CN107694347B

CN107694347B - Micropore array filter membrane and preparation method and application thereof

Info

Publication number: CN107694347B
Application number: CN201710679586.2A
Authority: CN
Inventors: 王玮; 刘姚萍
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2016-08-30
Filing date: 2017-08-10
Publication date: 2020-11-13
Anticipated expiration: 2037-08-10
Also published as: CN107694347A

Abstract

The invention discloses a microporous array filter membrane and a preparation method and application thereof, wherein the microporous array filter membrane which has a working area of more than 1 square centimeter and can accurately regulate and control pore diameter and pore gap is manufactured based on a micro electro mechanical system process (such as a Parylene MEMS process) of a conformal deposition polymer, has 2.5-dimensional characteristics and ultrahigh porosity, and can realize cell enrichment and separation of ultra-large volume flux.

Description

Micropore array filter membrane and preparation method and application thereof

Technical Field

The invention relates to the field of biological medicine and advanced manufacturing, in particular to an ultrahigh-porosity microporous array filter membrane which is manufactured based on a flexible micro-electro-mechanical system (flexible MEMS) process, has a large working area and can accurately regulate and control the aperture and the micropore gap, and a preparation method and application thereof.

Background

In recent years, Liquid Biopsy technology (i.e., capturing and recovering trace (down to single) exfoliated tumor cells and molecular markers such as cancer-associated DNA from non-invasively obtained clinical samples such as blood, urine or other body fluids, clinical lavage fluids, etc., without tissue puncture) has become an emerging important method for cancer diagnosis. In the liquid biopsy technology, the direct obtaining of exfoliated tumor cells from clinical samples is an important sample source for the existing pathological detection of tumor cytology and also a key basis for improving the sensitivity and specificity of related molecular markers or gene detection, but the main problems at present are that the detection rate of target tumor cells is low, the clinical diagnosis positive rate is low, and therefore the exfoliated tumor cells cannot be widely applied to cancer diagnosis. The realization of the liquid biopsy technology with high specificity and positive rate of the detection of the desquamated tumor cells has more important significance for realizing early accurate diagnosis of cancer.

In the liquid biopsy study of exfoliated Tumor cells, the most widely studied object is the isolation of Circulating Tumor Cells (CTCs) from blood, and in particular, the capture of CTCs in highly sensitive whole blood based on micro-nano technology has become possible since 2007, and the technology of capturing as low as a single CTC from 7.5mL of whole blood has begun to be applied in the clinical diagnosis and treatment study of cancer. Besides blood, trace exfoliated tumor cells can be enriched and separated from clinical samples such as alveolar lavage fluid, urine, saliva, tissue digestive fluid, cerebrospinal fluid, peritoneal lavage fluid and the like, and compared with CTC capture in blood, the exfoliated tumor cells captured from the clinical samples have stronger pertinence and tumor part directivity; but at the same time, the difficulty and challenge is greater, mainly due to: (1) compared with blood, the clinical samples have various cell sources, and part of the samples have more non-cell impurities; (2) more importantly, the volume of the clinical sample is usually large, for example, the alveolar lavage fluid is usually 20-50mL, the urine is 50-100mL, the peritoneal lavage fluid is usually up to 500-1000 mL, and the conventional liquid biopsy technology for CTC is difficult to handle. In order to realize a liquid biopsy system of trace exfoliated tumor cells in a large volume of clinical samples, the most critical technical difficulty lies in how to increase the volume flux so as to be able to complete the capture and separation of trace exfoliated tumor cells in the large volume of clinical samples within a proper time range (even during the operation). Among the many methods for the capture and separation of CTCs in whole blood, the most promising method for achieving a large volume flux is microfiltration membrane filtration, which is recognized from the viewpoint of hydrodynamics.

U.S. Pat. No. 5,324,328 (Membrane filter for capturing circulating tumor cells, US 7846393B2) describes a method for capturing circulating tumor cells in blood using a microporous Membrane, but the patent discloses a microporous array Membrane with large micropore gaps (hereinafter, referred to as the shortest distance between the edges of two adjacent micropores), and large shearing force of fluid on cells during filtration, resulting in poor activity of captured cells; another patent (Method and apparatus for microfiltration to performance cell separation, US 20090188864A1) designed 3-dimensional double-layer microporous array filters that use the lower membrane to provide a supporting force to the cells captured by the upper membrane to increase the activity of the captured cells. However, the processing method of the 3-dimensional double-layer filter membrane is complex and high in cost. In addition, the microporous filter membranes utilized in the two patents have large micropore gaps, low porosity and small filtration flux, and thus trace cells in large-volume clinical samples cannot be efficiently captured, so that a convenient large-scale high-porosity microporous array filter membrane processing method is still in urgent need of development.

Two key problems remain to be further solved in the microporous membrane method reported at present: (1) non-specific cell adhesion, in view of ensuring the mechanical strength of the filter membrane structure, the micro-pore gaps of the micro-pore filter membranes reported and commercialized in the prior art are large (>10 μm; mainly because the manufacturing process difficulty of the small micro-pore gap filter membrane is large and the yield of large-area suspension micro-pore arrays is low), non-target cells smaller than or close to the size can be adhered to the micro-pore gap supporting structure with a high probability, so that the purity of target exfoliated tumor cells obtained by enrichment and separation is low, and interference is brought to subsequent imaging analysis, further culture amplification and drug screening; (2) the activity of the captured exfoliated tumor cells is low, and due to the large micropore gaps and the small porosity of the microporous filter membrane, the shearing force of liquid flow to the captured target cells in the filtering process is large, so that the cells are reduced in activity and even completely cracked after being mechanically damaged, and further amplification culture and subsequent biochemical/molecular level analysis and research of the target cells cannot be carried out.

Disclosure of Invention

Aiming at the problems that the prior microporous array filter membrane technology still faces low recovery purity, influences on cell activity and the like, the invention provides a microporous array filter membrane, a preparation method and application thereof, wherein the microporous array filter membrane has the 2.5-dimensional characteristic and ultrahigh porosity, can overcome the defects of the prior microporous filter membrane in cell enrichment and separation, and is particularly suitable for the enrichment and separation of high-flux cells and particles of large-volume liquid.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a microporous array filter membrane, wherein the microporous array is a hollowed area on the filter membrane, the microporous gaps are filled with conformally deposited polymer and connected to form the filter membrane, the complete working area of the filter membrane is not less than 5mm x 5mm, and the membrane thickness is greater than the microporous gaps.

Further, the shape of the micropores can be designed into one or more of circular, rectangular, regular hexagonal and other geometric shapes or a combination of two or more different geometric shapes according to requirements.

Preferably, the conformally deposited polymer is parylene.

Preferably, the porosity of the filter membrane can be designed, typically ≧ 40%.

The preparation method of the microporous array filter membrane comprises the following steps:

1) preparing an inverse micro-column array on a substrate, wherein the characteristic dimension of the cross section of each micro-column is consistent with the micropore of the micropore array filter membrane to be prepared, and the height of the micro-column is consistent with the thickness of the micropore array filter membrane to be prepared;

2) conformally depositing the polymer on the micro-column array until the micro-column gaps are closed;

3) removing the polymer above the top end surface of the micro-column array until the top end surface of the micro-column array is completely exposed, and obtaining a micropore array structure of the polymer;

4) releasing the polymer micropore array structure to obtain the micropore array filter membrane.

Further, in step 2), the thickness of the deposited polymer is greater than half of the gap of the microcolumn.

Further, in the step 3), the polymer above the top end surface of the micro-column array is removed by oxygen plasma dry etching back.

Further, the polymer microwell array structure is released in step 4) by removing or etching the substrate.

The invention also provides application of the microporous array filter membrane in enriching and separating spherical or nearly spherical targets.

Furthermore, the distance between the opposite sides of the micropores of the micropore array filter membrane is less than or equal to the diameter of the spherical or nearly spherical target object.

Furthermore, the micropore gap of the micropore array filter membrane is smaller than the radius of the background object of the spherical or nearly spherical object, and is preferably less than or equal to 4 μm.

Further, the spherical or near spherical target includes a mass of cells, particles, or aggregates thereof.

The beneficial effect of the invention is that,

the microporous array filter membrane can keep the large working area of a complete unsupported structure while ensuring the mechanical strength of the membrane, and the precision error of a pore structure is 0.05-0.3 mu m.

(II) the characteristics of both 2-dimensional film and 3-dimensional structure: the effective continuous working area of the filter membrane is far larger than the membrane thickness, so that the filter membrane integrally presents the characteristic of a 2-dimensional membrane; the membrane thickness is larger than the micropore clearance, thereby showing 3-dimensional characteristic near the local micropores and simultaneously ensuring the mechanical strength of the filter membrane structure in the filtering process.

The shape of the micropores of the micropore array filter membrane is preferably regular hexagon or a structure close to regular hexagon, so that the uniform micropore gaps of the adjacent micropores in the whole filter membrane are ensured, and the difference between the diameters of the circumscribed circle and the inscribed circle of the micropores is smaller.

(IV) the distance between the opposite sides of the micropores is less than or equal to the diameter of the lumps of the target cells, the particles or the aggregates thereof and the like, so as to ensure the high recovery rate capture of the target cells; the filter has the advantages that the adjacent micropore gap with the radius smaller than that of background cells, particles or lumps is formed, so that objects such as the background cells and the like are easier to be taken away by fluid in the filtering process, the non-specific adhesion is obviously reduced, meanwhile, the shearing force of the fluid on the cells is also greatly reduced, and the volume flux, the purity and the activity of the cell capture operation are greatly improved. And for PBS, urine, lavage fluid and other clinical samples with low viscosity, the work flux is up to more than 120 mL/min.

And (V) the filter membrane can be used for capturing blood circulation tumor cells, enriching cells, particles or lumps in a blood sample and being used for subsequent detection or re-culture.

Drawings

FIG. 1(a) is a schematic view of a filter membrane of a micropore array in which the shape of micropores is regular hexagon according to the present invention and a partially enlarged portion thereof; FIG. 1(b) is a schematic sectional view taken along the side of the regular hexagon shown in FIG. 1 (a); fig. 1(c) is a schematic cross-sectional view taken along a diagonal line of the regular hexagon shown in fig. 1 (a).

FIG. 2 is a typical fabrication process of a microporous array filter membrane of the present invention, wherein (a) a silicon microcolumn array is prepared; (b) depositing parylene on the array of silicon micropillars; (c) etching back by the oxygen plasma dry method until the top end of the silicon micro-column is exposed; (d) HNA corrodes the silicon substrate, and the parylene micropore array structure is released.

FIG. 3(a) is a photograph of a physical embodiment of the microporous array filter according to the present invention; FIG. 3(b) is a partial magnified electron micrograph of the filter of the micropore array.

FIG. 4(a) is a schematic view of a slit-shaped microwell array filter according to the present invention and a partially enlarged portion thereof; FIG. 4(b) is a schematic diagram of different shapes and sizes of composite microporous array filter membranes; FIG. 4(c) is a schematic view of a high aspect ratio small-size micro-well array filter and a partially enlarged portion thereof.

FIG. 5(a) is a schematic diagram of the principle of testing a CCI non-contact optical platform for mechanical property testing of a microporous array filter membrane, wherein: 1-a mechanical sensor, 2-a micropore array filter membrane, 3-a PMMA carrier, 4-a surface gold reflection coating, 5-a chromatic aberration lens, 6-a beam splitter, 7-a pinhole, and 8-a spectrometer; FIG. 5(b) is a typical force-deformation curve diagram of a microporous array filter membrane measured when the pore gaps of the filter membrane are the same and the sizes of the micropores are different; FIG. 5(c) is a graph of a typical force-strain curve for a microwell array filter membrane measured for the same membrane microwell size and different microwell spacing; FIG. 5(d) equivalent Young's moduli of different porosity filters; FIG. 5(e) stress and pore size lateral deformation of different porosity filters during high throughput (>100mL/min) filtration.

FIG. 6 is a pellet particle via test based on different size filter membranes.

FIG. 7(a) shows the lateral stress experienced by 10 μm beads at the pore spacing of a 12 μm micropore; FIG. 7(b) shows the pressures applied to the beads on the surfaces of the filter membranes with different pore gap sizes; FIG. 7(c) shows the pressure and shear stress applied to the surface of the filter membrane with different pore gap sizes.

FIG. 8(a) is the results of T24 cell filtration/centrifugation controls diluted in 10mLPBS buffer solution; fig. 8(b) is a549 cells filtration/centrifugation control diluted in 10mLPBS buffer solution.

FIG. 9(a) a fluorescent label map of MC3T3 cells captured by a microwell array filter of the present invention, 5, 6-fluorescently labeled MC3T3 cells; FIG. 9(B) is a fluorescent photograph of MC3T3 cells cultured in situ on a microwell array filter of the present invention, 7, 8-DAPI labeled nuclei, 9, 10-Phalloidin-Rhodamine B labeled microwires; FIG. 9(c) in situ culture on locally enlarged cell membrane; FIG. 9(d) effect of different pore spacing on activity of reservoired cells after capture.

FIG. 10A comparison of the flux of filtered whole blood and PBS samples of the present invention compared to the flux of filtered whole blood of a reported response porosity product.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

Examples

The design of the microporous array filter membrane with the shape of the regular hexagon is shown in figures 1(a) - (c), wherein the gap regions of the micropores are filled with parylene and connected to form the filter membrane, and the regular hexagon micropores are hollow regions on the filter membrane. The main characteristics of the microporous array filter membrane are as follows:

(1) has 2.5-dimensional geometric characteristics;

(2) has larger effective filtration area (not less than 13mm multiplied by 13mm) and can realize super-large volume flux (not less than 150 mL/min);

(3) has precise pore diameter/micropore gap, and can realize high-performance cell filtration;

(4) the shape of the micropores is regular hexagon, so that the width of the gap between adjacent micropores is ensured to be consistent with that of the whole filter membrane, and the diameter difference between the circumscribed circle and the inscribed circle of the micropores is minimum;

(5) the distance between the opposite sides of the regular hexagonal micropores is smaller than the diameter of the target cells, so that the high recovery rate capture of the target cells is ensured;

(6) the gap between adjacent micropores (less than or equal to 4 μm) with the radius smaller than that of the background cells ensures that the background cells are easier to be taken away by fluid in the flowing process of the filtrate, reduces non-specific adhesion, and simultaneously reduces the shearing force of the fluid for capturing the cells on the micropores, thereby improving the activity of the cells.

FIG. 2 is a schematic diagram of a typical method of manufacturing a microwell array filter according to the present invention. The method comprises the following specific steps:

1) preparing an inverse micro-column array (which can also be realized by processes such as injection molding) on a silicon substrate, wherein the silicon micro-column is in a regular hexagon shape, and the characteristic size of the silicon micro-column is consistent with that of the micro-pores of the final filter membrane to be prepared as shown in figure 2 (a);

2) depositing parylene on the silicon micropillar array until the micropillar gap is closed (the parylene deposition thickness is more than half of the micropillar gap because parylene grows uniformly in all directions, the deposition thickness is related to deposition time, and the film thickness is also related to the structure, i.e. if the deposition thickness is less than half of the micropillar gap, the film micropore gap is not completely filled, the structure is not completely closed, and thus a slit is formed; the gap can be filled only if the deposition thickness is more than half of the gap of the microcolumn), as shown in fig. 2 (b);

3) performing dry etching back etching by using oxygen plasma to remove the parylene above the top surface of the microcolumn until the top surface of the silicon microcolumn array is completely exposed again to obtain a parylene microporous structure, as shown in fig. 2 (c);

4) the parylene microporous structure is released by etching the silicon substrate, as shown in fig. 2(d), to obtain an ultra-large volume flux of 2.5-dimensional cell enrichment separation filter.

A sample of the micropore array filter prepared by the above method is shown in FIG. 3(a), and the filter has a total area of 20 mm. times.20 mm, an effective filtration area of 13 mm. times.13 mm, and a thickness of 10 μm.

The photograph of the filter with the micropore array, which was observed and photographed under an electron microscope (JEOL, JSM-7500F), is shown in FIG. 3(b), and the filter shown in FIG. 3(a) had a porosity of 70.3%, a micropore gap of 2 μm, a membrane thickness of 10 μm, a side-to-side distance of regular hexagonal micropores of 10.3 μm, and a diagonal length of 12 μm.

The microporous structure of the filter membrane can be designed into various structures of round, rectangular, hexagonal and the like with different sizes according to the requirement, wherein, figure 4(a) is a slit-shaped high-porosity microporous array filter membrane (2 Mum multiplied by 8 Mum slit, 2 Mum long axis direction slit gap, 8 Mum short axis direction slit gap, 10 Mum membrane thickness), figure 4(b) is a multi-structure or multi-scale microporous array filter membrane (membrane thickness 10 Mum, microporous gap 16.46 +/-0.17 Mum, large pore diameter 11.21 +/-0.11 Mum, small pore diameter 3.46 +/-0.27 Mum), figure 4(c) is a high aspect ratio (>2) microporous array filter membrane, the microporous gap is 2 Mum, pore diameter (microporous diagonal length) is 1.39 +/-0.07 Mum, microporous depth (membrane thickness) is 10.45 +/-0.21 Mum, and aspect ratio (pore diameter/pore depth) is 7.5.

Experimental example 1: filter membrane performance testing and simulation

1-a force sensor, 2-a micropore array filter membrane, 3-a PMMA carrier, 4-a surface gold reflection coating, 5-a chromatic aberration lens, 6-a beam splitter, 7-a pinhole, and 8-a spectrometer; the mechanical properties of the filter membrane were tested using a non-contact optical method with CCI. The test principle is shown in fig. 5(a), and mainly comprises three parts of mechanical loading, optical imaging and signal processing: the mechanical loading part applies acting force to the microporous array filter membrane 2 through the mechanical sensor 1, the filter membrane deforms under the action of external force, the lower surface position of the filter membrane changes in the vertical direction (namely longitudinal deformation is generated), the lower surface position of the filter membrane is detected by the optical imaging system, and corresponding image information is converted into an optical imaging signal on the lower surface of the filter membrane after signal processing.

The optical imaging part adopts a chromatic aberration lens 5 to carry out continuous monochromatic light imaging on a white light point light source. When the surface is within the range of the distribution image, the light is scattered back to the chromatically poor lens 5 and finally reaches the pinhole 7. While and only a single wavelength image is completely on the detected surface (i.e. the lower surface of the filter) while all other wavelengths are filtered out of the pinhole 7.

The signal processing section: the optical imaging information is transmitted to the spectrometer 8 of the signal processing section, and the spectrometer 8 can calculate the position of the measurement point on the surface by analyzing the collected light. Along with the loading change of the force, the surface position calculated by the spectrometer changes, and the variable quantity is the longitudinal deformation quantity of the microporous array filter membrane.

In order to enhance the light reflection performance of the surface of the parylene filter membrane, the surface of the filter membrane is sprayed with gold (Ion beam coater ETD 2000, Ion current 15mA, 3min) before mechanical measurement. The thickness of the gold reflective coating 4 coated on the surface of the Parylene is 3nm, the Young modulus of gold is 79GPa (bulk material data), the thickness of the micropore array filter membrane 2 is 10 μm, and the Young modulus of Parylene is 2.8GPa, so that the influence of the ultra-thin gold reflective coating for auxiliary measurement on the deformation of the filter membrane can be completely ignored.

Typical force-deformation curves for filters obtained by a Color Confocal Imaging (CCI) based mechanical testing apparatus shown in fig. 5(a) are shown in fig. 5(b), (c), where the force applied by the mechanical sensor is f (n) and the longitudinal deformation of the filter is Δ L (μm).

Wherein FIG. 5(b) is a force-deformation curve of a regular hexagonal microporous filter membrane having the same membrane thickness (10 μm), the same micropore gap (4 μm), and different micropore diameters (herein referred to as micropore diagonal lengths), the graph showing that the larger the pore diameter (the higher the porosity), the larger the deformation of the filter membrane; FIG. 5(c) is a force-strain curve of a regular hexagonal microporous filter membrane having the same membrane thickness (10 μm), the same pore diameter (here, the diagonal length of the micropores is 12 μm), and different pore gaps, and the graph shows that the larger the pore gap (the lower the porosity), the smaller the strain of the filter membrane.

Further, in the experimental example, the experimental data obtained by the mechanical test is analyzed by using a large deformation circular plate theory proposed by s.timoshenko. Assuming that the filter membrane is clamped at the edge and a concentrated load F is applied to the center of the filter membrane, the relationship between the longitudinal deformation Δ L of the filter membrane and the load can be expressed by equation (1):

where μ represents the Poisson's ratio of parylene, here calculated using 0.5; h is the membrane thickness, a is the radius of the membrane area, and E is the equivalent Young's modulus of a 2.5 dimensional parylene micropore array membrane. The experimental data were fitted to equation (1) using Matlab to extract the equivalent young's modulus E of each test sample filter.

To examine the accuracy of equation (1) in deriving the equivalent Young's modulus of the microporous array membrane, it was first verified that the non-porous parylene film exhibited good consistency with the actual experimental test points of the non-porous parylene film and the values derived from equation (1), and the Young's modulus value calculated by fitting was 2.65. + -. 0.04GPa, which was consistent with the properties of parylene materials provided by the raw material supplier (https:// sccoating. com /), indicating that it is reasonable to derive the equivalent Young's modulus of the microporous array membrane from the data points obtained by mechanical testing based on CCI through equation (1). The equivalent Young's modulus of the microporous array filter membranes with different porosities is shown in FIG. 5(d), and the equivalent Young's modulus of the parylene microporous array filter membranes decreases with increasing porosity.

And further substituting the membrane equivalent Young modulus value into a filter membrane stress simulation model under high flux (100mL/min) liquid filtration to obtain the pressure applied to the filter membranes with different porosities and the deformation of the microporous structure caused by the pressure. As shown in FIG. 5(e), the microporous membrane with 70% porosity in the example of the present invention has a micropore deformation of less than 33 nm. Therefore, in the process of high-flux cell capture, the deformation of the micropores caused by the fluid pressure cannot influence the effect of micron-sized cell separation.

Experimental example 2: rigid microsphere via hole test for filter membrane filtration performance

Assembling 4 microporous array filter membranes into a multistage filtration system from top to bottom according to the sequence of the sizes of micropores from large to small, wherein the pore diameters/gaps (the sizes actually measured according to an electron microscope photograph after the processing) of the parylene regular hexagonal microporous array filter membranes with the film thickness of 10 mu m used from top to bottom are as follows: 14.52 +/-0.20 mu m/4.43 +/-0.06 mu m, 11.21 +/-0.11 mu m/4.77 +/-0.15 mu m, 9.13 +/-0.09 mu m/4.69 +/-0.24 mu m and 7.51 +/-0.13 mu m/4.46 +/-0.09 mu m. 10mL of deionized water suspended with polystyrene microspheres (6-1-2200, Tianjin BeisLei chromatography technical development center) with a diameter range of 6-22 μm was filtered through an assembled multi-layer filtration system. After filtration, the parylene filter membrane is taken down and placed overnight at room temperature, gold (Ion beam coater ETD 2000, Ion current 15mA, 3min, thickness-3 nm) is sprayed on the surface of the filter membrane capturing the microspheres, then the microspheres captured on the filter membrane are observed and photographed by using a scanning electron microscope (SEM, JSM-7500F, JEOL), the diameter of the microspheres on each filter membrane is measured according to the electron microscope photograph, and the specific result is shown in FIG. 6.

The diameter distribution range of the microspheres obtained by separation on the filter membrane is between the diameter (d ') of the inscribed circle of the micropores of the membrane and the diameter (d') of the inscribed circle of the micropores of the previous membrane, and further verifies that the pore diameter of the micropore array filter membrane can not be changed in the filtering process, so that the size resolution of microsphere filtering can not be influenced. Meanwhile, the microsphere multistage filtration separation result shows a good size truncation effect, and the high size resolution and the separation purity of the microporous array filter membrane based on size separation are fully proved.

Wherein, the simulation of the stress condition of the microspheres in the surface flow field of the microporous filter membrane in fig. 7(a) reveals some reasons of the high-efficiency separation and the excellent size cut-off effect of the present invention. The structure of the pore space greatly reduces the possibility that cells or particles stay in the micropore gaps, and the calculation proves that in the cell scale range, the structure that the micropore gaps are smaller than the film thickness effectively ensures that the nonspecific recovery phenomenon that the cells or the particles stay in the micropore gaps is reduced.

Experimental example 3: cell capture in liquid

The control results of trace amounts of urothelial cancer cells (T24) and lung cancer cells (a549) in large volume samples detected by the filter filtration method of the present invention and the conventional centrifugation method are shown in fig. 8(a) and fig. 8(b), respectively. Using the recovery efficiency results of A549 cells in FIG. 8(b) as an example, the number of cells in 10mLPBS was 10 in the conventional centrifugation method³In one occasion, the recovery efficiency is lower than 10 percent (9.6 +/-1.5 percent) by using the traditional centrifugal conditions (3500rpm, 20min), andthe regular hexagon micropore array filter membrane with the membrane thickness of 10 mu m, the micropore diameter of 10 mu m (regular hexagon diagonal line) and the micropore gap of 4 mu m prepared by the invention is adopted for filtration, and the recovery efficiency is 10 when the number of cells is 10³、10⁴The cell recovery capacity of the method is respectively as high as 76.7 +/-16.8% and 90.4 +/-9.5%, which is far higher than that of the traditional centrifugation method. Because the traditional centrifugal method has low cell recovery efficiency, when the cell number in 10mL of liquid is less than 10²Control experiments for recovery by centrifugation were not performed. The liquid biopsy method of the invention has the cell number of 10²And still has very high recovery efficiency for 10, which is 86.7 +/-16.8 percent and 71.3 +/-5.1 percent respectively; even when the number of cells is as low as 1, the recovery efficiency is still higher than 55.6% (the recovery rate reduction is also possible to be caused by the cell loss caused by the micro-pipette experiment error in the initial experiment), therefore, the recovery capacity of the ultra-large volume flux liquid biopsy method established on the basis of the ultra-high porosity microporous array filter membrane is at least 2 orders of magnitude higher than that of the traditional centrifugation method in the recovery capacity of the trace cells in a large volume sample.

Experimental example 4: re-culture after recovery of cells in liquid

A certain amount of activated fluorescent dye (Cell Tracker staining, Molecular Probes) counted/diluted by a hemocytometer was added^TM) The labeled model cells (mouse osteoblasts, MC3T3) were added to 10mL Phosphate Buffered Saline (PBS) to prepare a cell suspension to be filtered. After the cell suspension is filtered through the regular hexagon micropore array filter membrane with the pore size of 12 mu m micropores (regular hexagon diagonal line), the gap of 4 mu m micropores and the membrane thickness of 10 mu m, the filter membrane is taken down and moved to a cell culture environment for cultivation. Wherein, the fluorescence of in situ labeling of cells captured by the micropore array filter according to the present invention is shown in FIG. 9(a), and the morphology and spread state of MC3T3 captured and separated on the micropore array filter with a 4 μm micropore gap cultured in situ through a three-day filter are shown in FIG. 9(B) and FIG. 9(c), wherein DAPI-labeled

nuclei

7 and 8 are shown to be blue fluorescence and Phalloidin-Rhodamine B-labeled

microwires

9 and 10 are shown to be red fluorescence under a fluorescence microscope. Laser confocal microscope (Laser Scanni)ng confocal microscopical, TCS SP5X, Leica) photo shows that the spreading state of cells on the filter membrane is good, experiments prove that the micropore array filter membrane with 4-micron micropore gaps provided by the invention can ensure the activity of the captured and separated cells, and the filter membrane is expected to be used for deep research in the aspects of biochemical/gene mechanism, tumor drug screening/curative effect detection and the like through subsequent culture and amplification.

Meanwhile, in the process of separating cells by using the ultra-high porosity filter membrane, the key of reducing the micropore clearance and reducing the low stress borne by the cells is the maintenance of the cell activity. As shown in FIG. 9(d), the re-chaperoning activity of cells after being captured by filters of different micropore gaps was examined using (EthD-1/Calcein AM) dead/live cell double stain kit (L3224, Invitrogen, Thermofoisher). The kit comprises two components, one component is ethidium homimimer-1 labeled with red fluorescence, and in a normal condition, the ethidium homimimer-1 cannot penetrate the intact cell membrane of a living cell, and when the cell loses activity, the integrity of the cell membrane is destroyed, the permeability is improved, so that the ethidium homimimer-1 can enter the cell and specifically bind to nucleic acids inside the cell, and therefore, dead cells bound with the ethidium homimimer-1 are red under a fluorescence microscope (circled by white dotted lines in FIG. 9 d). The other is calcin-AM labeled with green fluorescence, which has living cell membrane permeability and can specifically bind with lipase in cells by permeating into cell membranes, and the living cells combined with the calcin-AM show green fluorescence under a fluorescence microscope. The smaller the proportion of dead cells in the total cell count circled by the white dotted line, the higher the proportion of active cells, and the higher the activity of the cells captured by the corresponding filter. Therefore, as shown in FIG. 9(d), as the pore space of the membrane increases, specifically, the pore space changes from 2 μm to 16 μm, the activity of the recovered cells tends to decrease, and when the pore space increases to 8 μm, the corresponding cell activity decreases to less than 50%. Further demonstrated by modeling simulation of the surface flow of the microporous filter membrane, as shown in fig. 7(b) and 7(c), under the condition that the pore size of the filter structure is fixed, the values of the shearing force and the pressure applied to the rigid ball (8 μm) passing through the micropore and the rigid ball (16 μm) captured at the micropore are increased along with the increase of the micropore gap, and the increase of the shearing force and the pressure means that the damage degree of the cell is increased during the filtration process, which may lead to the reduction of the cell activity. Therefore, to increase the activity of the cells, it is necessary to decrease the micropore spacing of the filter of the micropore array, and the simulation result is also consistent with the experimental result of FIG. 9 (d). The experimental example shows that the invention provides a simple and effective means for efficiently capturing target cells in a low-stress environment and culturing the target cells in situ.

Experimental example 5: application of microporous filter membrane in tumor cell separation of clinical liquid sample

The invention has a high-flux high-precision 2.5-dimensional micropore array structure, and is suitable for the application of cell enrichment and separation in clinical samples such as blood and the like, particularly large-volume clinical samples (such as urine, various lavage fluids and the like). The highest flux for filtering blood with the filter membrane with different porosities reported in the prior report and literature is 2mL/min (the flux is under the condition of an additional injection pump or negative pressure drive). The working flux of the whole blood sample filtration is more than 20mL/min under the condition of not needing external power and only using the gravity of the liquid to drive. For clinical samples with low viscosity such as PBS, urine, lavage fluid and the like, the working flux is up to more than 120mL/min, and the product has obvious advantages in the aspect of working flux compared with similar products, and relevant statistics are shown in FIG. 10.

The above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and those skilled in the art can make modifications or equivalent substitutions to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and the scope of the present invention should be determined by the claims.

Claims

1. A micropore array filter membrane is characterized in that a micropore array is a hollow area on the filter membrane, micropore gaps are filled with conformally deposited polymers and connected to form the filter membrane, the complete working area of the filter membrane is more than or equal to 5mm multiplied by 5mm, and the membrane thickness is larger than the micropore gaps; the micropore array filter membrane ensures the mechanical strength of the membrane and simultaneously keeps a large working area of a complete unsupported structure.

2. A microwell array membrane according to claim 1, wherein said conformally deposited polymer is parylene.

3. The filter membrane according to claim 1, wherein the porosity of the filter membrane is not less than 40%.

4. The method for preparing a filter membrane according to any of claims 1 to 3, comprising the steps of:

5. The method of claim 4, wherein in the step 2), the polymer is deposited to a thickness greater than half of the gap between the microcolumns.

6. The method of claim 4, wherein the polymer above the top surface of the micropillar array is removed in step 3) by dry etching back with oxygen plasma.

7. The method of claim 4, wherein the polymer microwell array structure is released in step 4) by removing or etching the substrate.

8. Use of the microwell array filtration membrane of any one of claims 1 to 3 for enrichment and separation of spherical or subsphaeroidal targets.

9. The use of claim 8, wherein the microwells have a distance across less than or equal to the diameter of a spherical or near-spherical object.

10. The use of claim 8, wherein the micropore gap is smaller than the radius of a background object for a spherical or near spherical object.