CN107694347A - A kind of microwell array filter membrane and its preparation method and application - Google Patents
A kind of microwell array filter membrane and its preparation method and application Download PDFInfo
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- CN107694347A CN107694347A CN201710679586.2A CN201710679586A CN107694347A CN 107694347 A CN107694347 A CN 107694347A CN 201710679586 A CN201710679586 A CN 201710679586A CN 107694347 A CN107694347 A CN 107694347A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
- B01D69/02—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor characterised by their properties
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/14—Ultrafiltration; Microfiltration
- B01D61/147—Microfiltration
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
- B01D67/0002—Organic membrane manufacture
- B01D67/0023—Organic membrane manufacture by inducing porosity into non porous precursor membranes
- B01D67/0032—Organic membrane manufacture by inducing porosity into non porous precursor membranes by elimination of segments of the precursor, e.g. nucleation-track membranes, lithography or laser methods
- B01D67/0034—Organic membrane manufacture by inducing porosity into non porous precursor membranes by elimination of segments of the precursor, e.g. nucleation-track membranes, lithography or laser methods by micromachining techniques, e.g. using masking and etching steps, photolithography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
- B01D71/06—Organic material
- B01D71/72—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, not provided for in a single one of the groups B01D71/46 - B01D71/70 and B01D71/701 - B01D71/702
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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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
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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
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2325/00—Details relating to properties of membranes
- B01D2325/02—Details relating to pores or porosity of the membranes
- B01D2325/021—Pore shapes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2325/00—Details relating to properties of membranes
- B01D2325/24—Mechanical properties, e.g. strength
Abstract
The invention discloses a kind of microwell array filter membrane and its preparation method and application, the MEMS technique (such as Parylene MEMS technologies) of polymer based on conformal deposited manufactured with more than 1 square centimeter work area, can accuracy controlling aperture and interporal lacuna microwell array filter membrane, the microwell array filter membrane has 2.5 dimension features and superelevation porosity, can realize the cell enrichment separation of ultra-large volume flux.
Description
Technical field
The present invention relates to biological medicine and advanced manufacturing field, and in particular to one kind is based on flexible micro-electromechanical system process
(flexible MEMS technique) manufacture have larger work area, can accuracy controlling aperture and micropore gap superelevation porosity it is micro-
Hole array filter membrane and its preparation method and application.
Background technology
In recent years, liquid Biopsy (Liquid Biopsy, i.e., without tissue penetration, from blood, urine or other bodies
Liquid, clinical irrigating solution etc. it is non-invade property materials clinical sample capture, recovery trace (as little as single) Exfoliated tumor cells and
Cancer correlation DNA equimoleculars label) as an emerging important method of cancer diagnosis technology.In liquid Biopsy,
The important sample source that Exfoliated tumor cells are existing oncocytology pathological examinations is directly obtained from clinical sample,
And the key foundation of related molecular marker thing or genetic test sensitivity and specificity is improved, but current subject matter exists
Relatively low in the recall rate of target tumor, clinical diagnosis positive rate is low, thus can not obtain extensively should in cancer diagnosis
With.Realize and be capable of the detection specificity of high Exfoliated tumor cells and the liquid Biopsy of positive rate to realizing cancer early stage essence
Make a definite diagnosis to break and there is even more important meaning.
In the liquid biopsy research of Exfoliated tumor cells, most commonly used research object is that circulation is separated from blood
Tumour cell (Circulating Tumor Cell, CTC), particularly since 2007, has been realized based on nanometer technique
Hypersensitivity whole blood in CTC capture be possibly realized, captured from 7.5mL whole bloods as little as single CTC technology have begun to should
In being studied for cancer clinical diagnosis and treatment.In addition to blood, trace Exfoliated tumor cells can also be from bronchoalveolar lavage fluid, urine, saliva
CTC is captured in concentration and separation, with blood in the clinical samples such as liquid, tissue digestion liquid, cerebrospinal fluid and peritoneal lavage fluid
Compare, Exfoliated tumor cells are captured from above-mentioned clinical sample has stronger specific aim and tumor locus directive property；It is but same
When, difficulty and challenge are also bigger, main reason is that：(1) compared with blood, the cell derived of above-mentioned clinical sample is various, portion
Divide acellular impurity in sample also more；(2) volume of the above-mentioned clinical sample of what is more important is generally larger, as alveolar fills
Washing lotion is usually 20-50mL, urine 50-100mL, and peritoneal lavage fluid generally up to 500-1000 mL, existing CTC liquid
Body Biopsy is difficult to handle.To realize the liquid biopsy syste of trace Exfoliated tumor cells in large volume clinical sample, most close
The technological difficulties of key are how to improve volume flux, so as in suitable time range (or even in surgical procedure
In) complete large volume clinical sample in trace Exfoliated tumor cells capture separation.Capture separation CTC's is many in whole blood
In method, from fluid-mechanics point of view, it is recognized that the method for being most expected to realize large volume flux be filtering with microporous membrane.
United States Patent (USP) (Membrane filter for capturing circulating tumor cells, US
The method that circulating tumor cell in blood is captured using miillpore filter 7846393B2) is described, but the patent microwell array is filtered
Greatly, fluid is to cell in filter process in the micropore gap (referring both to the beeline between the edge of adjacent two micropore below) of film
Shearing force it is big, cause capture cell poor activity；Another patent (Method and apparatus for
Microfiltration to perform cell separation, US 20090188864A1) to devise 3-dimensional bilayer micro-
Hole array filter membrane, the cell captured to upper layer film using lower membrane provide a holding power, with improve capture cell work
Property.But the processing method of 3-dimensional double-layered filtration film is complicated, cost is high.And the miillpore filter utilized in this two patents due to
Micropore gap is big, porosity is low, and filtration flux is small, can not efficiently realize the capture of trace cell in large volume clinical sample, because
Easily extensive high porosity microwell array filter membrane processing method is still urgently developed for this.
At present it has been reported that miillpore filter method still suffer from two key issues and need further to be solved：(1) it is non-specific
Property cell adherence, collateral security filter membrane construction machine intensity considers, at present document report and commercialized miillpore filter micropore
Gap it is big (>10μm；Mainly due to small micropore gap filter membrane manufacturing process difficulty is big, large area suspension microwell array yield rate
It is low), it is less than or has greater probability close to the untargeted cells of the size and be adhered in the supporting construction of micropore gap so that
The target Exfoliated tumor cells purity that concentration and separation obtains is low, to follow-up imaging analysis and further culture amplification and medicine
Thing screening zone is disturbed；(2) capture gained Exfoliated tumor cells is active low, because micropore gap is big, the hole of miillpore filter
Rate is small, and liquid stream is big to the target cell shearing force being captured in filter process so that activity reduces after cell is mechanically damaged
Even split completely, so as to which the analysis of the amplification cultivation and subsequent biochemical/molecular level that can not further carry out target cell is ground
Study carefully.
The content of the invention
Still it is faced with that recovery purity is not high, cytoactive is affected etc. asks for existing microwell array filter film technology
Topic, the present invention propose a kind of microwell array filter membrane and its preparation method and application, and the microwell array filter membrane has 2.5 Wei Te
Point and superelevation porosity, the shortcomings that existing miillpore filter is in cell enrichment separation can be overcome, be particularly suitable for use in large volume
The high-flux cell of liquid and the concentration and separation of particle.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of microwell array filter membrane, wherein, microwell array is the void region on filter membrane, and micropore gap is by conformal deposited
Polymer-filled and be connected to form filter membrane, complete job area >=5mm × 5mm of the filter membrane, and film thickness be more than micropore between
Gap.
Further, the shape of micropore may be designed as in the geometries such as circle, rectangle, regular hexagon as needed
The combination of two or more one or more of different geometries.
Preferably, the polymer of the conformal deposited is Parylene.
Preferably, the porosity of the filter membrane can design, generally >=40%.
The preparation method of above-mentioned microwell array filter membrane comprises the following steps：
1) prepare the micro-pillar array of transoid on substrate, the characteristic size of the cross section of each microtrabeculae and need to prepare
The micropore of microwell array filter membrane is consistent, the consistency of thickness of the height of microtrabeculae and the microwell array filter membrane that need to be prepared；
2) polymer described in conformal deposited in micro-pillar array, until closing microtrabeculae gap；
3) polymer more than micro-pillar array top end surface is removed, until micro-pillar array top end surface all exposes, is obtained
The microwell array structure of the polymer；
4) the polymer micro array structure is discharged, obtains microwell array filter membrane.
Further, in step 2), the thickness of the polymer of deposit is more than the half in microtrabeculae gap.
Further, return to carve by oxygen plasma dry etching in step 3) and remove more than micro-pillar array top end surface
Polymer.
Further, by removing or corroding substrate release polymers microwell array structure in step 4).
Present invention also offers above-mentioned microwell array filter membrane concentration and separation is spherical or subsphaeroidal object in application.
Further, the opposite side distance of the micropore of above-mentioned microwell array filter membrane is less than or equal to spherical or subsphaeroidal object
Diameter.
Further, the micropore gap of above-mentioned microwell array filter membrane is less than the background objects of spherical or subsphaeroidal object
Radius, preferably≤4 μm.
Further, described spherical or subsphaeroidal object includes the agglomerates such as cell, particle or its aggregation.
The invention has the advantages that
(1) this microwell array filter membrane can keep the big of complete unbraced structure while film mechanical strength is ensured
Work area, 0.05-0.3 μm of pore structure trueness error.
(2) while there is the characteristic of 2 dimension films and 3-dimensional structure：The effective continuous face product of filter membrane is far longer than thickness, makes
Obtain the characteristic that 2 dimension films are integrally presented in filter membrane；Thickness is more than micropore gap, so as to which 3-dimensional characteristic be presented near local micropore, together
When ensure that mechanical strength of the filter membrane structure in filter process.
(3) the preferred regular hexagon of the micro-pore shape of microwell array filter membrane or close to regular hexagon structure, is ensured whole with this
The micropore gap of adjacent cells is consistent in individual filter membrane, and the circumscribed circle of micropore and the difference of inscribed circle diameter are smaller.
(4) micropore opposite side distance is less than or equal to the agglomerate diameters such as target cell, particle or its aggregation, ensures mesh with this
Mark the high-recovery capture of cell；Adjacent cells gap with less than background cells, particle or agglomerate radius, is ensured with this
The objects such as background cells are easier to be carried away by the flow in filter process, so as to significantly reduce non-specific adhesion, flow simultaneously
Body also significantly reduces to the shearing force of cell, so as to greatly improve volume flux, purity and the activity of cell sequestration operation, this
Invention without additionaling power, only with liquid weight-driven itself under the conditions of, the working flux of filtering whole blood sample is 20mL/
More than min.And for clinical samples such as the less PBS of viscosity, urine, irrigating solutions, its working flux be up to 120mL/min with
On.
(5) filter membrane can be used for capture blood circulation tumour cell, cell, particle or the group being enriched with blood sample
Block, for subsequent detection or it is further cultured for.
Brief description of the drawings
Fig. 1 (a) is that micro-pore shape of the present invention is the microwell array filter membrane of regular hexagon and its showing for enlarged fragmentary portion
It is intended to；Fig. 1 (b) is the edge direction schematic cross-section along regular hexagon shown in Fig. 1 (a)；Fig. 1 (c) is along shown in Fig. 1 (a) positive six
The diagonal schematic cross-section of side shape.
Fig. 2 is the exemplary manufacturing method of microwell array filter membrane of the present invention, wherein, it is prepared by (a) silicon micro-pillar array；(b) silicon is micro-
Parylene is deposited on post array；(c) oxygen plasma dry etching is returned to carve to silicon microtrabeculae top and exposed；(d) HNA corrodes
Silicon substrate, discharge Parylene microwell array structure.
Fig. 3 (a) is microwell array filter membrane material object photo of the present invention；Fig. 3 (b) is microwell array filter membrane partial enlargement Electronic Speculum
Photo.
Fig. 4 (a) is the microwell array filter membrane and its enlarged fragmentary portion schematic diagram of slit-shaped of the present invention；Fig. 4 (b) is not
The compound microwell array filter membrane schematic diagram of similar shape, different sizes；Fig. 4 (c) be high-aspect-ratio small size microwell array filter membrane and
The schematic diagram of its enlarged fragmentary portion.
Fig. 5 (a) is the test philosophy signal that CCI non-contact opticals platform is used for microwell array filter membrane Mechanics Performance Testing
Figure, wherein：1-mechanics sensor, 2-microwell array filter membrane, 3-PMMA carriers, 4-surface gold reflected coat layer, 5-aberration
Lens, 6-beam splitter, 7-pin hole, 8-spectrometer；Fig. 5 (b) filter membrane micropores gap is identical, measures during pore size difference
The exemplary force of microwell array filter membrane-inflection curves figure；Fig. 5 (c) filter membrane pore sizes are identical, measure during the difference of micropore gap
The exemplary force of microwell array filter membrane-inflection curves figure；The equivalent Young's modulus of Fig. 5 (d) different porosities filter membranes；Fig. 5 (e) is high
Flux (>100mL/min) stress of different porosities filter membrane and pore size transverse deformation in filter process.
Fig. 6 is the small spherical particles via test based on different size filter membranes.
Fig. 7 (a) is lateral stress suffered by 10 μm of beads stop at the interporal lacuna of 12 μm of micropores；Fig. 7 (b) is different holes
Pressure suffered by gap size filter membrane surface bead；Fig. 7 (c) is pressure suffered by different interporal lacuna size filter membrane surface beads and cut
Stress.
Fig. 8 (a) is the T24 cell filtrations/centrifugation results of comparison being diluted in 10mLPBS cushioning liquid；Fig. 8 (b) is dilute
Release A549 cell filtrations/centrifugation results of comparison in 10mLPBS cushioning liquid.
The MC3T3 cell fluorescence mark figures that Fig. 9 (a) microwell array filter membranes of the present invention are captured, 5,6-fluorescence labeling
MC3T3 cells；Fig. 9 (b) is the fluorescence photo of the MC3T3 cells of Culture in situ on microwell array filter membrane of the present invention, 7,8-
The nucleus of DAPI marks, the microfilament of 9,10-Phalloidin-Rhodamine B marks；The cell of Fig. 9 (c) partial enlargements
Culture in situ on film；The different micropore gaps of Fig. 9 (d) to accompanying the influence of foster cytoactive again after capture.
Figure 10 filtering whole blood samples of the present invention and PBS samples are with having been reported the logical of response porosity product filtering whole blood
Amount compares.
Embodiment
Hereafter by specific embodiment and coordinate accompanying drawing, the present invention is described in detail.
Embodiment
Micro-pore shape of the present invention is shown for microwell array filter membrane design such as Fig. 1 (a)-(c) of regular hexagon, wherein micropore
Gap area is filled and is connected by Parylene, maintains to form filter membrane, and regular hexagon micropore is the void region on filter membrane.
The principal character of the microwell array filter membrane is as follows：
(1) there are 2.5 dimension geometrical features；
(2) there is larger effective filtration area (>=13mm × 13mm), it is possible to achieve ultra-large volume flux (>=
150mL/min)；
(3) there is accurate aperture/micropore gap, it is possible to achieve high performance cell filtration；
(4) micro-pore shape is regular hexagon, ensures that the whole filter membrane of adjacent cells gap width is consistent with this, and micropore
Circumscribed circle differs minimum with inscribed circle diameter；
(5) regular hexagon micropore opposite side distance is less than target cell diameter, ensures that the high-recovery of target cell is captureed with this
Obtain；
(6) there is the adjacent cells gap (≤4 μm) less than background cells radius, ensure background cells in filtrate with this
It is easier to be carried away by the flow in flow process, and reduces non-specific adhesion, while the fluid for reducing capture cell on micropore is cut
Stress is cut, so as to improve its activity.
Fig. 2 is the exemplary manufacturing method of microwell array filter membrane of the present invention.It is specific as follows：
1) micro-pillar array (can also be realized by techniques such as injection moldings) of transoid is prepared on a silicon substrate, such as Fig. 2 (a)
Shown, silicon microtrabeculae is regular hexagon, and the micropore of filter membrane of the characteristic size with need to finally prepare is consistent；
2) Parylene is deposited in silicon micro-pillar array, until (Parylene deposition thickness is big in closing microtrabeculae gap
Half in microtrabeculae gap, this is due to that Parylene is all directions homoepitaxial, and deposition thickness and deposition time have
Close, and film thickness is also relevant with structure, i.e., if deposition thickness is less than microtrabeculae gap half, then membrane micropore gap is not yet filled out
Charge entirely, structure does not close completely, so as to have slit；It can just be filled out when only deposition thickness is more than the half in microtrabeculae gap
Full gap), as shown in Fig. 2 (b)；
3) returned and carved by oxygen plasma dry etching, remove Parylene more than microtrabeculae top end surface, until silicon
Micro-pillar array top end surface all exposes again, Parylene microcellular structure is obtained, as shown in Fig. 2 (c)；
4) Parylene microcellular structure is discharged by corroding silicon substrate, as shown in Fig. 2 (d), obtains ultra-large volume and lead to
2.5 dimension cell enrichment separation filter membranes of amount.
The microwell array filter membrane being prepared using the above method is in kind as shown in Fig. 3 (a), filter membrane gross area 20mm
× 20mm, effective filtration area 13mm × 13mm, thickness are 10 μm.
Photo such as Fig. 3 (b) institutes that the microwell array filter membrane is observed and shot under Electronic Speculum (JEOL, JSM-7500F)
Show, the porosity of filter membrane shown in Fig. 3 (a) is 70.3%, and micropore gap is 2 μm, and thickness is 10 μm, pair of regular hexagon micropore
Back gauge is 10.3 μm, and catercorner length is 12 μm.
Filter membrane microcellular structure can be designed as the various structures such as various sizes of circle, rectangle, hexagon as needed, its
Middle Fig. 4 (a) for slit-shaped high porosity microwell array filter membrane (2 8 μm of μ m slits, 2 μm of long axis direction slit gaps, 8 μm
Short-axis direction slit gap, 10 μm of thickness), Fig. 4 (b) is more structures or multiple dimensioned microwell array filter membrane (10 μm of thickness, micropore
16.46 ± 0.17 μm of gap, 11.21 ± 0.11 μm of macropore diameter, 3.46 ± 0.27 μm of small aperture), Fig. 4 (c) is advanced wide
Than (>2) microwell array filter membrane, its micropore gap are 2 μm, and aperture (micropore catercorner length) is 1.39 ± 0.07 μm, and micropore is deep
Spend 10.45 ± 0.21 μm of (film thickness), depth-to-width ratio (aperture/hole depth) is 7.5.
Experimental example 1：Filter membrane performance test and emulation
1-force snesor, 2-microwell array filter membrane, 3-PMMA carriers, 4-surface gold reflected coat layer, 5-aberration are saturating
Mirror, 6-beam splitter, 7-pin hole, 8-spectrometer；This experiment uses CCI Non-contact optical methods to the mechanical property of filter membrane
Tested.Shown in test philosophy such as Fig. 5 (a), mainly including Mechanical loading, optical imagery and signal transacting three parts：Mechanics
Loading section applies active force by mechanics sensor 1 to microwell array filter membrane 2, and in the presence of external force, shape occurs for filter membrane
Becoming, filter membrane lower surface position changes in vertical direction and (produces longitudinal deformation), and is optically imaged system detectio and arrives,
Respective image information is changed into filter membrane lower surface optical imagery signal after signal processing.
Optical imaging moieties, continuous monochromatic photoimaging is carried out to a white light spot light using aberration lens 5.Work as table
When face is located in the range of distributed image, light is scattered back aberration lens 5 and eventually arrives at pin hole 7.And only single wavelength
Image completely on the surface (i.e. filter membrane lower surface) detected, and every other wavelength is filtered out from pin hole 7.
Signal processing：For optical imagery information transmission to the spectrometer 8 of signal processing, spectrometer 8 passes through analysis
Collected light, the position of measurement point on surface can be calculated.Change with the loading puted forth effort, the table that spectrometer calculates
Face position changes, and variable quantity is the longitudinal deformation amount of microwell array filter membrane.
In order to strengthen the reflective function of Parylene filter membrane surface, before mechanical meaurement first to filter membrane lower surface metal spraying at
Manage (Ion beam coater ETD 2000, gas current 15mA, 3min).The gold reflection plating of Parylene surface coating
Layer 4 thickness~3nm, golden Young's modulus is 79GPa (body material data), and the thickness of microwell array filter membrane 2 is 10 μm,
Parylene Young's modulus is 2.8GPa, therefore the influence that the ultra-thin golden reflecting layer of subsidiary can be to filter membrane deformation is complete
It can ignore entirely.
By shown in Fig. 5 (a) based on colored co-focusing imaging (chromatic confocal imaging, CCI) power
Exemplary force-inflection curves such as the Fig. 5 (b) for the filter membrane that test device obtains is learned, shown in (c), the power of mechanics sensor loading is F
(N), the longitudinal deformation of filter membrane is Δ L (μm).
Wherein Fig. 5 (b) be thickness identical (10 μm), micropore gap identical (4 μm), micropore size (referred to micropore it is diagonal
Line length) different regular hexagon miillpore filter power-inflection curves, curve map shows that aperture is bigger (porosity is higher), filter
The deformation of film is bigger；Fig. 5 (c) is that identical (referred to micropore catercorner length is 12 μ for thickness identical (10 μm), micropore size
M), power-inflection curves of the different regular hexagon miillpore filter in micropore gap, curve map show the bigger (porosity in micropore gap
It is lower), the deformation of filter membrane is smaller.
Further, the large deformation plectane theory that this experimental example is proposed using S.Timoshenko obtains to mechanical test
To experimental data analyzed.Assuming that filter membrane edge is clamped, and concentrfated load F is applied to the center of filter membrane, and filter membrane is indulged
Equation (1) can be used to represent to the relation between deformation Δ L and load：
Wherein μ represents the Poisson's ratio of Parylene, is calculated here using 0.5；H is filter membrane thickness, and a is filter membrane face
Long-pending radius, E are the equivalent Young's modulus of 2.5 dimension Parylene microwell array filter membranes.Experimental data is intended using Matlab
Close in equation (1), so as to extract the equivalent Young's modulus E of each test sample filter membrane.
To examine equation (1) to derive the accuracy of microwell array filter membrane equivalent Young's modulus, first by imporous poly-
The flat film of paraxylene verified, the actual experiment test point of the flat film of imporosity Parylene and is derived according to equation (1)
The value gone out shows good uniformity, and the Young's modulus numerical value gone out by the Fitting Calculation is 2.65 ± 0.04GPa, with former material
Expect the consistent (https of Parylene material attribute that supply company provides://scscoatings.com/), illustrate to be based on CCI
It is rational to carry out the equivalent Young's modulus that the data point that mechanical test obtains derives microwell array filter membrane by equation (1).No
With porosity microwell array filter membrane equivalent Young's modulus such as Fig. 5 (d) shown in, Parylene microwell array filter membrane it is equivalent
Young's modulus reduces with the increase of porosity.
Film equivalent Young's modulus value is further substituted into the filter membrane stress under high flux under the filtering of (100mL/min) liquid
Simulation model, obtain pressure and the pressure deformation to caused by microcellular structure that the filter membrane of different porosities is subject to.Such as Fig. 5 (e)
Shown, by taking the miillpore filter of 70% porosity in the embodiment of the present invention as an example, its micropore deformation is less than 33nm.Therefore in high pass
During measuring cell capture, caused by by Fluid pressure micropore deformation do not interfere with the effect of micron-sized cell separation.
Experimental example 2：The rigid microspheres via test of membrane filtration performance
4 microwell array filter membranes are assembled into multistage filtering system from top to bottom according to the order of pore size from big to small
System, Parylene regular hexagon microwell array filter sizes/gap of used 10 μm of thickness (processes from top to bottom
Into rear according to the actual size measured of electromicroscopic photograph) it is followed successively by：14.52 ± 0.20 μm/4.43 ± 0.06 μm, 11.21 ±
0.11 μm/4.77 ± 0.15 μm, 9.13 ± 0.09 μm/4.69 ± 0.24 μm, 7.51 ± 0.13 μm/4.46 ± 0.09 μm.Will be outstanding
There is the 10mL for the polystyrene microsphere (happy chromatographic technique development centre is thought in 6-1-2200, Tianjin again) that diameter range is 6-22 μm
Deionized water is filtered by the multiple filtration system assembled.After filtering, Parylene filter membrane is removed into rear ambient temperature overnight and put
Put, there is filter membrane surface metal spraying (Ion beam coater ETD 2000, gas current 15mA, 3min, the thickness of microballoon to capture
~3 nm of degree) microballoon captured on filter membrane is seen using SEM (SEM, JSM-7500F, JEOL) afterwards
Examine and take pictures, and the diameter of microballoon on every filter membrane is measured according to electromicroscopic photograph, concrete outcome is as shown in Figure 6.
Isolated microsphere diameter distribution is in the tunic micropore inscribed circle diameter (d ') and last layer on filter membrane
Between membrane micropore inscribed circle diameter (d '), further demonstrating microwell array filter membrane aperture in filter process will not become
Change, therefore do not interfere with the size resolution of microballoon filtering.Meanwhile microballoon multistage filtering separating resulting shows good chi
Very little truncation effect also fully demonstrates the high size resolution and separation purity that microwell array filter membrane is separated based on size.
Wherein, stressing conditions emulation of Fig. 7 (a) microballoon in microporous membrane surface flow field discloses of the invention efficient
The reason in part for separation and excellent size truncation effect.The structure of small pitch of holes makes cell or particle entrapment in micropore gap
Possibility greatly reduce, calculating is demonstrated in cell dimensions scope, and previously mentioned micropore gap is less than the knot of thickness
Structure, it is effectively guaranteed the non-specific recovery phenomenon for reducing cell or particle entrapment in micropore gap.
Experimental example 3：Cell capture in liquid
Trace bladder transitional cell carcinoma in bulk sample is detected by the membrane filtration method and conventional centrifugal method of the present invention
The results of comparison of cell (T24) and lung carcinoma cell (A549) is respectively as shown in Fig. 8 (a) and Fig. 8 (b).It is thin with A549 in Fig. 8 (b)
Analyzed exemplified by the organic efficiency result of born of the same parents, the method for the conventional centrifugal cell number in 10mLPBS is 103When individual, tradition is used
Centrifugal condition (3500rpm, 20min), organic efficiency are less than 10% (9.6 ± 1.5%), and using 10 μm prepared of the invention
Thickness, 10 μm of micropore sizes (regular hexagon diagonal), the regular hexagon microwell array membrane filtration in 4 μm of micropore gaps, recovery
Efficiency is 10 in cell number3、104When respectively be up to 76.7 ± 16.8%, 90.4 ± 9.5%, far above conventional centrifugal method
Cell recovery ability.Because conventional centrifugal method cell organic efficiency is low, when cell number is less than 10 in 10mL liquid2It is individual and
The control experiment of centrifugal method recovery can not be set using below.And liquid biopsy method of the present invention is 10 in cell number2、
Still there is very high organic efficiency at 10, respectively 86.7 ± 16.8%, 71.3 ± 5.1%；It is even low in cell number
During to 1, organic efficiency is still higher than 55.6%, and (rate of recovery reduces micro pipette when being also likely to be initialization experiment and tested
Error causes caused by cell loss), therefore say, the super large body that the present invention is established based on superelevation porosity microwell array filter membrane
Product flux liquid biopsy method is at least higher by bulk sample in the recovery ability of trace cell than traditional centrifugal method
It is more than 2 orders of magnitude.
Experimental example 4：It is further cultured in liquid after cell recovery
To count/dilute by blood counting chamber obtain it is a number of through reactive fluorescent dye (Cell Tracker
staining,Molecular Probes TM) mark pattern cell (mouse bone-forming cell, MC3T3) be added to 10mL phosphoric acid
In salt buffer (Phosphate buffer, PBS), cell suspension to be filtered is prepared.Cell suspension is filtered through this hair
12 μm of micropore sizes (regular hexagon diagonal) of bright preparation, 4 μm of micropore gaps, the regular hexagon microwell array of 10 μm of thickness
After filter membrane, filter membrane is removed, move in cell culture environment accompany again it is foster.Wherein, cell is captured through microwell array filter membrane of the present invention
Shown in labeled in situ fluorescence such as Fig. 9 (a), on the microwell array filter membrane by 4 μm of micropore gaps of the filter membrane Culture in situ of three days
Capture the MC3T3 patterns of separation and sprawl shown in state such as Fig. 9 (b) and Fig. 9 (c), wherein see under fluorescence microscope
The nucleus 7 and 8 of DAPI marks is shown as blue-fluorescence, and the microfilament 9 of Phalloidin-Rhodamine B marks and 10 show
It is shown as red fluorescence.Laser confocal microscope (Laser scanning confocal microscope, TCS SP5X,
Leica) photo shows that cell is good in filter membrane upper berth spread state, and experiment demonstrates the micro- of 4 μm of micropore gaps provided by the invention
Hole array filter membrane can ensure that a point cellifugal activity is captured in filtering, and biochemistry/gene is expected to be used for by follow-up cultivation amplification
The further investigation of mechanism and tumor drug screening/examination of curative effect etc..
Meanwhile in the superelevation porosity filter membrane using the present invention during cell is separated, reducing micropore gap reduces
Low stress suffered by cell is to maintain the key of cytoactive.It is dead using (EthD-1/Calcein AM) as shown in Fig. 9 (d)
The double transfection reagent boxes (L3224, Invitrogen, Thermofisher) of cell/living cells detect cell by different micropore gaps
Accompanying again after filter membrane capture supports implementations.The kit includes two kinds of compositions, and a kind of composition is marked with red fluorescence
Ethidium homodimer-1, under normal circumstances, it is completely thin that ethidium homodimer-1 cannot pass through living cells
After birth, and after cell loses activity, cell membrane integrity is destroyed, and permeability improves so that ethidium
Homodimer-1 can enter cell and be combined with the nucleic acid specificity of cell interior, therefore, combine ethidium
Homodimer-1 dead cell takes on a red color under fluorescence microscope and (irised out in Fig. 9 d with white dashed line).Another kind is to be marked with
The calcin-AM of green fluorescence, calcin-AM have living cells membrane permeability in itself, can be by penetrating into cell membrane
Specifically bound with intracellular active lipase, combine calcin-AM living cells presented under fluorescence microscope it is green
Color fluorescence.Ratio of the dead cell that white dashed line is irised out in total cell number is fewer, then the ratio of competent cell is higher, right
Answer the activity of the cell of filter membrane capture high.Therefore, as shown in Fig. 9 (d), with the increase in membrane micropore gap, specifically between micropore
Gap changes to 16 μm from 2 μm, and cell is active on a declining curve after recovery, and when micropore gap increases to 8 μm, it is corresponding thin
Cytoactive drops to less than 50%.The further proof of modeling and simulating through microporous membrane surface flow, such as Fig. 7 (b), Fig. 7
(c) shown in, in the case of being fixed in filtration aperture, by the rigid ball (8 μm) of micropore and it is trapped in firm at micropore
The numerical value of shearing force and pressure suffered by property ball (16 μm) all increases, shearing force and pressure with the increase in micropore gap
Increase mean cell in filter process the extent of damage increase, cytoactive may be caused to reduce.Therefore, to improve thin
For the activity of born of the same parents, it is necessary to reduce the micropore gap of microwell array filter membrane, the simulation result is also consistent with Fig. 9 (d) experimental results.Should
Experimental example shows the present invention as efficient capture target cell under low stress environment, and Culture in situ, there is provided simple and effective
Means.
Experimental example 5：The application that miillpore filter of the present invention separates in the tumour cell of clinical liquid sample
There is the present invention high flux high accuracy 2.5 to tie up microwell array structure, particularly big suitable for clinical samples such as blood
The application of cell enrichment and separation in volume clinical sample (such as urine, all kinds of irrigating solutions).It is related in existing report and document
The highest flux of the membrane filtration blood of different porosities is that (flux is that additional syringe pump or negative pressure drive to 2mL/min
Under the conditions of).And the present invention is without additionaling power, only with liquid weight-driven itself under the conditions of, the work of filtering whole blood sample
Flux is more than 20mL/min.And it is up to for the clinical samples such as the less PBS of viscosity, urine, irrigating solution, its working flux
More than 120mL/min, had a clear superiority in terms of working flux compared with like product, ASSOCIATE STATISTICS is as shown in Figure 10.
The above embodiments are merely illustrative of the technical solutions of the present invention rather than is limited, those skilled in the art
Technical scheme can be modified by member or equivalent substitution, without departing from the spirit and scope of the present invention, this hair
Bright protection domain should be to be defined described in claim.
Claims (10)
1. a kind of microwell array filter membrane, wherein, microwell array is the void region on filter membrane, and micropore gap is by the poly- of conformal deposited
Compound is filled and is connected to form filter membrane, complete job area >=5mm × 5mm of the filter membrane, and film thickness is more than micropore gap.
2. a kind of microwell array filter membrane as claimed in claim 1, it is characterised in that the polymer of the conformal deposited is poly- pair
Dimethylbenzene.
A kind of 3. microwell array filter membrane as claimed in claim 1, it is characterised in that the porosity of=40% of the filter membrane.
4. the preparation method of any described microwell array filter membranes of claim 1-3, comprises the following steps：
1) micro-pillar array of transoid, the characteristic size of the cross section of each microtrabeculae and the micropore battle array that need to be prepared are prepared on substrate
The micropore of row filter membrane is consistent, the consistency of thickness of the height of microtrabeculae and the microwell array filter membrane that need to be prepared；
2) polymer described in conformal deposited in micro-pillar array, until closing microtrabeculae gap；
3) polymer more than micro-pillar array top end surface is removed, until micro-pillar array top end surface all exposes, it is poly- to obtain this
The microwell array structure of compound；
4) the polymer micro array structure is discharged, obtains microwell array filter membrane.
5. preparation method as claimed in claim 4, it is characterised in that in step 2), the thickness of the polymer of deposit is more than micro-
The half of intercolumniation gap.
6. preparation method as claimed in claim 4, it is characterised in that returned and carved by oxygen plasma dry etching in step 3)
Remove polymer more than micro-pillar array top end surface.
7. preparation method as claimed in claim 4, it is characterised in that by removing or corroding substrate release polymerization in step 4)
Thing microwell array structure.
8. any described microwell array filter membranes of claim 1-3 concentration and separation is spherical or subsphaeroidal object in application.
9. application as claimed in claim 8, it is characterised in that the opposite side distance of the micropore is less than or equal to spherical or near ball
The diameter of shape object.
10. application as claimed in claim 8, it is characterised in that the micropore gap is less than spherical or subsphaeroidal object
The radius of background objects.
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