its thickness can be betweenBetween.In addition, in order to strengthen Au electrodes and Pt electrodes and glass base
The combined with firmness at bottom, at both between substrate of glass, also deposition has titanium (Ti) tack coat.
In addition, the material of tack coat can be titanium (Ti) or chromium (Cr), as long as can strengthen electrode and glass base
The adhesiveness at bottom, also, be using non-glass substrate, can be without using tack coat;
It will be apparent to those skilled in the art that the pattern of each small interior of cut, powered electrode and cut electrode does not limit to
Shape in Fig. 2 shown in B figures, as long as leaving the glass surface of no electrode covering between cut electrode and powered electrode,
Realize the present invention.But generally the area of cut electrode is less than powered electrode.
The preparation method of micro-fluidic chip shown in Fig. 2 is illustrated below, refer to Fig. 3, the system of the micro-fluidic chip
Preparation Method includes:
Step S102:Make PDMS cuts cell array;
Step S102 is specific can be included again:
Sub-step S102a:PDMS formpistons are prepared by way of 3D printing;
The graphics of formpiston is drawn with three-dimensional drawing software first, inputs 3D printer software, printer will be beaten automatically
Go out the formpiston shown in A figures in Fig. 4.
It is understood that cast PDMS formpiston, a kind of not only method of 3D printing, used material is not yet
ABS plastic is confined to, any method that can form columnar arrays and material may serve to make PDMS formpistons, such as make metal
Formpiston.
Sub-step S102b:PDMS is poured into a mould in PDMS formpistons:
After PDMS mixes with curing agent 12: 1, stir, pour on the formpiston of 3D printing, 80 DEG C of bakings make it solid overnight
Change, as shown in B figures in Fig. 4.
Sub-step S102c:Overmolded obtains the PDMS functional layers with cut cell array:
PDMS overmoldeds after solidification, obtain 384 hole arrays, are for cut cell, as shown in C figures in Fig. 4.
Step S104:Cut electrode and powered electrode are formed on the glass substrate;
Step S104 is specific can be included again:
Sub-step S104a:The AZ1500 formpistons of Au electrodes are made on the glass substrate;
Sheet glass cleans successively in acetone, ethanol and deionized water, and surface Rotating with Uniform applies one layer after drying
AZ1500, put mask plate exposure.After developing in MIF300 developer solutions, glass table is exposed needing the position of sputtering electrode
Face, the position without sputtering electrode leaves the sacrifice layer of AZ1500 formation, as shown in D figures in Fig. 4.
Sub-step S104b:Ti/Au films are sputtered in the substrate of glass with AZ1500 formpistons;
One layer of Ti bottoming is first sputtered as tack coat, strengthens the adsorptivity of Au electrodes, layer of Au is then sputtered, such as E in Fig. 4
Shown in figure.
Sub-step S104c:The Ti/Au films outside cut electrode position are removed, form cut electrode;
Sheet glass is placed in acetone and soaked, ultrasound, until metal level is completely exfoliated, completes the making of Au electrodes, such as Fig. 4
Shown in middle F figures.
Sub-step S104d:Above-mentioned sub-step S104a~S104c is repeated, prepares the powered electrode of Ti/Pt films.
Step S106:The PDMS functional layers made and the substrate of glass for making Au electrodes and Pt electrodes are bonded, obtained
Complete chip, as shown in G figures in Fig. 4;
Step S108:In cut cell mercaptan is modified on cut electrode;
Modify before mercaptan, complete chip is first played into oxygen processing, to get rid of the impurity on Au surfaces, enhancing mercaptan modification
Effect.Then, as shown in H figures in Fig. 4, in each cut small indoor dropwise addition ethanol, deionized water or phosphate buffer
(PBS) thiol solution of dissolving, immersion a period of time, Au electrodes is reacted with mercaptan, form one layer of PEG.
Wherein, mercaptan can be combined only with gold electrode, form PEG.After PEG is formed, cell will only not have optionally
PEG surface adherent growth.
So far, the facture of microchip shown in Fig. 2 for the migration observation of big sequence cell cut is completed.
The observation procedure that big sequence cell migration observation is carried out using micro-fluidic chip shown in Fig. 2 is illustrated below.
Fig. 5 is refer to, the observation procedure includes:
Step S202:With culture medium vitellophag to suspended state, and the cell of suspended state is seeded in the volley of rifle fire micro-
Each cut cell of fluidic chip, as shown in A figures in Fig. 6;
Step S204:The small indoor cell of cut is cultivated, until place patch of the cell beyond cut electrode (Au) surface
Wall grows, as shown in B figures in Fig. 6.
Under normal circumstances, a subculture is changed after cell attachment, removes not adherent cell, specifically changing the liquid time regards
Depending on the different situations of cell.
Now the growth conditions of cell are:In the surface of cut electrode (Au) not adherent growth, and substrate of glass and plus
The surface of electrode (Pt) can adherent growth.
Step S206:Apply voltage, total powered electrode between total cut electrode of micro-fluidic chip and total powered electrode
Positive pole is connected, total cut electrode connects negative pole, and continues preset time, makes the golden sulfur bonds on cut electrode;
Before general progress scratch experiment, it will treat that cell covers with whole surface and carried out again.In the present embodiment, it is ensured that each
There is cell culture medium the small interior of cut, to build the environment of electrochemical reaction.The operating method of cut is to take a 2.5V direct currents
Source, the negative electrode of power supply is connected on total electrode of Au electrodes, the positive electrode of power supply is connected on total electrode of Pt electrodes, and is continued
30s.Then, golden sulfur bonds, the thiol molecule for having PEG dissociate in the solution, as shown in C figures in Fig. 6 again.Au electrode surfaces do not have
There is PEG, cell with adherent growth or can be migrated to Au surfaces again.
It will be apparent to those skilled in the art that in scratch experiment, connect electrode with 2.5V dc sources and continue 30s not
It is preset parameter, direct current power source voltage can be 0.5V-2.5V in theory, and the time that switches on power can be 10s-10min.
Step S208:Go after the completion of mercaptan step, first remove the culture medium for having thiol molecule, needed according still further to specific experiment
Ask, have the culture medium of variety classes or concentration chemotactic factor (CF) in the small indoor addition of different cuts, photograph to record thin after certain time
Situation from born of the same parents to cut electrode (Au) surface migration, as shown in D figures in Fig. 6.
The observation procedure introduction of micro-fluidic chip finishes, into data processing stage, i.e.,:By cell to Au surface migrations
Situation microphoto carry out data processing, to obtain the design parameter of cell migration.The method of analysis can use image
Identification software, the area that identification Au surfaces are not occupied by cell, then the migration results by variety classes or concentration chemotactic factor (CF)
Between contrast, obtain experiment conclusion.
It should be noted that during experiment, can according to being actually needed the inoculating cell in the hole of respective numbers,
Without 384 holes are used in once testing completely.Moreover, in the step of mercaptan is removed in power-up, can also remove in partial hole
Culture medium, and then destroy electrochemical reaction condition, the mercaptan in this some holes is not removed, flexibly it is controllable.
So far, the embodiment of the present invention is described in detail combined accompanying drawing.According to above description, art technology
Personnel should have clear understanding to micro-fluidic chip of the present invention and its observation procedure.
In addition, the above-mentioned definition to each element and method is not limited in various concrete structures, the shape mentioned in embodiment
Shape or mode, those of ordinary skill in the art simply can be changed or replaced to it, such as:
(1) platinum electrode of powered electrode can be substituted with gold electrode:The original gold electrode as negative electrode is also after replacement
Negative electrode, the gold electrode that instead of platinum electrode are used as positive electrode;
(2) substrate can also use other transparent materials outside glass, such as organic plastics, Al2O3Or prepared by MgO etc.,
And functional layer can also use the other materials outside PDMS, such as polymethyl methacrylate (PMMA) or resin to prepare, only
Functional layer is wanted to be incorporated into by bonding pattern above substrate;
(3) above-described embodiment is by taking the scratch experiment of Real Time Observation cell migration as an example can illustrate, and in fact, this
Invention is given in the other biological experiment that micro-fluidic chip can also be applied to outside scratch experiment, is had the characteristics of its big flux
There is the advantage of uniqueness;
(4) demonstration of the parameter comprising particular value can be provided herein, but these parameters are worth accordingly without being definitely equal to, and
It is that can be similar to analog value in acceptable error margin or design constraint;
(5) direction term mentioned in embodiment, such as " on ", " under ", "front", "rear", "left", "right" etc., only it is ginseng
The direction of accompanying drawing is examined, is not used for limiting the scope of the invention;
(6) unless specifically described or the step of must sequentially occur, each step in above-mentioned preparation method and observation procedure
Execution sequence have no be limited to it is listed above, and can according to it is required design and change or rearrange.
In summary, the invention provides it is a kind of for big sequence cell cut migration observation micro-fluidic chip and its
Observation procedure, it can once realize multigroup parallel laboratory test, and flux is high, so as to greatly reduce experimental cost;In addition, drawing
In trace experiment, cut border is controlled with electrode, removes mercaptan with alive method, it is ensured that cut one between each cut cell
Cause, and cell and matrix are not damaged in the process, therefore migration results are precisely credible, have preferable application prospect.
Particular embodiments described above, the purpose of the present invention, technical scheme and beneficial effect are carried out further in detail
Describe in detail it is bright, should be understood that the foregoing is only the present invention specific embodiment, be not intended to limit the invention, it is all
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvements done etc., it should be included in the guarantor of the present invention
Within the scope of shield.
Claims (12)
- A kind of 1. micro-fluidic chip for the migration observation of big sequence cell cut, it is characterised in that including:Substrate and fixation Functional layer above substrate;Wherein, function cell array, each function cell bottom substrate in the function cell array are formed in the functional layer Partial upper surface is formed with the first electrode and second electrode electrically isolated;The first electrode is cut electrode, described Two electrodes are powered electrode;The first electrode is located at the middle position of function cell;It is rounded, and the connection electrode of the left and right sides is in strip-form, should The width of strip-form is less than the circular diameter;The second electrode is located at front side and the rear side of the first electrode, pre-determined distance is separated with the first electrode, in bar Band shape;Molecule of the modification with anti-protein adsorption functional group on the cut electrode, the small interior of function hangs with cell Liquid;The molecule with anti-protein adsorption functional group prevents cell from being adsorbed on cut electrode;When powered electrode and cut It is that the molecule of anti-protein adsorption departs from from cut electrode, and cell can be inhaled on cut electrode when adding forward voltage between electrode It is attached.
- 2. micro-fluidic chip according to claim 1, it is characterised in that the molecule is thiol molecule, is had thereon anti- Protein adsorption functional group, the cut electrode are prepared by the metal material that can combine thiol molecule.
- 3. micro-fluidic chip according to claim 2, it is characterised in that the metal material that thiol molecule can be combined For gold.
- 4. micro-fluidic chip according to claim 2, it is characterised in that the anti-protein adsorption functional group is poly- second two Alcohol(PEG)Long-chain-.
- 5. micro-fluidic chip according to claim 1, it is characterised in that pre- between powered electrode and the cut electrode If distance is between 50 μm~1mm.
- 6. micro-fluidic chip according to claim 1, it is characterised in that:The cut electrode is electrically connected to the cut electrode of colleague's adjacent functional cell by the connection electrode of the left and right sides, together The cut electrode of row function cell is sequentially connected, and in the cut of the side of the micro-fluidic chip and other row function cells electricity Pole is electrically connected with, and forms total cut electrode;Both sides extend the powered electrode to the left and right, are electrically connected with the powered electrode for other function cells of going together, function of going together The powered electrode of cell is sequentially connected, and the powered electrode of the opposite side and other row function cells in the micro-fluidic chip It is electrically connected with, forms total powered electrode.
- 7. micro-fluidic chip according to any one of claim 1 to 6, it is characterised in that the powered electrode by gold, Silver, platinum or copper it is standby, its thickness betweenBetween.
- 8. micro-fluidic chip according to any one of claim 1 to 6, it is characterised in that the cut electrode and power-up Between electrode and substrate, there is tack coat, with cohesive force of both enhancings between substrate.
- 9. micro-fluidic chip according to any one of claim 1 to 6, it is characterised in that the function cell it is transversal Face is shaped as square, rectangle, circle or triangle.
- 10. micro-fluidic chip according to any one of claim 1 to 6, it is characterised in that the material of the substrate is glass Glass, organic plastics, Al2O3Or MgO;The functional layer is prepared using dimethyl silicone polymer (PDMS), polymethyl methacrylate (PMMA) or resin material, It is fixed on by way of bonding above substrate.
- 11. a kind of observation procedure, it is characterised in that for utilizing the micro-fluidic chip any one of claim 1 to 10 Cell migration observation is carried out, including:Step S202:With trypsin digestion and cell, and it is resuspended, and the cell of suspended state is seeded to micro-fluidic with culture medium The function cell of chip;Step S204:The small indoor cell of function is cultivated, until local adherent growth of the cell beyond cut electrode;Step S206:Apply voltage between the cut electrode and powered electrode of cut cell, and continue preset time, make to have The molecule of anti-protein adsorption functional group departs from from cut electrode;Step S208:The culture medium containing anti-protein adsorption molecule is removed, is replaced with the training of the chemotactic factor (CF) needed containing experiment Base is supported, photographs to record after preset time cell to the situation of cut electrode transfer.
- 12. observation procedure according to claim 11, it is characterised in that described in the cut electrode of cut cell and power-up In the step of applying voltage between electrode, and continuing preset time:The powered electrode connects positive pole, and cut electrode connects negative pole, and the voltage is described default between 0.5V-2.5V Time is between 10s-10min.
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