Filter base system and method for separating clumped particles

Abstract

Embodiments of the disclosed technology provide a separation device for separating agglomerated particles. The separation device can include an inlet designed to receive a fluid and an outlet designed to output the fluid. The fluid can include a plurality of non-agglomerated particles and a plurality of agglomerated particles. The separation device can include a plurality of micropores. Each micropore can have a plurality of sidewalls and a bottom surface having a mesh capture region. The mesh capture region can capture the plurality of agglomerated particles while allowing the non-agglomerated particles to pass through. The output fluid can include the plurality of non-agglomerated particles and substantially free of the plurality of agglomerated particles.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority and other benefits to U.S. Provisional Patent Application No. 62 / 862,211, filed June 17, 2019, pursuant to 35 USC § 119(e), as described below, which is incorporated herein by reference in its entirety. Technical Field

[0003] The disclosed technology relates primarily to systems and methods for separating agglomerated particles in fluids, and more specifically to systems and methods for separating agglomerated particles in fluids at high volumetric flow rates without dispersing the agglomerated particles. Background Technology

[0004] Aggregates enriched in the blood of cancer patients include circulating tumor cell (CTC) clusters and other forms of cancer cell clusters. These aggregates can provide valuable information about the stage of the disease, enabling minimally invasive prediction and diagnosis, enhancing the understanding of metastasis, and ultimately improving cancer treatment.

[0005] Specifically, neutrophil clusters of CTCs may have a metastatic tendency up to 100 times higher than that of a single CTC. This high metastatic tendency may be associated with reduced apoptosis and prolonged survival. Furthermore, in patients with advanced breast cancer, neutrophil clusters of CTCs may have an increased potential for metastasis, with neutrophil-protected CTC clusters demonstrating higher expression levels of the proliferation marker protein (Ki67) and genes related to cell cycle progression. Clinical studies have shown that the presence of CTC clusters may be associated with shorter developmental survival and overall survival in patients.

[0006] While existing separation techniques designed for detecting single cells (such as individual CTCs) can be applied to detect aggregate particles, these techniques may have low sensitivity and specificity for capturing aggregate particles. Although microfiltration techniques may be simple, they may not be suitable for enriching certain aggregate particles. For example, CTC clusters can pass through smaller confinement areas by reorganizing into a single-stranded chain structure to reduce their hydraulic resistance, especially under the higher pressures typically used in conventional filter media systems. Additionally, the high shear forces experienced within filter media systems in most cases may disperse aggregate particles into single cells, thus preventing effective enrichment. Alternatively, antibody-based enrichment systems can be used to separate single cells and aggregate particles. However, this technique may be difficult to succeed when attempting to separate heterogeneous CTC single cells and cell clusters because it relies on specific cell surface antigens. The small surface area to volume ratio of CTC clusters negatively impacts the capture efficiency of these antibody-based techniques, making them less efficient as CTC cluster enrichment platforms.

[0007] In addition, a two-stage continuous flow microfluidic chip has been developed to isolate CTC clusters from whole blood by utilizing a modified deterministic lateral displacement (DLD) method. However, this technology can have a low flow throughput of less than 2.5 mL / hour. This low flow throughput can limit its use in clinical applications, as large volumes of blood need to be processed due to the extreme rarity of cell clusters. In addition, this technology cannot isolate cell clusters of 2 or 3 cells, which constitute a large portion of the CTC clusters observed in cancer patients. Non-equilibrium inertial focusing array (NISA) can have a competitive operational flow rate. However, due to the microfluidic channel size limitations, cell clusters consisting of more than 5-6 cells can be susceptible to high shear stress, which can damage and disperse these relatively larger cell clusters. Finally, the significantly larger clusters observed in patient samples can cause microfluidic channel clogging.

[0008] Therefore, there is a need for systems and methods to isolate clustered particles at high volumetric flow rates without causing dispersion of the clustered particles. SUMMARY

[0009] The present invention relates to an apparatus for isolating clustered particles from a fluid sample. The isolation apparatus can include a plurality of microwells having a bottom surface with a mesh capture region. The mesh capture region can be partitioned into a plurality of pores by one or more partition lines. As a fluid sample including unclustered particles and clustered particles is passed through the isolation apparatus, the fluid can flow into the microwells. The pores can be sized such that the unclustered particles can pass through the pores while the clustered particles can be captured within the mesh capture region. Once captured, the clustered particles can be recovered from the mesh capture region for molecular and functional analysis.

[0010] The disclosed technology can include an apparatus for isolating clustered particles. The apparatus can include an inlet designed to receive a fluid, a plurality of microwells, and an outlet designed to output the fluid. The fluid can include a plurality of unclustered particles and a plurality of clustered particles. Each microwell can include a plurality of sidewalls and a bottom surface having a mesh capture region. The mesh capture region can be designed to capture the plurality of clustered particles and pass the plurality of unclustered particles. The output fluid can include the plurality of unclustered particles and substantially no plurality of clustered particles.

[0011] In any of the embodiments disclosed herein, the fluid can be blood, the unclustered particles can include unclustered cells, and the clustered particles can include cell clusters.

[0012] In any of the embodiments disclosed herein, the fluid can be urine, the unclustered particles can include unclustered cells, and the clustered particles can include cell clusters.

[0013] In any of the embodiments disclosed herein, the device can be designed to provide a volumetric flow rate of about 20-100 mL / h through the inlet and outlet.

[0014] In any of the embodiments disclosed herein, each microwell can have a depth of about 10-500 microns.

[0015] In any of the embodiments disclosed herein, each sidewall can be at least partially sloped.

[0016] In any of the embodiments disclosed herein, the device can comprise about 40-280 microwells per mm2.

[0017] In any of the embodiments disclosed herein, the mesh capture zone can comprise one or more partition lines.

[0018] In any of the embodiments disclosed herein, the one or more partition lines can define a plurality of apertures.

[0019] In any of the embodiments disclosed herein, the plurality of apertures can divide fluid flow into a plurality of flow paths.

[0020] In any of the embodiments disclosed herein, the plurality of apertures can be arranged in an array.

[0021] In any of the embodiments disclosed herein, each aperture of the plurality of apertures can be sized such that unaggregated particles can pass through the aperture and aggregated particles can not pass through the aperture.

[0022] In any of the embodiments disclosed herein, each aperture of the plurality of apertures can be square. Each square aperture of the plurality of apertures can have a side length of about 10-17 microns.

[0023] In any of the embodiments disclosed herein, each aperture of the plurality of apertures can be circular.

[0024] In any of the embodiments disclosed herein, each aperture of the plurality of apertures can be elliptical.

[0025] In any of the embodiments disclosed herein, each aperture of the plurality of apertures can be polygonal.

[0026] In any of the embodiments disclosed herein, each aperture of the plurality of apertures can have the same shape.

[0027] In any of the embodiments disclosed herein, the aggregated particles can be unlabeled.

[0028] In any of the embodiments disclosed herein, the aggregated particles can be labeled.

[0029] In any of the embodiments disclosed herein, the device can have a diameter of about 5-300 millimeters.

[0030] In any of the embodiments disclosed herein, the device can comprise a fluorine-based polymer.

[0031] In any of the embodiments disclosed herein, the device can comprise a perfluoropolyether-based polymer.

[0032] In any of the embodiments disclosed herein, the device can comprise a heat-curable polymer.

[0033] In any of the embodiments disclosed herein, the device can comprise a UV-curable polymer.

[0034] In any of the embodiments disclosed herein, the device can comprise a metal.

[0035] In any of the embodiments disclosed herein, the device can comprise a semiconductor.

[0036] The disclosed technology can also include a method for making a separation device for separating clustered particles, comprising: making a silicon mold on a silicon wafer; making a polymer mold; making a separation device; and releasing the separation device.

[0037] In any of the embodiments disclosed herein, making the silicon mold on the silicon wafer can comprise: depositing a first photoresist layer on the silicon wafer; patterning the first photoresist layer; etching the silicon wafer to form a plurality of posts; depositing a nitride layer on the silicon wafer; depositing a second photoresist layer; patterning the second photoresist layer and the nitride layer; etching the silicon wafer to form a sloped sidewall extending into each of the plurality of posts; depositing a third photoresist layer; patterning the third photoresist layer; and etching the silicon wafer to form the silicon mold.

[0038] In any of the embodiments disclosed herein, making the polymer mold can comprise: coating the silicon wafer with silane; depositing a first polymer layer on the silicon wafer; curing the first polymer layer to form a first polymer mold; removing the first polymer mold from the silicon wafer; coating the first polymer mold with silane; depositing a second polymer layer on the first polymer mold; and curing the second polymer layer to form a second polymer mold.

[0039] In any of the embodiments disclosed herein, the first and second polymer layers can comprise polydimethylsiloxane (PDMS).

[0040] In any of the embodiments disclosed herein, making the polymer mold can further comprise removing the second polymer mold from the first polymer mold.

[0041] In any of the embodiments disclosed herein, preparing the separation device can comprise: securing the second polymer mold to the substrate; filling the second polymer mold with the UV-curable polymer; exposing the UV-curable polymer to UV light; and curing the UV-curable polymer.

[0042] In any of the embodiments disclosed herein, a vacuum pump can be applied to fill the second polymer mold with the UV-curable polymer.

[0043] In any of the embodiments disclosed herein, the substrate can be a vinyl cutting tape.

[0044] In any of the embodiments disclosed herein, the substrate can be an acetate sheet.

[0045] In any of the embodiments disclosed herein, the substrate can be a PET sheet.

[0046] In any of the embodiments disclosed herein, filling the second polymer mold with the heat-curable polymer can be performed on a thermoelectric chiller.

[0047] In any of the embodiments disclosed herein, the UV-curable polymer can be a heat-curable polymer.

[0048] In any of the embodiments disclosed herein, releasing the separation chip can comprise: removing the second polymer mold; and removing the separation chip from the substrate.

[0049] The disclosed technology can also include a method for separating clumped particles, comprising: providing a separation device comprising a plurality of microwells, wherein each microwell can comprise a plurality of sidewalls and a bottom surface having a meshed trapping region; passing a fluid through the separation device, the fluid comprising a plurality of clumped particles and a plurality of unclumped particles; trapping the plurality of clumped particles within the meshed trapping region; and outputting the fluid, the outputted fluid comprising the plurality of unclumped particles.

[0050] In any of the embodiments disclosed herein, the fluid can be blood, the unclumped particles can be cells, and the clumped particles can be cell clumps.

[0051] In any of the embodiments disclosed herein, the fluid can be urine, the unclumped particles can be cells, and the clumped particles can be cell clumps.

[0052] In any of the embodiments disclosed herein, the method for separating clumped particles can further comprise placing the separation device within a filtration holder.

[0053] In any of the embodiments disclosed herein, passing the fluid through the separation device can be performed at a flow rate of about 20-100 mL / h.

[0054] In any of the embodiments disclosed herein, the output fluid can be substantially free of clumped particles.

[0055] In any of the embodiments disclosed herein, the method for isolating clumped particles can further comprise recovering the clumped particles from the mesh capture zone.

[0056] In any of the embodiments disclosed herein, recovering the clumped particles from the mesh capture zone can comprise washing the clumped particles with PBS and transporting the cell clusters to a receiving container.

[0057] In any of the embodiments disclosed herein, the micro-operators can directly recover the cell clusters from the mesh capture zone.

[0058] In any of the embodiments disclosed herein, the method for isolating clumped particles can further comprise analyzing the cell clusters.

[0059] In any of the embodiments disclosed herein, the clumped particles can comprise circulating tumor cell clusters.

[0060] In any of the embodiments disclosed herein, the clumped particles can comprise exfoliated cancer cells in urine.

[0061] In any of the embodiments disclosed herein, the method for isolating clumped particles can further comprise coating the isolation device with a growth medium.

[0062] In any of the embodiments disclosed herein, the captured clumped particles can be grown on the coated isolation device.

[0063] In any of the embodiments disclosed herein, the grown clumped particles can be analyzed directly on the coated isolation device.

[0064] In any of the embodiments disclosed herein, the method for isolating clumped particles can further comprise coating the isolation device with an inorganic material.

[0065] In any of the embodiments disclosed herein, the method for isolating clumped particles can further comprise coating the isolation device with an organic material.

[0066] The disclosed technology can further comprise a method of filtering an unprocessed blood sample using the device of claim 1.

[0067] The disclosed technology can further comprise a method of filtering a blood sample in-line using the device of claim 1.

[0068] The disclosed technology can further comprise a method of detecting clots using the device of claim 1.

[0069] The disclosed technology can further comprise a method of dispersing clumped particles using the device of claim 1.

[0070] These and other aspects of the invention are described in the following detailed description and accompanying drawings. Other aspects and features of the invention will become more apparent to those skilled in the art after reviewing the description of specific exemplary embodiments of the invention in conjunction with the accompanying drawings. While features of the invention may be discussed with respect to certain embodiments and drawings, all embodiments of the invention may include one or more features discussed herein. Furthermore, while one or more embodiments may be discussed as including certain advantageous features, one or more such features may also be used in various embodiments of the invention discussed herein. Similarly, while exemplary embodiments are discussed below as embodiments of apparatus, systems, or methods, it should be understood that such exemplary embodiments can be implemented in various apparatuses, systems, and methods of the invention. Attached Figure Description

[0071] Please refer to the attached diagram below. The diagram is not necessarily drawn to scale.

[0072] Figure 1A This is a top view of the separation device according to some aspects of the present invention.

[0073] Figure 1B This is a bottom view of the separation device according to some aspects of the present invention.

[0074] Figure 2 This is a schematic diagram of a separation device within a filter support according to some aspects of the present invention.

[0075] Figure 3A The illustration shows a plurality of micropores in a separation device according to some aspects of the present invention.

[0076] Figure 3B The illustration shows forces acting on captured agglomerated particles according to some aspects of the invention.

[0077] Figure 3C The illustration shows micropores containing trapped agglomerated particles according to some aspects of the present invention.

[0078] Figures 4A-4D Variations in the mesh trapping zone of the micropores according to some aspects of the present invention are described.

[0079] Figure 5A A cross-sectional view of a plurality of micropores according to some aspects of the present invention is described.

[0080] Figure 5B Some aspects of the invention have been described. Figure 5A A top view of multiple micropores.

[0081] Figure 6A A cross-sectional view of a plurality of micropores according to some aspects of the present invention is described.

[0082] Figure 6B A top view of a plurality of microwells according to some aspects of the application is depicted. Figure 6A

[0083] Figure 7A A cross-sectional view of a microwell according to some aspects of the application is depicted.

[0084] Figure 7B A top view of a plurality of microwells according to some aspects of the application is depicted. Figure 7A

[0085] Figure 8A A cross-sectional view of a microwell according to some aspects of the application is depicted.

[0086] Figure 8B A top view of a microwell according to some aspects of the application is depicted. Figure 8A

[0087] A flow chart outlining a method of preparing a separation device according to some aspects of the application is depicted. Figure 9

[0088] A method of preparing a silicon mold according to some aspects of the application is depicted. Figures 10A-10I

[0089] A method of preparing a polymer mold according to some aspects of the application is depicted. Figures 11A-11C

[0090] A method of preparing and releasing a separation device according to some aspects of the application is depicted. Figures 12A-12C

[0091] A method of separating clustered particles according to some aspects of the application is depicted. Figure 13 DETAILED DESCRIPTION

[0092] The present application relates to a separation device for separating clustered particles from a fluid sample comprising unclustered particles and clustered particles. The separation device can comprise a plurality of microwells having a bottom surface with a meshed trapping region. The meshed trapping region can be divided into a plurality of pores by one or more partition lines. The fluid sample can flow into the microwells when the fluid sample is passed through the separation device at a high volumetric flow rate. The pores can be sized such that the unclustered particles can pass through the pores while the clustered particles are gently trapped within the meshed trapping region. Once trapped, the clustered particles can be recovered from the meshed trapping region for further molecular and functional analysis. By separating and analyzing the trapped clustered particles, valuable diagnostic information and insights about possible therapeutic procedures can be obtained.

[0093] ​​​The disclosed technology will be described more fully below with reference to the accompanying drawings. However, the disclosed technology may be embodied in many different forms and should not be construed as limited to the examples described herein. The components constituting the elements of the disclosed technology described below are descriptive and not limiting. Many suitable components that have the same or similar functions as those described herein should be included within the scope of the disclosed electronic devices and methods. Other components not described herein may include, but are not limited to, those developed after the development of the disclosed technology.

[0094] Numerous specific details are described below. However, it should be understood that embodiments of the disclosed technology can be implemented without these specific details. In other instances, well-known methods, structures, and techniques are not detailed for clarity of the description. When referring to “one embodiment,” “implementation,” “exemplary embodiment,” “some embodiments,” “certain embodiments,” “various embodiments,” etc., it means that an embodiment of the described disclosed technology may include a particular feature, structure, or characteristic, but not every embodiment must include that particular feature, structure, or characteristic. Furthermore, repeated use of the phrase “in one embodiment” does not necessarily refer to the same embodiment, although it may.

[0095] Throughout the specification and claims, unless the context clearly indicates otherwise, the following terms shall have at least the meaning explicitly relevant herein. The term “or” is used to mean an inclusive “or”. Additionally, both the indefinite and definite articles refer to one or more, unless otherwise specified or clearly indicated by the context as singular.

[0096] Unless otherwise stated, the use of ordinal numbers such as “first,” “second,” “third,” etc., to describe the same type of objects merely indicates different circumstances of the objects referred to, and does not imply that the objects described are ranked in time or space or are in any other given order.

[0097] Unless otherwise stated, the term "agglomerated particles" refers to any cluster consisting of two or more particles, including micron-sized particles and nano-sized particles.

[0098] Unless otherwise stated, the term "cell cluster" includes any cluster of two or more cells, wherein the cells can be of any type, including but not limited to circulating tumor cells, exfoliated tumor cells, erythrocytes, and synthetic nanoparticles and microparticles.

[0099] Figure 1AThis is a top view of the separation device 100. The separation device 100 may have an inlet 112 designed to receive fluid. The separation device 100 may include a plurality of micropores 102 designed to trap agglomerated particles. The micropores 102 may be recesses of the separation device 100. The micropores 102 may include a plurality of sidewalls 104. The sidewalls 104 may extend from the upper surface of the micropores 102 to the lower surface. The micropores 102 may have any depth. The depth of the micropores 102 can facilitate the separation and trapping of agglomerated particles. The depth of the micropores 102 may be based on the purpose of the separation device 100 and the size of the agglomerated particles trapped by the separation device 100. In some embodiments, the depth of the micropores 102 may be about 10-500 micrometers. In some embodiments, the separation device 100 may be applied to trap nanoparticle-scale agglomerated particles or membrane vesicle agglomerated particles. In this application, the depth of the micropores 102 may be submicrometer. The bottom surface of the separation device 100 may include a mesh trapping area 106. Multiple fine dividing lines 110 can divide the mesh trapping area 106 into multiple pores 108.

[0100] Figure 1B This is a bottom view of the separation device 100. The separation device 100 may include an outlet 114 designed to discharge fluid. The inlet 112 and outlet 114 can be any type of inlet or outlet designed to deliver fluid to and from the micropores 102. In some embodiments, the inlet 112 may be an open surface above the micropores 102. In some embodiments, the outlet 114 may be an open surface adjacent to the pores 108 of the mesh trapping zone 106.

[0101] Figure 2 A separation device 100 located within a filter holder 202 is described. The filter holder 202 can be any commercially available filter holder. The filter holder 202 can be customized based on the required size and shape of the separation device 100 and the intended use of the separation device 100.

[0102] The separating device 100 can have any size and any shape. In some embodiments, such as Figure 1A and 1B As shown, the separation device 100 can be substantially rectangular. In some embodiments, such as Figure 2 As shown, the separation device 100 can be substantially circular. For example... Figure 1A , 1B As shown in Figure 2, the separation device 100 may have a diameter D. Figure 1A and 1BThe diameter D of the separation device 100 can be the length of the separation device 100 with respect to the longitudinal axis. The diameter D can be based on the diameter of the substrate (e.g., silicon wafer) used in the method of making the separation device 100. The diameter D of the separation device 100 can be about 5-300 millimeters. The diameter D of the separation device 100 can be based on the use for which the separation device 100 is applied. The diameter D of the separation device 100 can be larger in uses requiring a volumetric flow rate of greater than 1000 mL / h, as compared to uses requiring a volumetric flow rate of 20-100 mL / h.

[0103] The flow rate of the fluid through the separation device 100 can depend on the diameter D of the separation device 100 and the use for which the separation device 100 is applied. In some embodiments, the fluid can flow through the separation device 100 at a flow rate of about 20-100 mL / h. At this volumetric flow rate, the diameter D of the separation device 100 can be about 25 millimeters or greater, and can be effective to separate and trap cluster particles. In some embodiments, the fluid can flow through the separation device 100 at a volumetric flow rate of greater than 1000 mL / h. At this volumetric flow rate, the diameter D of the separation device 100 can be about 150-300 millimeters, and can be effective to separate and trap cluster particles.

[0104] The velocity of the fluid through the separation device 100 can similarly depend on the size of the separation device 100 and the use for which the separation device 100 is applied. In some embodiments, the fluid can flow through the separation device 100 at a velocity of about 20-260 micrometers / second.

[0105] The separation device 100 can include any number of micropores 102. The number of micropores 102 can depend on the surface area of the separation device 100. The number of micropores 102 can depend on the size of the cluster particles to be separated by the separation device 100. In some embodiments, the separation device 100 can have about 40-280 micropores per square millimeter. When the separation device 100 is used to separate nanometer cluster particles, the separation device 100 can have about 40,000-280,000 micropores 102 per square millimeter, wherein each micropore 102 has a nanometer scale size.

[0106] The separation device 100 can be made of any material that can flow and subsequently solidify as desired, and can be micro-patterned and / or nano-patterned. In some embodiments, the separation device 100 can be made substantially of a polymer. The polymer can be a UV-curable polymer. Alternatively or additionally, the polymer can be a heat-curable polymer. The polymer can be a fluorine-based polymer, such as a perfluoropolyether-based polymer. The fluorine-based polymer can facilitate the release of the separation device 100 from various molds during the fabrication of the separation device 100. In some embodiments, the separation device 100 can be made substantially of a metal. In some embodiments, the separation device can be made substantially of a semiconductor.

[0107] Figure 3A A plurality of microwells 102 of the separation device 100 is depicted. A fluid sample can pass through the inlet 112 of the separation device 100. The fluid can include a plurality of un-clustered particles 302 and a plurality of clustered particles 304. The fluid can vary depending on the use for which the separation device 100 is employed. In some embodiments, the fluid can be blood. Alternatively, in some embodiments, the fluid can be urine. The un-clustered particles 302 can include un-clustered cells, such as single red blood cells and white blood cells. In some embodiments, the un-clustered particles 302 can include single cancer cells 306, such as single circulating tumor cells. The clustered particles 304 can include cell clusters. The clustered particles 304 can be any number of cells clustered together, including but not limited to 2-cell clusters, 3-cell clusters, and 10-cell clusters. The clustered particles 304 can be unlabeled. Alternatively, the clustered particles 304 can be labeled. The label can include a molecular label, such as a fluorescent imaging or a bead-based label. The cell clusters can be cancer cell clusters. For example, the cell clusters can include circulating tumor cell (CTC) clusters, ovarian cancer cell clusters, breast cancer cell clusters, prostate cell clusters, etc. In some embodiments, the cell clusters can include blood cell clusters, indicating potential blood clots. In some embodiments, the clustered particles 304 can include nanoparticle clustered particles. In some embodiments, the clustered particles 304 can include membrane bleb clusters.

[0108] As the fluid sample passes through the inlet 112 of the separation device 100, the microwells 102 can introduce the un-clustered particles 302 and the clustered particles 304 to the mesh trapping region 106. As shown, the sidewalls 104 of the microwells 102 can have a sloped portion 104a. The sloped sidewalls 104a can be sloped at any angle, including positive angles, negative angles, and 0 degree angles. The sloped sidewalls 104a can facilitate the entry of the un-clustered particles 302 and the clustered particles 304 to the mesh trapping region 106. The sloped sidewalls 104a can also reduce the movement of the trapped clustered particles 304, such that the trapped clustered particles 304 can be securely retained within the microwells 102. Figure 3A As the fluid sample passes through the inlet 112 of the separation device 100, the microwells 102 can introduce the un-clustered particles 302 and the clustered particles 304 to the mesh trapping region 106. As shown, the sidewalls 104 of the microwells 102 can have a sloped portion 104a. The sloped sidewalls 104a can be sloped at any angle, including positive angles, negative angles, and 0 degree angles. The sloped sidewalls 104a can facilitate the entry of the un-clustered particles 302 and the clustered particles 304 to the mesh trapping region 106. The sloped sidewalls 104a can also reduce the movement of the trapped clustered particles 304, such that the trapped clustered particles 304 can be securely retained within the microwells 102.

[0109] The partition lines 110 forming the apertures 108 can divide the fluid stream into multiple flow paths. The apertures 108 can be sized according to the intended use of the separation device 100, such that unaggregated particles 302 can pass through the apertures 108 and out the outlet 114. The geometry of the aggregated particles 304 relative to the size of the apertures 108 can prevent the aggregated particles 304 from passing through the apertures 108. In some embodiments, the apertures 108 can be about 100-300 square microns in size. In some embodiments, the apertures 108 can be sized accordingly when the separation device 100 is used to capture nanoparticle aggregated particles. The size of the apertures 108 can be optimized such that the micropores 102 can capture 2- and 3-cell aggregated particles 304 while reducing unwanted white blood cell capture. Because the unaggregated particles 302 can easily pass through the apertures 108 without interference, the separation device 100 can process large volumes of fluid, including untreated whole blood, without the risk of clogging the separation device 100. With the reduced risk of clogging, the separation device 100 can be an ideal choice for clinical settings.

[0110] Figure 3B The forces that can act on the aggregated particles 304 within the mesh capture zone 106 of the micropore 102 are described. When the fluid sample passes through the micropore 102, a Dean drag force F D may be exerted on the aggregated particles 304 due to the flow of the fluid sample. R A counteracting force F R may be generated when the aggregated particles 304 encounter the partition lines 110 of the mesh capture zone 106. Additionally, a frictional force F F may be generated when the aggregated particles 304 encounter the sloped side wall 104a. The combination of these forces can allow the micropore 102 to gently immobilize the aggregated particles 304 without causing the aggregated particles 304 to disperse.

[0111] Figure 3C is an additional description of the aggregated particles 304 captured within the micropore 102. The configuration of the mesh capture zone 106 can allow the micropore 102 to gently capture the aggregated particles 304. This gentle capture can reduce the dispersion of the aggregated particles 304. Because the aggregated particles 304 can be relatively rare in the fluid sample and can provide valuable information upon analysis, it is important to prevent the aggregated particles 304 from dispersing.

[0112] Figures 4A-4D Various configurations of the mesh capture zone 106 are described. Each mesh capture zone 106 can include one or more partition lines 110 designed to partition the mesh capture zone 106 into multiple apertures 108 and support the captured aggregated particles 304. As Figure 4AAs shown, the partition lines 110 can divide the mesh capture zone 106 into four square-shaped apertures 108. The apertures 108 can be arranged in a 2 x 2 array. In some embodiments, each square-shaped aperture 108 can have a side length of about 10-17 microns. As shown, Figure 4B As shown, the mesh capture zone 106 can be divided into four substantially circular-shaped apertures 108. As shown, Figure 4C As shown, the mesh capture zone 106 can be divided into four substantially elliptical-shaped apertures 108. As shown, Figure 4D As shown, the mesh capture zone 106 can be divided into five substantially polygonal-shaped apertures 108. In some embodiments, each aperture 108 can be hexagonal-shaped.

[0113] While Figures 4A-4D While various variations of the mesh capture zone 106 are described, it is contemplated that the mesh capture zone 106 can include any number of partition lines 110 to create any number of apertures 108 having any geometric shape. The size and shape of the apertures 108 can be based on the size and shape of the aggregate particles 304 and the use for which the separation device 100 is being used. In some embodiments, the apertures 108 can have the same geometric shape and size. In some embodiments, the apertures 108 can have different geometric shapes and sizes. The apertures 108 can be sized accordingly when the separation device 100 is used to capture nano-particle aggregate particles and / or membrane bubble aggregate particles.

[0114] Figures 5A-8B Cross-sectional and top views of an example structure of a plurality of microwells 102 are described.

[0115] Figure 5A And 5B Cross-sectional and top views of a plurality of microwells 102 are described. The microwells 102 can include sloped sidewalls 104a designed to direct fluid into the mesh capture zone 106. The partition lines 110 can divide the mesh capture zone 106 into a plurality of square-shaped apertures 108 arranged in a 2 x 2 aperture array. The microwells 102 can be separated from each other by a flat portion of the upper surface of the separation device 100.

[0116] In contrast to the plurality of microwells described in Figure 5A And 5B Figure 6A And 6B Cross-sectional and top views of a plurality of microwells 102 having a tapered top are described. Adjacent microwells 102 can be connected to each other, such that a substantially pointed top can be created. The partition lines 110 can divide the mesh capture zone 106 of each microwell 102 into four square-shaped apertures 108 arranged in a 2 x 2 aperture array. The microwells 102 can include sloped sidewalls 104a to facilitate the collection and capture of aggregate particles 304 within the mesh capture zone 106. ​

[0117] Figure 7A and 7B Cross-sectional and top views of a plurality of micropores 102 having a linear array of pores 108 are described respectively. A dividing line 110 separates the mesh trapping zone 106 of each micropore 102 into 12 pores 108. The pores 108 can be arranged in a 2 x 6 pore array, such that the array is substantially linear. The micropores 102 may include inclined sidewalls 104a to facilitate the collection and trapping of aggregated particles 304 within the mesh trapping zone 106. The micropores 102 can be separated from each other by a flat portion of the upper surface of the separation device 100.

[0118] Figure 8A and 8B Cross-sectional and top views of multiple micropores 102, which are essentially a mesh structure, are described respectively. Separating lines 110 divide the mesh-like trapping zone 106 of the micropores 102 into 36 pores. The pores 108 can be arranged in a 9 x 4 pore array. The micropores 102 may include inclined sidewalls 104a to facilitate the collection and trapping of aggregated particles 304 within the mesh-like trapping zone 106.

[0119] Although Figures 5A-8B Exemplary variations of the micropores 102 are described, but it is contemplated that the micropores 102 can have any configuration. The mesh trapping area 106 can include any array of pores 108. The array of pores 108 can be any number of pores x any number of pores, including but not limited to 2 x 2 pore arrays, 3 x 5 pore arrays, 4 x 6 pore arrays, and 5 x 10 pore arrays.

[0120] The disclosed technology may also include a method 900 for preparing the separation device 100. For example... Figure 9 As shown, the method 900 may include preparing a silicon mold 902 on a silicon wafer, preparing a polymer mold 904, preparing a separation device 906, and releasing a separation device 908. The preparation method 900 of the separation device 100 can be carried out in a cleanroom-less environment, thereby reducing labor costs and time.

[0121] Figures 10A-10I The fabrication method of the silicon mold 1012 is described. As... Figure 10A As shown, a silicon wafer 1002 can be provided. In some embodiments, the thickness of the silicon wafer 1002 can be about 300-600 micrometers.

[0122] exist Figure 10B and 10C In this process, a first photoresist layer 1004 can be deposited on a silicon wafer 1002. The photoresist layer 1002 can be spun and patterned. The patterned photoresist layer 1004 can serve as the basis for a desired array of apertures 108 in the mesh trapping region 106.

[0123] In Figure 10D some embodiments, the silicon wafer 1002 can be etched to form the pillars 1006. A deep reactive ion etch can be applied to etch the silicon wafer 1002 about 10 microns deep.

[0124] In Figure 10E some embodiments, a nitride layer 1006 can be deposited. The nitride layer 1006 can be about 300 nanometers thick. The nitride layer 1006 can be deposited in a low pressure chemical vapor deposition furnace. The nitride layer 1006 can be coated with a second photoresist layer 1008. As Figure 10F shown, the nitride layer 1006 and the second photoresist layer 1008 can be patterned. In some embodiments, the second photoresist layer 1008 can be exposed by a maskless aligner.

[0125] As Figure 10G shown, a reactive ion etch can be applied to etch the nitride layer 1006 to form a hard mask, and the silicon wafer 1002 can be anisotropically etched in a 45% KOH solution at about 80°C for about 10-20 minutes. The etching of the silicon wafer 1002 can result in sloped walls. The sloped walls can extend to the plurality of pillars. The formation of the sloped walls can be the basis for the formation of the sloped sidewalls 104a of the separation device 100.

[0126] As Figure 10H shown, a third photoresist layer 1010 can be deposited on the silicon wafer 1002 and patterned. A deep reactive ion etch can be applied to etch the silicon wafer 1002 about 50 microns deep. The etching of the silicon wafer 1002 can form a silicon mold 1012.

[0127] Figures 11A-11C A method of making a polymer mold is described. The method of making a polymer mold can include double molding of a polymer. Prior to making the polymer mold, the silicon mold 1012 can be coated with silane under vacuum conditions for 8 hours. Coating the silicon mold 1012 with silane can facilitate removal of the first polymer mold 1102 from the silicon mold 1012. In some embodiments, metal layer sputtering, including gold layer sputtering, can also be applied to reduce and / or eliminate the 8 hour wait time. Figure 11A A first polymer mold 1102 is described. A first polymer layer can be cast onto the silicon mold 1012. The first polymer layer can be degassed in a desiccator for 1 hour and then cured in an oven to form the first polymer mold 1102. As Figure 11B shown, the cured first polymer mold 1102 can be peeled from the silicon mold 1012. Oxygen plasma can be applied to activate the surface of the first polymer mold 1102 and coated with silane for about 8 hours. As Figure 11CAs shown, the first polymer mold 1102 can be used as a mold to make the second polymer mold 1104. A second polymer layer can be cast onto the first polymer mold 1102 and cured to form the second polymer mold 1104. After the second polymer mold 1104 is made, the second polymer mold 1104 can be removed from the first polymer mold 1102.

[0128] In some embodiments, the first polymer layer and the second polymer layer can comprise polydimethylsiloxane (PDMS).

[0129] Figures 12A-12C Methods of making and releasing the separation device 100 are described. As shown, Figure 12A As shown, the second polymer mold 1104 can be secured to a substrate 1202. In some embodiments, the second polymer mold 1104 can be secured to the non-adhesive side of a vinyl cutting tape. Alternatively, the substrate 1202 can comprise an acetate sheet, a PET sheet, or other similar material. After the second polymer mold 1104 is secured to the substrate 1202, the second polymer mold 1104 can be filled with a UV-curable polymer. The UV-curable polymer can be inserted through the inlet of the second polymer mold 1104. A vacuum can be applied to the outlet end to facilitate filling of the second polymer mold 1104 with the UV-curable polymer. Once the second polymer mold 1104 is filled with the UV-curable polymer, the UV-curable polymer can be exposed to UV light, thereby curing the UV-curable polymer to form the separation device 100. In some embodiments, the wavelength of the UV light can be about 365 nanometers. In some embodiments, the second polymer mold 1104 can be filled with the UV-curable polymer on top of a thermoelectric cooler. The thermoelectric cooler can lower the temperature of the UV-curable polymer, thereby increasing the viscosity of the UV-curable polymer. By increasing the viscosity of the UV-curable polymer, a higher vacuum can be used without creating air bubbles, thereby increasing the yield of manufacturing.

[0130] As shown, Figure 12B Once the UV-curable polymer has been cured, the second polymer mold 1104 can be peeled off of the separation device 100. The separation device 100 can then be released from the substrate 1202, as shown. Figure 12C In some embodiments, the separation device 100 can be located on a thermoelectric cooler to facilitate releasing the separation device 100.

[0131] In some embodiments, the UV-curable polymer can be a fluorine-based polymer, including a perfluoropolyether-based polymer. In some embodiments, the UV-curable polymer can be a heat-curable polymer. For example, a heat-curable polymer such as PDMS can be used to form the separation device 100 when exposure to UV light is not desired.

[0132] Although Figures 10A-12C Examples of methods for preparing the separation device 100 have been described, but other methods are also considered. In some embodiments, the separation device 100, which is essentially made of a polymer, can be prepared by thermoforming. Thermoforming can be a low-cost and scalable technique for preparing the separation device 100, thus making it suitable for a variety of applications. In this technique, the polymer may include polymethyl methacrylate, cyclic olefin copolymers, polycarbonate, polyethylene, etc. The technique typically includes heating, molding, and demolding. The polymer can be softened by heating it to a temperature above its glass transition temperature. Pressure can be applied to soften the polymer to form the shape of a basic mold. In the demolding step, the polymer can be cooled and released from the mold. The polymer can then be perforated to form pores 108 in the separation device 100. The temperature, pressure, and selection of the polymer can be varied depending on the application of the separation device 100 and desired parameters such as thickness.

[0133] Additionally, in some embodiments, the separation device 100, which is essentially made of metal, can be prepared using conventional electroplating and electroless plating. This technique may include the preparation of the second polymer mold 1104 described herein. A metal seed layer can be deposited on the surface of the second polymer mold 1104 using an electron beam evaporator under high vacuum. Metal ions can attach to the surface of the second polymer mold 1104 and subsequently grow. The grown metal can have substantially the same shape as the second polymer mold 1104. The strength and flexibility of the separation device 100 can be varied by changing the thickness of the electroplated metal.

[0134] In some implementations, silicon micromachining can be used to fabricate the separation device 100. Figures 10A-12C Unlike fabrication methods that can be performed without a cleanroom, silicon micromachining allows the separation device 100 to be fabricated from a silicon wafer within a cleanroom. For this technique, a silicon nitride layer can be deposited on the silicon wafer. Reactive ion etching can be applied to pattern the silicon nitride layer, and a KOH (or TMAH) solution can be used to etch the silicon wafer. Backside photolithography can be performed, and subsequently, plasma etching can be performed on the nitride layer to form the separation device 100.

[0135] Figure 13 A method 1300 for separating agglomerated particles is described. Method 1300 may include 1302 providing a separation device 100 comprising a plurality of micropores 102. Each micropore 102 may include a plurality of sidewalls 104 and a bottom surface 106 having a mesh-like trapping zone. The separation device 100 may also include any features discussed herein.

[0136] Method 1300 can include 1304 passing a fluid through separation device 100. The fluid can include a plurality of unclustered particles 302 and a plurality of clustered particles 304. Unclustered particles 302 and clustered particles 304 can be introduced into microwells 102 as the fluid passes through separation device 100.

[0137] Method 1300 can include 1306 trapping a plurality of clustered particles 304 within mesh trapping region 106.

[0138] Method 1300 can include 1308 outputting a sample of the fluid. The outputted sample includes a plurality of unclustered particles 302. The outputted sample is substantially free of clustered particles 304, as clustered particles 304 can be trapped within microwells 102.

[0139] Method 1300 can also include recovering clustered particles 304 from mesh trapping region 106. To recover clustered particles 304, clustered particles 304 can be washed with PBS. After washing with PBS, trapped clustered particles 304 can be released at a different relative counterflow rate relative to the volumetric flow rate of the fluid through separation device 100. The released clustered particles 304 can then be transferred to a receiving vessel. Alternatively, clustered particles 304 can be recovered directly from mesh trapping region 106. In some embodiments, a micromanipulator can be applied to recover clustered particles 304 directly from mesh trapping region 106. Unlike conventional well filters, where trapped clustered particles 304 can adhere to the surface of the filter, the recessed location of mesh trapping region 106 within microwells 102 can allow separation device 100 to be moved to a system or device designed for analysis without the risk of losing trapped clustered particles 304.

[0140] Recovered clustered particles can be imaged and subjected to any form of molecular and functional analysis. By analyzing clustered particles 304, valuable information about clustered particles 304 can be obtained, including the origin of cancer and mutations of cells. Additionally, possible treatment procedures can also be explored. In some embodiments, possible drugs and / or other forms of therapy can be applied to treat clustered particles 304. The results of these drugs and treatments can help improve individualized treatment.

[0141] In some embodiments, method 1300 of isolating clustered particles can include coating separation device 100 with an organic coating or an inorganic coating. In some embodiments, the inorganic coating can enhance the surface adhesion properties of separation device 100. The inorganic coating can include antibodies with specific affinities so that separation device 100 can trap clustered particles 304. In some embodiments, the organic coating, such as a PEG or BSA coating, can reduce non-specific adhesion so that trapped clustered particles 304 can be released.

[0142] In some embodiments, the method 1300 of isolating aggregate particles can include coating the isolation device 100 with a growth medium. When the isolation device 100 is coated with a growth medium, the captured aggregate particles 304 can grow directly on the isolation device 100. In this sense, the isolation device 100 can act similar to human organs and / or tissues. Due to the continuous flow of fluid (e.g., blood flow), the fluid flow can provide a continuous source of nutrients, and the captured aggregate particles 304 can easily survive. The grown aggregate particles 304 can then be further analyzed by various techniques. In some embodiments, the grown aggregate particles 304 can be released and cultured to form new cell lines or to develop new drug therapies.

[0143] The isolation device 100 and / or method 1300 of isolating aggregate particles can be used for a variety of other applications. For example, urinary cytology is a technique that can examine abnormal cells in urine under a microscope to diagnose urinary tract cancers, including bladder cancer. This technique can require enrichment of rare exfoliated cancer cells from a large sample of urine obtained from a patient. Instead of current centrifugation and cell spinning methods, the isolation device 100 can be applied to filter a large urine sample without damaging or losing a large number of rare exfoliated cancer cells. After the exfoliated cancer cells are captured, fluorescence and Papanicolaou staining can be applied to characterize the cells.

[0144] In some embodiments, the isolation device 100 can be applied to filter an untreated blood sample. Additionally, the isolation device 100 can be applied in an online blood purification system. Since circulating tumor cell clusters have a higher metastatic tendency than individual circulating tumor cells, it is important to remove CTC clusters from blood. In this application, blood can be removed from a patient. The blood can be directed directly to a blood pump and an anticoagulant can be added. The blood can be passed through the isolation device 100. CTC clusters can be gently captured within the micropores 102 of the isolation device 100, while individual red blood cells, white blood cells, and individual CTCs can pass through the isolation device 100. Clean blood substantially free of CTC clusters can be directed back to the patient. This technique can be performed continuously using a portable system and / or intermittently for a period of time depending on the severity of the patient.

[0145] In some embodiments, the isolation device 100 can be applied to break up aggregate particles. In this technique, the volumetric flow rate of a blood sample through the isolation device 100 can be increased, thereby also increasing the shear force on the captured aggregate particles. The increase in shear force can cause the aggregate particles 304 to disperse into un-aggregated particles 302. For example, CTC clusters can be dispersed into individual CTCs. Since individual CTCs have been found to be less metastatic, this technique can aid in therapeutic intervention and improve the course of treatment.

[0146] It is to be understood that the embodiments and claims disclosed herein are not limited in their application to the details of construction and the arrangements of components set forth in the description and illustrated in the drawings. Rather, the description and drawings merely provide examples of the contemplated embodiments. Embodiments disclosed herein and claims can be implemented in other embodiments and can be practiced or carried out in various ways. It is also to be understood that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting.

[0147] As such, those skilled in the art will appreciate that the conception, upon which this disclosure and claims are based, can readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present embodiments and claims. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar they do not depart from the spirit and scope of the present embodiments and claims. It will be clear to those skilled in the art that the present embodiments and claims can be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments and claims are therefore considered to cover any and all such equivalent forms, which would be within the scope of the embodiments and claims.

[0148] Furthermore, the purpose of the abstract is to enable the U.S. Patent and Trademark Office and the public generally, especially those not familiar with patent or legal terms or phraseology, to determine quickly from a cursory inspection the nature and essence of the technical disclosure. The abstract is not intended to be limiting as to the scope or appurtenance of the embodiments. The claims define the scope of the embodiments.

Claims

1. A separation device for separating agglomerated particles, comprising: The inlet is designed to receive fluid in the direction of flow, the fluid comprising multiple unagglomerated particles and multiple agglomerated particles; Downstream of the inlet are multiple micropores, each micropore comprising multiple sidewalls and a bottom surface having a mesh trapping zone, at least one of the sidewalls and the mesh trapping zone being designed to trap multiple agglomerated particles and allow multiple unagglomerated particles to pass through, wherein the mesh trapping zone includes multiple dividing lines defining multiple pores, the multiple pores being designed to divide the flow of the fluid into multiple channels, wherein the multiple pores are arranged in a 2×2 array, and wherein the micropores are recesses of the separation device; and The outlet is designed to output fluid downstream of the multiple micropores, and the output fluid includes multiple unagglomerated particles and is substantially free of multiple agglomerated particles. Each of the said sidewalls is at least partially inclined.

2. The separation device of claim 1, wherein the fluid is blood, the unaggregated particles comprise unaggregated cells, and the aggregated particles comprise cell clusters.

3. The separation device of claim 1, wherein the fluid is urine, the unaggregated particles comprise unaggregated cells, and the aggregated particles comprise cell clusters.

4. The separation device of claim 1, wherein the device is designed to provide a volumetric flow rate of 20-100 mL / h through the inlet and outlet.

5. The separation device according to any one of claims 1-4, wherein the depth of each micropore is 10-500 micrometers.

6. The separation device according to any one of claims 1-4, wherein the device comprises 40-280 micropores per square millimeter.

7. The separation apparatus of any one of claims 1-4, wherein each of the plurality of pores is sized such that unagglomerated particles pass through the pores while agglomerated particles do not.

8. The separating device according to any one of claims 1-4, wherein each of the plurality of pores is square, circular, elliptical or polygonal.

9. The separation device of claim 8, wherein each of the plurality of pores is square, and the side length of each square pore is 10-17 micrometers.

10. The separation device according to any one of claims 1-4, wherein each of the plurality of pores has the same shape.

11. The separation apparatus of any one of claims 1-4, wherein the agglomerate particles are unmarked.

12. The separation apparatus of any one of claims 1-4, wherein the agglomerates are marked.

13. The separation device according to any one of claims 1-4, wherein the diameter of the device is 5-300 mm.

14. The separation device according to any one of claims 1-4, wherein the device is made of a material selected from: fluoropolymers, thermosetting polymers, UV-curable polymers, metals, and semiconductors.

15. The separation apparatus of claim 14, wherein the fluoropolymer is a perfluoropolyether polymer.

16. An apparatus for separating agglomerated particles, comprising: The inlet is designed to receive fluid in the direction of flow, the fluid including unagglomerated particles and agglomerated particles; Downstream of the inlet, a micropore includes sidewalls with inclined portions and a bottom surface with a mesh trapping zone, wherein the micropore is a recess of the separation device, and the micropore is designed to funnel-introduce unagglomerated and agglomerated particles into the mesh trapping zone, the mesh trapping zone being formed of pores having defined pore sizes, wherein the pores are arranged in a 2×2 array, at least one of the sidewalls and the mesh trapping zone being designed to trap agglomerated particles with particle sizes larger than the pore sizes, and to allow unagglomerated particles with particle sizes smaller than the pore sizes to pass through therethrough; and At the outlet downstream of the micropore, the outlet is designed to output fluid downstream of the mesh trapping zone, the fluid downstream of the mesh trapping zone comprising unaggregated particles.

17. A method for preparing a separation apparatus for separating agglomerated particles according to any one of claims 1-16, comprising: Fabricating silicon molds on silicon wafers; A polymer mold is prepared from the silicon mold; The separation device is prepared from the polymer mold; and The separation device is then disengaged.

18. The method of claim 17, wherein the preparation of the separation device from the polymer mold comprises: The polymer mold is fixed onto the substrate; Fill the polymer mold with polymer; and The polymer is cured to form the device; and Disengaging the separation device includes detaching the separation device from the substrate.

19. The method of claim 17, wherein fabricating a silicon mold on a silicon wafer comprises: A first photoresist layer is deposited on a silicon wafer; Pattern the first photoresist layer; Etching a silicon wafer to form multiple pillars; Depositing a nitride layer on a silicon wafer; Deposit a second photoresist layer; Pattern the second photoresist layer and the nitride layer; The silicon wafer is etched to form sloping sidewalls extending to each of the plurality of pillars; Deposit the third photoresist layer; Pattern the third photoresist layer; and Etching silicon wafers to form silicon molds.

20. The method of claim 18, wherein preparing the polymer mold comprises: Coating a silicon mold with silane; A first polymer layer is deposited on a silicon mold; The first polymer layer is cured to form a first polymer mold; Remove the first polymer mold from the silicon mold; The first polymer mold was coated with silane; A second polymer layer is deposited on the first polymer mold; and The second polymer layer is cured to form a second polymer mold.

21. The method of claim 20, further comprising removing the second polymer mold from the first polymer mold; The first polymer layer and the second polymer layer comprise polydimethylsiloxane (PDMS).

22. The method of claim 20, wherein the preparation of the separation apparatus further comprises: Expose the polymer to UV light; The polymer mold thereon is the second polymer mold; and The polymer mentioned therein is a UV-curable polymer.

23. The method of claim 22, wherein a vacuum pump is used to fill the second polymer mold with a UV-curable polymer.

24. The method of claim 18, wherein the matrix is ​​a vinyl cut tape, an acetate sheet, or a PET sheet.

25. The method of claim 22, wherein the second polymer mold is filled with a UV-curable polymer and carried out on a thermoelectric cooler.

26. The method of claim 22, wherein the UV-curable polymer is a thermocurable polymer.

27. The method of claim 20, wherein disengaging the separating device comprises: Remove the second polymer mold; and Remove the separation device from the substrate.

28. A method for separating aggregated particles, comprising: Passing a fluid through the separation device of any one of claims 1-16, the fluid comprising a plurality of aggregated particles and a plurality of unaggregated particles; Multiple aggregated particles are captured within the mesh-like trapping zone of the multiple micropores in the separation device; and The fluid downstream of the mesh trapping zone is output, and the output fluid includes multiple unagglomerated particles and substantially no agglomerated particles.

29. The method of claim 28, wherein the fluid is blood, the unaggregated particles are cells, and the aggregated particles are cell clusters.

30. The method of claim 28, wherein the fluid is urine, the unaggregated particles comprise unaggregated cells, and the aggregated particles comprise cell clusters.

31. The method of any one of claims 28-30, further comprising placing the separation device within a filter support.

32. The method of any one of claims 28-30, wherein the fluid is passed through the separation device at a flow rate of 20-100 mL / h.

33. The method of any one of claims 28-30, further comprising recovering agglomerated particles from the mesh-like trapping zone of the plurality of micropores of the separating device.

34. The method of claim 33, wherein recovering agglomerated particles from the mesh-like trapping zone of the plurality of micropores of the separation device comprises: Wash the aggregated particles with PBS; and The aggregated particles are conveyed to the container.

35. The method of claim 33, wherein the micromanipulator directly recovers the agglomerated particles from the mesh-like trapping zone of the plurality of micropores of the separation device.

36. The method of any one of claims 28-30, further comprising analyzing the agglomerates.

37. The method of claim 29, wherein the aggregated particles comprise circulating tumor cell clusters.

38. The method of claim 30, wherein the aggregated particles comprise cancer cells shed in urine.

39. The method of any one of claims 28-30, further comprising coating the separation device with a growth culture.

40. The method of claim 39, wherein the captured agglomerates are grown on a coated separation device.

41. The method of claim 40 further includes analyzing the grown agglomerates directly on the coated separation device.

42. The method of any one of claims 28-30, further comprising coating the separation device with an inorganic material.

43. The method of any one of claims 28-30, further comprising coating the separation device with an organic material.

44. A method for filtering untreated blood samples using the separation apparatus of any one of claims 1-16.

45. A method for filtering blood samples online using the separation device of any one of claims 1-16.

46. ​​A method for detecting clumps using the separation apparatus of any one of claims 1-16.

47. A method for separating agglomerated particles using the separating apparatus of any one of claims 1-16.

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