CN109554285A - A kind of flexible microporous film and the cell separation apparatus using its preparation - Google Patents
A kind of flexible microporous film and the cell separation apparatus using its preparation Download PDFInfo
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- CN109554285A CN109554285A CN201811643014.XA CN201811643014A CN109554285A CN 109554285 A CN109554285 A CN 109554285A CN 201811643014 A CN201811643014 A CN 201811643014A CN 109554285 A CN109554285 A CN 109554285A
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- film
- micropore
- flexible microporous
- microporous film
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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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- 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
- C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
Abstract
The present invention relates to biological medicines and advanced manufacturing technology field, and in particular to a kind of flexible microporous film and the cell separation apparatus using its preparation；Flexible microporous film of the present invention includes micropore size >=15 μm, and the working region of porosity of=50% further includes supporting zone, and the supporting zone assigns working region rigidity, hardness and mechanical strength, it is avoided to deform；The present invention also provides a kind of cell separation apparatus, the cell separation apparatus includes at least one primary seperation film and at least one final stage seperation film, the primary seperation film uses flexible microporous film of the present invention, bulk background component and cell component of the primary seperation film to separate complex fluid sample, prevent separation the blocking of film, increase flux, the final stage separates membrane aperture≤15 μm, porosity of=25%, after filtering retention fluid sample, primary seperation film and final stage seperation film all can be in microscopically observations, increase object positive rate, practicability is good.
Description
Technical field
The present invention relates to biological medicines and advanced manufacturing technology field, and in particular to a kind of flexible microporous film and application its system
Standby cell separation apparatus.
Background technique
In recent years, liquid Biopsy becomes tumour cell detection field one emerging important method.Relative to general
Logical tumour cell detection technique, liquid Biopsy do not need tissue penetration, reduce wound, by collecting blood, urine, facing
Circulation or Exfoliated tumor cells in the clinical samples such as bed irrigating solution, can be detected tumour cell through separation, capture and detection, lead to
Source of the target cell as follow-up study and test can be also further amplified by crossing in situ or release culture.Due to these clinics
Sample generally includes blood, urine, bronchoalveolar lavage fluid, saliva, tissue digestion liquid, cerebrospinal fluid or peritoneal lavage fluid, sample volume
Flux is big, and circulation or Exfoliated tumor cells content therein are few, and containing complicated structural constituent, mucus and interferes cell,
And viability is poor after circulation or Exfoliated tumor cells separation, leads to the capture rate of target cell and detection in liquid Biopsy
Positive rate is low.
In order to overcome this technical problem, in whole blood in all multi-methods of capture separation circulating tumor cell, from fluid
The angle of mechanics is set out, it is recognized that the method for being most expected to realize large volume flux is filtering with microporous membrane, United States Patent (USP)
US7846393B2 describes the method using circulating tumor cell in miillpore filter capture blood, but the patent microwell array is filtered
The micropore gap size of film is big, and fluid is big to the shearing force of cell in filter process, leads to the poor activity of cell, and micropore volume
It is easily organized into a point blocking, influences flux and detection positive rate.
Chinese patent CN107694347 discloses a kind of microwell array filter membrane, passes through the side of conformal deposited Parylene
Formula increases porosity and the mode of reduction adjacent cells gap size increases flux, reduces section of structural constituent and background cells
It stays, and increases the viability of circulating tumor cell, but tested by a large amount of clinical sample, find to face in face of complicated
Bed sample, such as bronchoalveolar lavage fluid, hydrothorax or ascites, the structural constituent and cell component of bulk are all retained down, and flux is anxious
Drastic change is small, it may occur that blocking, to can not achieve the detection of the circulation or Exfoliated tumor cells for complex sample.
The common gauze of clinic, steel wire, nylon wire go to separate the tissue of these bulks and mucus ingredient, then again through array
Miillpore filter is separated as secondary seperation film retention, but defect of both the presence of this separation method, on the one hand, gauze,
Steel wire or nylon wire micropore size are too big and homogeneity is poor, limited, the secondary separation of the bulk tissue or mucus ingredient blocked
Film still has very big probability to block, second aspect, there is very that maximum probability finds tumour cell in the structural constituent of bulk, and
The thickness of gauze, steel wire or nylon wire is very thick and out-of-flatness, is difficult through native staining directly in microscopic observation to capture
Target cell.
If by increase Parylene array miillpore filter aperture come separate bulk structural constituent and cell at
Point, do not change its thickness and homogeneity in order to which native staining is in the tumour cell of microscopic observation capture, such as make this micro-
Work as porosity of=since viability of the porosity for cell has a significant impact in micropore size >=15 μm of hole array filter membrane
When 50%, micropore gap is sufficiently small, and the microwell array filter membrane for meeting this micropore size and porosity meets cell separation process
The demand to cells viability, but its rigidity and mechanical strength be remarkably decreased, flexibility dramatically increases, and leads to microwell array film
Deformation, curling or fold, cannot form, the practicability is poor.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is that overcoming filter membrane in the prior art with large aperture array micropore
It is flexible excessive, it cannot form, the technological deficiency without practicability, to provide a kind of with larger aperture high porosity micropore
The flexible microporous film of array is faced by increasing supporting zone with suitable rigidity and mechanical strength to separate complexity
Bulk and high viscosity background component and cell component in bed fluid sample；
The technical problem to be solved in the present invention also resides in the micropore battle array for overcoming and using micropore size≤15 μm in the prior art
The technological deficiency of blockage of the micro orifice occurs for column filter membrane detection of complex clinical fluid samples, to provide a kind of with multi-stage separation film
Cell separation apparatus, the detection of object is tested to complex fluid sample flux with higher and can increase through clinical practice
Rate.
For this purpose, the present invention provides a kind of flexible microporous film, the flexible microporous film includes working region and is located at work
The supporting zone of region surrounding, the working region have micropore, and the micropore is arranged in array, micropore size >=15 μ
M, porosity of=50%, the flexible microporous film thickness are greater than the gap of micropore.
Preferably, the micropore size of the working region is 25-100 μm, porosity of=71.2%.
Preferably, the flexible microporous film is Parylene microporous barrier.
Preferably, the supporting zone micropore is arranged in array, micropore rate < 50%.
Preferably, the supporting zone is the membrane structure of not micropore.
The invention also discloses a kind of preparation methods of above-mentioned flexible microporous film, comprising the following steps:
Preparation micro-pillar array: preparing the transoid micro-pillar array of the flexible microporous membrane micropore on substrate, the microtrabeculae
Size is consistent with the pore size of flexible microporous film of required preparation；
Deposit polymer: Parylene is deposited in micro-pillar array, until closing microtrabeculae gap；
Except polymer: polymer more than removal micro-pillar array surface obtains until micro-pillar array top end surface all exposes
Obtain the microwell array structure of the polymer；
Release: release flexible microporous film.
Preferably, the height of the transoid microtrabeculae is identical as the thickness of the flexible microporous film；Pass through oxygen plasma soma
Method etches back the polymer on the surface for carving removal micro-pillar array top or more；By removing or corroding substrate release polymers micropore
Array structure.
The flexibility prepared the invention also discloses a kind of above-mentioned flexible microporous film or above-mentioned flexible microporous membrane preparation method is micro-
Application of the pore membrane in cell separation field.
The invention also discloses a kind of cell separation apparatus, including,
Filter chamber, including injection port and outlet；
Separating film module is set in filter chamber, including,
At least one above-mentioned flexible microporous film or the flexible microporous film of above-mentioned flexible microporous membrane preparation method preparation are as just
Grade seperation film, close to the injection port；
With,
At least one final stage seperation film, close to the outlet, micropore size≤15 μm, porosity of=25%；
Wherein, the thickness of the primary seperation film and the final stage seperation film is all larger than its micropore gap.
Preferably, the micropore in the final stage seperation film is arranged in array.
Preferably, the final stage seperation film is microwell array filter membrane.
Preferably, including, sample feeding pipe, connecting tube and sample outlet pipe, the sample feeding pipe, connecting tube and sample outlet pipe are sequentially connected with shape
At the filter chamber, the primary seperation film is set to the junction of the sample feeding pipe and the connecting tube, the final stage separation
Film is set to the junction of the sample outlet pipe and the connecting tube.
Preferably, the primary seperation film and the final stage seperation film are fixed by seperation film fixture, the seperation film
Fixture includes magnetic coupling, passes through the fixed primary seperation film of magnetic force and the final stage seperation film.
Preferably, the seperation film fixture further includes fixed magnet ring, is sheathed on magnetic coupling and reinforces connection.
Technical solution of the present invention has the advantages that
1. flexible microporous film provided by the utility model is the microwell array film of 2.5 dimension of one kind, including working region, work
Micropore size >=15 μm for making region, due to the porosity of=50% of the flexible microporous film, micropore gap is small, therefore in this hole
Under diameter, working region flexibility it is excessive, rigidity and mechanical strength are all substantially reduced, can deform in actual use, crimp or
Fold, shape are difficult to maintain, therefore further include supporting zone, and the membrane structure of the supporting zone includes two kinds of situations, first is that branch
Support the membrane structure that region is not micropore；Second is that supporting zone is in microwell array shape, micropore rate < 50%；Make to support
Region has suitable mechanical strength and mechanical property, and the structure of these two types of supporting zones can play entire flexible microporous film
To supporting role, increase its rigidity and mechanical performance, its deformation, curling or fold is prevented, to improve the practicality.
2. the present invention prepares flexible microporous film of the supporting zone porosity less than 50% using transoid microtrabeculae conformal deposited method
When, the transoid micro-pillar array in preparation work region and supporting zone, makes after Parylene is deposited on microtrabeculae on substrate
It is standby to obtain the smooth supporting zone of marginal texture, since Parylene growth has non-directional, grown along micro-post surface,
Manufactured flexible microporous membrane micropore regular shape after removal microtrabeculae, accuracy is high, is particularly suitable for preparing supporting zone micropore gap
Lesser flexible microporous film, flexible microporous membrane fluid mechanical property obtained is good, and flux is high；And Parylene has well
Biocompatibility, be conducive to the viability of target cell being retained down；
3. the present invention prepares the not membrane structure of micropore or supporting zone micropore gap using traditional mask etching method
Biggish flexible microporous film, preparation method is simple, at low cost, and support strength is high.
4. a kind of cell separation apparatus provided by the invention has multi-stage separation system, primary seperation film is using the present invention
The flexible microporous film, aperture >=15 μm, porosity of=50% can efficiently separate bulk background component and cell component, cut
The background component of bulk is left, lower cell component is filtered, increases the liquid flux of entire separator, and of the present invention soft
Property microporous barrier, surfacing, the bulk background component of retention can be directly in the target of microscopic observation capture by native staining
Object increases positive rate；And final stage seperation film uses the microwell array filter membrane of aperture≤15 μm, porosity of=25%, this is micro-
Hole array filter membrane micropore size is greater than the diameter of target cell, under the conditions of the porosity, can retain lower target cell and filter down
Background cells can capture target cell in conjunction with primary seperation film to the maximum extent, improve positive rate.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is 4 gained flexible microporous membrane structure diagram of the embodiment of the present invention；
Fig. 2 is the partial enlargement of 4 gained flexible microporous film working region and supporting zone intersection of the embodiment of the present invention
Figure；
5 gained flexible microporous membrane structure diagram of Fig. 3 embodiment of the present invention；
Fig. 4 is the structural schematic diagram of cell separation apparatus described in the embodiment of the present invention 10；
Fig. 5 is the longitudinal section of cell separation apparatus described in the embodiment of the present invention 10；
Fig. 6 is the electron microscope of cell separation apparatus primary seperation film working region described in the embodiment of the present invention 10；
Fig. 7 is the partial enlarged view of Fig. 6；
Fig. 8 is the electron microscope of cell separation apparatus final stage seperation film described in the embodiment of the present invention 10；
Fig. 9 is the partial enlarged view of Fig. 8；
Figure 10 is the structural schematic diagram of cell separation apparatus final stage seperation film described in the embodiment of the present invention 10；
Figure 11 is magnetic coupling structural schematic diagram in cell separation apparatus described in the embodiment of the present invention 10；
Figure 12 is to fix magnet ring structure schematic diagram in cell separation apparatus described in the embodiment of the present invention 10；
Figure 13 is cell separation apparatus described in embodiment 10 in present invention evaluation example 3 to sample 1, sample 2 and sample 3
Average flux column diagram；
Figure 14 is in present invention evaluation example 3 using cell described in flexible microporous film alternative embodiment 10 described in embodiment 1
The real-time flux schematic diagram of liquid when primary seperation film is filtered separation to sample 1 in separator；
Figure 15 is in present invention evaluation example 3 using cell described in flexible microporous film alternative embodiment 10 described in embodiment 2
The real-time flux schematic diagram of liquid when primary seperation film is filtered separation to sample 1 in separator；
Figure 16 is in present invention evaluation example 3 using cell described in flexible microporous film alternative embodiment 10 described in embodiment 3
The real-time flux schematic diagram of liquid when primary seperation film is filtered separation to sample 1 in separator；
Figure 17 is in present invention evaluation example 3 using cell described in flexible microporous film alternative embodiment 10 described in embodiment 6
The real-time flux schematic diagram of liquid when primary seperation film is filtered separation to sample 1 in separator；
Figure 18 is in present invention evaluation example 3 using cell described in flexible microporous film alternative embodiment 10 described in embodiment 7
The real-time flux schematic diagram of liquid when primary seperation film is filtered separation to sample 1 in separator；
Figure 19 is that cell separation apparatus described in the embodiment of the present invention 10 filters primary seperation film progress original position HE obtained by hydrothorax
Testing result schematic diagram under microscope is put in after dyeing；
Figure 20 is that cell separation apparatus described in the embodiment of the present invention 10 filters primary seperation film progress obtained by bronchoalveolar lavage fluid
Testing result schematic diagram under microscope is put in after HE dyeing in situ；
Figure 21 is that cell separation apparatus described in the embodiment of the present invention 10 filters the progress of final stage seperation film obtained by bronchoalveolar lavage fluid
Testing result schematic diagram under microscope is put in after HE dyeing in situ；
Appended drawing reference
1- filter chamber, 11- injection port, 12- outlet, 2- primary seperation film, the working region 21-, 22- supporting zone, 23-
Micropore gap, 3- final stage seperation film, 4- sample feeding pipe, 5- connecting tube, 6- sample outlet pipe, 7- seperation film fixture, 71- magnetic coupling,
The connecting pin 711-, 712- fixing end, the fixed magnet ring of 72-.
Specific embodiment
There is provided following embodiments is to preferably further understand the present invention, it is not limited to the best embodiment party
Formula is not construed as limiting the contents of the present invention and protection scope, anyone under the inspiration of the present invention or by the present invention and its
The feature of his prior art is combined and any and identical or similar product of the present invention for obtaining, all falls within of the invention
Within protection scope.
Specific experiment step or condition person are not specified in embodiment, according to the literature in the art described routine experiment
The operation of step or condition can carry out.Reagents or instruments used without specified manufacturer, being can be by commercially available acquisition
Conventional reagent product.
Embodiment 1
The specific embodiment for present embodiments providing a kind of flexible microporous film, specifically comprises the following steps:
The transoid micro-pillar array and supporting zone micropore of flexible microporous film working region micropore are prepared on a silicon substrate
Transoid micro-pillar array, the transoid microtrabeculae cross section of the working region micropore is hexagon, and the opposite side distance of hexagon is 15 μ
The transoid microtrabeculae cross section of m, 2.6 μm of micropore gap, the supporting zone micropore are square, and side length is 4 μm, micropore gap
2.1 μm, the micropore gap is the shortest distance between two neighboring square, and the height of all microtrabeculaes is 10 μm；It will be poly- pair
Dimethylbenzene is deposited in micro-pillar array, until closing microtrabeculae gap；Micro-pillar array table is removed by oxygen plasma dry etching
Polymer more than face, until micro-pillar array top end surface all exposes；Corrosion silicon substrate obtains the microwell array of the polymer
Structure discharges flexible microporous film；The area of flexible microporous film working region described in the present embodiment be 12 × 12mm, working region and
The area of supporting zone and be 17 × 17mm, with a thickness of 10 μm.
Embodiment 2
The specific embodiment for present embodiments providing a kind of flexible microporous film, specifically comprises the following steps:
The transoid micro-pillar array and supporting zone micropore of flexible microporous film working region micropore are prepared on a silicon substrate
Transoid micro-pillar array, the transoid microtrabeculae cross section of the working region micropore is circle, and circular diameter is 25 μm, adjacent two
A circular circle center distance is 26.3 μm；The transoid microtrabeculae cross section of the supporting zone micropore is circle, and circular diameter is 4
μm, two neighboring circular circle center distance is 5.2 μm, and the height of all microtrabeculaes is 6 μm；Parylene is deposited on microtrabeculae battle array
On column, until closing microtrabeculae gap；The polymer on micro-pillar array surface or more is removed by oxygen plasma dry etching, until
Micro-pillar array top end surface all exposes；Corrosion silicon substrate obtains the microwell array structure of the polymer, discharges flexible microporous film；
The area of flexible microporous film working region described in the present embodiment is 10 × 10mm, the area of working region and supporting zone and be 15
× 15mm, with a thickness of 6 μm.
Embodiment 3
The specific embodiment for present embodiments providing a kind of flexible microporous film, specifically comprises the following steps:
The transoid micro-pillar array and supporting zone micropore of flexible microporous film working region micropore are prepared on a silicon substrate
Transoid micro-pillar array, the transoid microtrabeculae cross section of micropore is rectangle, and the long side of rectangle is 30 μm, and the short side of rectangle is 9 μm,
Micropore gap is 2.5 μm, and micropore gap is the shortest distance between being connected two rectangles, porosity 73%, the supporting zone
The transoid microtrabeculae cross section of micropore is rectangle, and the long side of rectangle is 8 μm, and the short side of rectangle is 2 μm, and micropore gap is 2.5 μm, hole
Gap rate is 35%, and the height of all microtrabeculaes is 10 μm；Parylene is deposited in micro-pillar array, until between closing microtrabeculae
Gap；The polymer on micro-pillar array surface or more is removed by oxygen plasma dry etching, until micro-pillar array top end surface is complete
Expose in portion；Corrosion silicon substrate obtains the microwell array structure of the polymer, discharges flexible microporous film；Flexibility described in the present embodiment is micro-
The area of pore membrane working region is 13 × 13mm, the area of working region and supporting zone and be 18 × 18mm, with a thickness of 10 μ
m。
Embodiment 4
The specific embodiment for present embodiments providing a kind of flexible microporous film, specifically comprises the following steps:
The transoid micro-pillar array and supporting zone micropore of flexible microporous film working region micropore are prepared on a silicon substrate
Transoid micro-pillar array, the transoid microtrabeculae cross section of the working region micropore is hexagon, and the opposite side distance of hexagon is 50 μ
M, micropore gap are 4.8 μm, the shortest distance of the micropore gap between two neighboring hexagon, and porosity 83.7% is described
The transoid microtrabeculae cross section of supporting zone micropore is square, and side length is 4 μm, and micropore gap is 4 μm, porosity 25%, institute
The height for having microtrabeculae is 10 μm；Parylene is deposited in micro-pillar array, until closing microtrabeculae gap；Pass through oxygen plasma
Body dry etching removes the polymer on micro-pillar array surface or more, until micro-pillar array top end surface all exposes；Corrode silicon lining
Bottom obtains the microwell array structure of the polymer, discharges flexible microporous film；Flexible microporous film working region described in the present embodiment
Area is 12 × 12mm, the area of working region and supporting zone and be 17 × 17mm, with a thickness of 10 μm, institute as shown in Figure 1, Figure 2
Show.
Embodiment 5
The specific embodiment for present embodiments providing a kind of flexible microporous film, specifically comprises the following steps: on substrate
A strata paraxylene is deposited, with a thickness of 10 μm；Parylene C Surface PVD (Physical vapor deposition,
Physical vapor deposition) depositPhotoetching and H2O2Patterned Ti exposure mask micro-structure is obtained after (1:50) corrosion,
Then the RIE of Parylene, the H for the use of volume fraction being 2% after RIE are carried out using Ti micro-structure as exposure mask2O2It is water-soluble
The exposure mask Ti of corrosion remnants；It is finally impregnated using deionized water, microwell array filter membrane is discharged from substrate；The workspace
The opposite side distance of domain micropore hexagon can be 50 μm, and micropore gap is 8 μm, porosity 74%；Supporting zone is no micropore
Membrane structure；The flexible microporous film thickness is 10 μm.
Embodiment 6
The specific embodiment for present embodiments providing a kind of flexible microporous film, specifically comprises the following steps:
The transoid micro-pillar array and supporting zone micropore of flexible microporous film working region micropore are prepared on a silicon substrate
Transoid micro-pillar array, the transoid microtrabeculae cross section of the working region micropore is rectangle, and the long side of rectangle is 70 μm, rectangle
Short side is 35 μm, and micropore gap is 4 μm, and the transoid microtrabeculae cross section of the supporting zone micropore is rectangle, and the long side of rectangle is
16 μm, the short side of rectangle is 4 μm, and micropore gap is 3 μm, and the height of all microtrabeculaes is 10 μm；Parylene is deposited on micro-
On column array, until closing microtrabeculae gap；The polymer on micro-pillar array surface or more is removed by oxygen plasma dry etching,
Until micro-pillar array top end surface all exposes；Corrosion silicon substrate obtains the microwell array structure of the polymer, and release is flexible micro-
Pore membrane；The area of flexible microporous film working region described in the present embodiment is 15 × 15mm, the area of working region and supporting zone
With for 19 × 19mm, with a thickness of 10 μm.
Embodiment 7
The specific embodiment for present embodiments providing a kind of flexible microporous film, specifically comprises the following steps:
The transoid micro-pillar array and supporting zone micropore of flexible microporous film working region micropore are prepared on a silicon substrate
Transoid micro-pillar array, the transoid microtrabeculae cross section of the working region micropore is regular hexagon, and opposite side distance is 100 μm,
Micropore gap is 5 μm, and the transoid microtrabeculae cross section of the supporting zone micropore is round, a diameter of 20 μm, two neighboring circle
The circle center distance of shape is 25 μm, porosity 49%；The height of all microtrabeculaes is 15 μm.Parylene is deposited on microtrabeculae battle array
On column, until closing microtrabeculae gap；The polymer on micro-pillar array surface or more is removed by oxygen plasma dry etching, until
Micro-pillar array top end surface all exposes；Corrosion silicon substrate obtains the microwell array structure of the polymer, discharges flexible microporous film；
The area of flexible microporous film working region described in the present embodiment is 16 × 16mm, the area of working region and supporting zone and be 20
× 20mm, with a thickness of 15 μm.
Embodiment 8
The specific embodiment for present embodiments providing a kind of final stage seperation film, specifically comprises the following steps: on substrate
A strata paraxylene is deposited, with a thickness of 12 μm；Parylene C Surface PVD (Physical vapor deposition,
Physical vapor deposition) depositThe H that photoetching and volume fraction are 2%2O2It is obtained after aqueous corrosion graphical
Ti exposure mask micro-structure, the RIE of Parylene is then carried out using Ti micro-structure as exposure mask, volume fraction is used after RIE
For 2% H2O2The exposure mask Ti of aqueous corrosion remnants；It is finally impregnated using deionized water, by microwell array filter membrane from substrate
Release；The opposite side distance for obtaining final stage separation membrane micropore hexagon is 15 μm, and micropore gap is 10 μm, porosity 36%,；Institute
Stating flexible microporous film thickness is 12 μm.
Embodiment 9
The specific embodiment for present embodiments providing a kind of final stage seperation film, using transoid microtrabeculae conformal deposited legal system
It is standby, specifically comprise the following steps:
The transoid micro-pillar array of the final stage separation membrane micropore, the transoid of the working region micropore are prepared on a silicon substrate
Microtrabeculae cross section is hexagon, and the opposite side distance of hexagon is 10 μm, and 10 μm of microtrabeculae gap, the height of all microtrabeculaes is 12 μm；
Parylene is deposited in micro-pillar array, until closing microtrabeculae gap；Microtrabeculae is removed by oxygen plasma dry etching
Polymer more than array surface, until micro-pillar array top end surface all exposes；Corrosion silicon substrate obtains the micro- of the polymer
Hole array structure discharges flexible microporous film；Obtain final stage separation 10 μm of opposite side distance of membrane micropore hexagon, porosity 25%,
12 μm of film thickness.
Embodiment 10
Present embodiment discloses a kind of cell separation apparatus, as shown in Figure 4 and Figure 5, including,
Filter chamber 1, the filter chamber include 11 outlet 12 of injection port；
Separating film module is set in filter chamber 1, including,
At least one flexible microporous film is as primary seperation film 2, and the flexible microporous film is close to the injection port 11, packet
Working region 21 and the supporting zone 22 positioned at 21 surrounding of working region are included, the working region 21 has micropore, the micropore
It arranges in array, micropore size >=15 μm, porosity of=50%；
With,
At least one final stage seperation film 3, close to the outlet 12, micropore size≤15 μm, porosity of=30%；
Wherein, the thickness of the primary seperation film 2 and the final stage seperation film 3 is all larger than the width in its micropore gap.
The cell separation apparatus includes two seperation films, and the primary seperation film 2 retains bulk background component, thin under filter
Born of the same parents' ingredient；Target component in the 3 entrapped cell ingredient of final stage seperation film, small background component in cell component under filtering, two
The effect simultaneously of a seperation film, which can not only improve flux, prevents filter membrane from blocking, and primary seperation film and final stage seperation film can
Observation detection target cell under the microscope, increases the positive rate of detection.
1 inner wall of filter chamber of the cell separation apparatus and fluid sample, which react, in order to prevent influences testing result,
For 1 inner wall of filter chamber using the inert material Teflon not reacted with fluid sample, the injection port 11 is funnel-form,
Convenient for accepting liquid clinical sample, and a guide functions make under liquid flow；It is thin under width at the outlet 12, it is convenient for liquid flow
Out, in practical application, the detachably connected waste fluid container of the outlet.
The flexible microporous membrane aperture is bigger, and porosity is higher, under comprehensive equal densities and thickness, the higher machinery of porosity
Intensity is smaller, and flexible microporous film becomes very soft at aperture >=15 μm, porosity of=50%, and curling, fold, no occurs
It can form, not have practicability, the presence of the supporting zone 22 can maintain the shape and mechanical strength of large aperture working region；
Therefore the flexible microporous film that the primary seperation film 2 in the present embodiment cell separation apparatus is provided using the embodiment of the present invention 4, institute
State the working region 21 and supporting zone 22 that flexible microporous film includes large aperture；The working region micropore is hexagon, six sides
The opposite side distance of shape is 50 μm, and the supporting zone micropore is square, and side length is 4 μm；The area of the working region is 12
The area of × 12mm, working region and supporting zone and be 17 × 17mm, with a thickness of 10 μm；As shown in Figure 1, Figure 2, Fig. 6 and Fig. 7 institute
Show.
For flexible microporous film as array miillpore filter, mechanical strength is related with porosity, when porosity is less than 50%
It is unlikely to deform with enough mechanical strengths with practicability, supporting zone square micropore side length described in the present embodiment is 4 μ
M, porosity 25%.
Since micropore size is smaller, and the thickness of flexible microporous film is thicker, and micropore has certain depth, in order to guarantee
The precision of micropore, it is conformal using transoid microtrabeculae also for the separating property and liquid flux for further increasing primary seperation film 2
Sedimentation prepares the present embodiment primary seperation film, and flexible microporous film inner wall obtained is smooth, and liquid flow is out-of-date not to be generated cell
Damage influence viability；The present embodiment transoid microtrabeculae conformal deposited uses Parylene for raw material, makes according to described in embodiment 4
It is prepared by Preparation Method.
When the micropore size of supporting zone 22 is sufficiently large, at 6 μm or more, mask etching method can also be used and prepared；Institute
Supporting zone 22 or the not membrane structure of micropore are stated, this kind primary seperation film 2 is prepared using conventional mask etching method,
It plays a supportive role to working region 21.
In order in actual use, increase the target cell being retained down to the rejection effect of target cell and raising
Viability, the final stage seperation film 3 are small pore size micro-porous array filter membrane, the shapes and sizes of the micropore, according to to be separated
Target cell separating effect depending on, cell separation apparatus described in the present embodiment use final stage seperation film 3 on micropore be in
Array arrangement, the shape of the micropore are hexagon, and the opposite side distance of hexagon is 10 μm, less than the diameter 15 of target cell
μm, guarantee the high-recovery of target cell with this, 3 μm of the width bits in adjacent cells gap, less than 4 μm of radius of background cells,
So that background cells are easier to be carried away by the flow during filtrate flows, and the fluid for reducing the cell captured on micropore is cut
Shear force improves the activity of capture cell, as seen in figs. 8-10.
In order to further increase to the liquid flux and separating effect in fluid sample separation process, the cell separation dress
The final stage seperation film 2 set is prepared using transoid microtrabeculae conformal deposited method.
For the ease of assembling and replacing separating film module, it further includes sample introduction that the cell separation apparatus, which is split type structure,
Pipe 4, connecting tube 5 and sample outlet pipe 6, the sample feeding pipe 4, connecting tube 5 and sample outlet pipe 6 are sequentially connected with to form the filter chamber 1, described
Primary seperation film 2 is set to the junction of the sample feeding pipe 4 and the connecting tube 5, and the final stage seperation film 3 is set to described
The junction of sample outlet pipe 6 and the connecting tube 5.
It is damaged in order to seperation film be connected in the cell separation apparatus while avoiding it occur, the primary seperation film
2 and the final stage seperation film 3 be fixed by seperation film fixture 7, the seperation film fixture 7 include magnetic coupling 71, pass through
The fixed primary seperation film 2 of magnetic force and the final stage seperation film 3 have circle among the magnetic coupling 71 as shown in figure 11
The cross section in hole, the circular hole is consistent with the cross section of filter chamber 1, forms a part of filter chamber 1, the magnetic coupling 71
One end is smooth, is fixing end 711, for passing through with another 71 fixing end 711 of magnetic coupling in same seperation film fixture 7
Magnetic force clamps flexible microporous film, and 71 other end of magnetic coupling is that connecting pin 712 will be separated by being connected with connecting tube 5
Film is fixed in filter chamber 1.
Since magnetic coupling forces are weaker, in order to reinforce connection, each magnetic coupling is additionally provided with a fixed magnet ring
42, as shown in figure 12, the fixed magnet ring 72 is sheathed on the connecting pin 712 of a magnetic coupling 71.
Comparative example 1
This comparative example provides a kind of specific embodiment of flexible microporous film.
This comparative example embodiment and 1 embodiment of embodiment are essentially identical, and difference is, this comparative example does not have Support
Domain, this comparative example gained flexible microporous film are crimped, cannot be formed.
Comparative example 2
This comparative example provides the specific embodiment of cell separation apparatus.
This comparative example embodiment and cell separation apparatus described in embodiment 10 are essentially identical, and difference is, this comparative example
Without containing primary seperation film, only one interior final stage seperation film of filter chamber.
Comparative example 3
This comparative example provides the specific embodiment of cell separation apparatus.
This comparative example embodiment and 10 embodiment of embodiment are essentially identical, and difference is, this comparative example is using identical
Seperation film be primary seperation film and final stage seperation film, the aperture of the seperation film is hexagon, and aperture is 15 μm, and porosity is
30%, with a thickness of 10 μm.
Evaluate 1 porosity test of example
Porosity uses densimetry, it is known that the density a of film raw material, by observing and can be calculated film density b (film quality
The ratio between amount and apparent volume), then porosity can be obtained by formula (a-b)/a.
1 embodiment 1-7 of table, 1 porosity test result of comparative example
| Embodiment | Working region interporal lacuna (%) | Supporting zone interporal lacuna (%) | Film form |
| 1 | 50 | 49 | Smooth no deformation |
| 2 | 71.2 | 45 | Smooth no deformation |
| 3 | 73 | 35 | Smooth no deformation |
| 4 | 83.7 | 25 | Smooth no deformation |
| 5 | 74 | / | Smooth no deformation |
| 6 | 84.5 | 47.2 | Smooth no deformation |
| 7 | 91.4 | 49 | Smooth no deformation |
| Comparative example 1 | 50 | / | Curling and deformation occurs |
Evaluate 2 deformation test of example
Colored co-focusing imaging (CCI) mechanics test device, which is based on, for 100ml/min in flow rate of liquid obtains the power-of filter membrane
Deformation tendency, fitting obtain the film stress of embodiment 1- embodiment 7 and the variable quantity of micropore size, as shown in table 2 below.
2 embodiment 1-7 flexible microporous membrane forces of table-deformation test result
| Embodiment | Film stress (× 10-2N) | Micropore size variable quantity (nm) |
| 1 | 2.1 | 21 |
| 2 | 1.9 | 26 |
| 3 | 1.9 | 30 |
| 4 | 1.8 | 35 |
| 5 | 1.8 | 28 |
| 6 | 1.7 | 41 |
| 7 | 1.5 | 48 |
Evaluate the test of 3 flux of example
Respectively using the separation dress of cell described in flexible microporous film alternative embodiment 10 described in embodiment 1-3 and embodiment 5-7
It sets obtained by middle primary seperation film, following three kinds of samples is filtered and is separated:
Sample 1: normal limpid bronchoalveolar lavage fluid；
Sample 2: the more muddy bronchoalveolar lavage fluid that bronchoscope repeated flushing lung tumors are collected；
Sample 3: the hemorrhagic bronchoalveolar lavage fluid that the lung tumors of the concurrent inflammation of bronchoscope repeated flushing are collected；
Using flexible microporous film described in embodiment 10, embodiment 1-3 and embodiment 5-7 as fraction first in embodiment 10
It is averaged using microwell array film described in embodiment 8 as final stage seperation film to sample 1-3 from cell separation apparatus obtained by film
Flux result is as shown in table 3 below；
It is as shown in figure 13 using average flux column diagram of the cell separation apparatus described in embodiment 10 to sample 1-3；
It is separated and is filled as cell described in embodiment 10 using flexible microporous film described in embodiment 1-3, embodiment 6, embodiment 7
The primary seperation film set, using microwell array film described in embodiment 9 as final stage seperation film, to the real-time flux result of sample 1
As seen in figs. 14-18；.
Flexible microporous film described in 3 embodiment 10 of table, embodiment 1-3 and embodiment 5-7 is as separation primary in embodiment 10
The average flux (ml/min) of cell separation apparatus obtained by film, comparative example 2 and comparative example 3 to sample 1-3
The tumour cell home position observation captured in evaluation 4 seperation film of example
The hydrothorax fluid sample containing tumour cell is filtered using cell separation apparatus described in embodiment 10, by first fraction
It is observed from being put under microscope after film progress HE dyeing, as a result as shown in figure 19, wherein appended drawing reference a is target tumor；
Contain tumour cell bronchoalveolar lavage fluid using the filtering of cell separation apparatus described in embodiment 10, removes primary separation
Film and final stage seperation film be put under microscope after original position HE is dyed and are observed, as a result as shown in Figure 20 and Figure 21, wherein attached drawing
Label b is target tumor.
50 bronchoalveolar lavage fluids containing tumour cell are filtered by cell separation apparatus described in embodiment 10 and
Detection, testing result positive accuracy rate reaches 90.91%, higher than the TCT method of conventional centrifugal smear.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments；It is right
For those of ordinary skill in the art, in more embodiments, in order to be obtained most further directed to different samples
Good separating effect, can prepare the seperation film of different pore size, using different membrane material and preparation method, the primary separation
New seperation film can also be increased between film and final stage seperation film, make other various forms of variations on the basis of the above description
Or it changes.There is no necessity and possibility to exhaust all the enbodiments.And obvious variation extended from this
Or it changes still within the protection scope of the invention.
Claims (14)
1. a kind of flexible microporous film, which is characterized in that the flexible microporous film include working region (21) and be located at working region
(21) supporting zone (22) of surrounding, the working region (21) have micropore, and the micropore is arranged in array, micropore hole
Diameter >=15 μm, porosity of=50%, the flexible microporous film thickness are greater than the gap (23) of micropore.
2. flexible microporous film according to claim 1, which is characterized in that the micropore size of the working region (21) is
25-100 μm, porosity of=71.2%.
3. flexible microporous film according to claim 1 or 2, which is characterized in that the flexible microporous film is Parylene
Microporous barrier.
4. flexible microporous film according to claim 1-3, which is characterized in that supporting zone (22) micropore is in
Array arrangement, micropore rate < 50%.
5. flexible microporous film according to claim 1-3, which is characterized in that the supporting zone (22) is not have
The membrane structure of micropore.
6. a kind of preparation method of any one of claim 1-5 flexible microporous film, which comprises the following steps:
Preparation micro-pillar array: the transoid micro-pillar array of the flexible microporous membrane micropore, the size of the microtrabeculae are prepared on substrate
It is consistent with the pore size of flexible microporous film of required preparation；
Deposit polymer: Parylene is deposited in micro-pillar array, until closing microtrabeculae gap；
Except polymer: polymer more than removal micro-pillar array surface is somebody's turn to do until micro-pillar array top end surface all exposes
The microwell array structure of polymer；
Release: release flexible microporous film.
7. the preparation method of flexible microporous film according to claim 6, which is characterized in that the height of the transoid microtrabeculae is big
In or equal to the flexible microporous film thickness；By oxygen plasma dry etching return carve removal micro-pillar array top end surface with
On polymer；Substrate release polymers microwell array structure is removed or corroded by part.
8. a kind of described in any item flexible microporous films of claim 1-5 or the flexible microporous film preparation side of claim 6 or 7
Application of the flexible microporous film of method preparation in cell separation field.
9. a kind of cell separation apparatus, which is characterized in that including,
Filter chamber (1), including injection port (11) and outlet (12)；
Separating film module is set in filter chamber (1), including,
At least one primary seperation film (2), close to the injection port (11), the primary seperation film (2) is appointed for claim 1-5
Flexible microporous film described in one or the flexible microporous film of the flexible microporous membrane preparation method preparation of claim 6 or 7；
With,
At least one final stage seperation film (3), close to the outlet (12), micropore size≤15 μm, porosity of=25%；
Wherein, the thickness of the primary seperation film (2) and the final stage seperation film (3) is all larger than its micropore gap.
10. cell separation apparatus according to claim 9, which is characterized in that the micropore on the final stage seperation film (3) is in
Array arrangement.
11. cell separation apparatus according to claim 9 or 10, which is characterized in that the final stage seperation film (3) is micropore
Array filter membrane.
12. cell separation apparatus according to claim 9, which is characterized in that including, sample feeding pipe (4), connecting tube (5) and
Sample outlet pipe (6), the sample feeding pipe (4), connecting tube (5) and sample outlet pipe (6) are sequentially connected with to be formed the filter chamber (3), described first
Grade seperation film (2) is set to the junction of two connecting tubes (5), and the final stage seperation film (3) is set to the sample outlet pipe
(6) and the junction of the connecting tube (5).
13. cell separation apparatus according to claim 9, which is characterized in that the primary seperation film (2) and the final stage
Seperation film (3) is fixed by seperation film fixture (7), and the seperation film fixture (7) includes magnetic coupling (71), passes through magnetic force
The fixed primary seperation film (2) and the final stage seperation film (3).
14. cell separation apparatus according to claim 13, which is characterized in that the seperation film fixture (7) further includes solid
Determine magnet ring (72), is sheathed on magnetic coupling (71) and reinforces connection.
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