Filtration-based systems and methods for separating clustered particles

Abstract

One embodiment of the disclosed technology provides an apparatus for separating clustered particles. The apparatus can include an inlet configured to receive a fluid and an outlet configured to output the fluid. The fluid can include a plurality of non-clustered particles and a plurality of clustered particles. The separation apparatus can include a plurality of microwells. Each microwell can have a plurality of sidewalls and a bottom surface having a meshed capture region. The meshed capture region can capture the plurality of clustered particles while allowing the non-clustered particles to pass through. The output fluid can include the plurality of non-clustered particles or can be substantially free of the plurality of clustered particles.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to, and the benefit under 35 U.S.C. §119(e) of, U.S. Provisional Patent Application No. 62 / 862,211, filed June 17, 2019, the entire contents of which are incorporated herein by reference as if fully set forth below.

[0002] (Technical field) The disclosed technology relates generally to systems and methods for separating clustered particles in a fluid, and more particularly to systems and methods for separating clustered particles in a fluid at high volumetric flow rates without dissociating the clustered particles. [Background technology]

[0003] Clustered particles, such as circulating cancer cell clusters (CTC clusters) enriched from the bloodstream of cancer patients, can provide valuable information about disease stage, enable minimally invasive prognosis and diagnosis, improve our understanding of metastasis, and ultimately help improve cancer treatment.

[0004] In particular, the metastatic propensity of CTC clusters is up to 100-fold greater than that of single CTCs. This increased propensity may be associated with reduced apoptosis and prolonged survival. Furthermore, in patients with advanced breast cancer, CTC-neutrophil clusters may increase the likelihood of metastasis, and neutrophil-engulfed CTC clusters exhibit elevated expression levels of proliferation marker proteins (Ki67) and genes associated with cell cycle progression. Clinical trials have shown that the presence of CTC clusters is associated with shorter progression-free and overall survival in patients.

[0005] Although clustered particles can be detected using existing separation techniques designed to detect single cells, such as single CTCs, existing separation techniques often lack sensitivity and specificity for capturing clustered particles. While microfiltration techniques can be simple, they may not be suitable for enriching certain clustered particles. For example, CTC clusters can pass through small constrictions by reorganizing into single-file, chain-like structures that reduce hydrodynamic resistance, especially at the high pressures typically encountered in conventional filtration-based systems. Furthermore, the high shear forces in filtration systems often dissociate clustered particles into single cells, preventing efficient enrichment. Antibody-based enrichment systems can also be used to isolate single cells and clustered particles. However, these techniques rely on specific cell surface antigens, which can be challenging when attempting to isolate heterogeneous CTC single cells or clusters. Because CTC clusters have a small surface area-to-volume ratio, these antibody-based techniques negatively impact capture efficiency, making them an ineffective platform for CTC cluster enrichment.

[0006] A two-stage continuous-flow microfluidic chip for separating CTC clusters from whole blood has also been developed using an improved deterministic lateral displacement (DLD) method. However, this technology can have a low throughput of less than 2.5 mL / h. This low throughput may limit its use in clinical applications that require processing large volumes of blood due to the extremely low number of clusters. Furthermore, this technology is unable to separate relatively small clusters of two or three cells, which account for the majority of CTC clusters observed in cancer patients. Non-equilibrium inertial separation arrays (NISAs) can operate at competitive flow rates. However, due to the size limitations of the microchannel, cell clusters consisting of more than five or six cells are susceptible to high shear stress, which can damage and dissociate these relatively large clusters. Finally, the significantly larger clusters observed in patient samples may lead to clogging of the microchannel.

[0007] Therefore, there is a need for a system and method for separating clustered particles at high volumetric flow rates without dissociating them. Summary of the Invention

[0008] The present disclosure relates to an apparatus for separating clustered particles from a fluid sample. The separation apparatus can include a plurality of microwells having a bottom surface with a meshed capture region. The meshed capture region can be divided into a plurality of openings using one or more partition lines. A fluid sample containing non-clustered particles and clustered particles can be passed through the separation apparatus, allowing the fluid to flow into the microwells. The openings can be sized so that non-clustered particles pass through the openings and the clustered particles are captured within the meshed capture region. Once captured, the clustered particles can be recovered from the meshed capture region for molecular and functional analysis.

[0009] The disclosed technology can include an apparatus for separating clustered particles. The apparatus can include an inlet configured to receive a fluid, a plurality of microwells, and an outlet configured to output the fluid. The fluid can include a plurality of non-clustered particles and a plurality of clustered particles. Each microwell can include a plurality of sidewalls and a bottom surface having a meshed capture region. The meshed capture region can be configured to capture the plurality of clustered particles and allow the plurality of non-clustered particles to pass through. The output fluid can include the plurality of non-clustered particles or can be substantially free of the plurality of clustered particles.

[0010] In any of the embodiments disclosed herein, the fluid may be blood, the non-clustered particles may include non-clustered cells, and the clustered particles may include cell clusters.

[0011] In any of the embodiments disclosed herein, the fluid may be urine, the non-clustered particles may include non-clustered cells, and the clustered particles may include cell clusters.

[0012] In any of the embodiments disclosed herein, the device can be configured to provide a volumetric flow rate through the inlet and outlet of between about 20 mL / h and about 100 mL / h.

[0013] In any of the embodiments disclosed herein, each microwell can have a depth of from about 10 microns to about 500 microns.

[0014] In any of the embodiments disclosed herein, at least a portion of each sidewall can be sloped.

[0015] In any of the embodiments disclosed herein, the device can contain from about 40 to about 280 microwells per square millimeter.

[0016] In any of the embodiments disclosed herein, the meshed capture area can include one or more septum lines.

[0017] In any of the embodiments disclosed herein, the one or more septum lines can define a plurality of openings.

[0018] In any of the embodiments disclosed herein, the multiple openings can divide the fluid flow into multiple flow paths.

[0019] In any of the embodiments disclosed herein, the plurality of apertures may be arranged in an array.

[0020] In any of the embodiments disclosed herein, each opening in the plurality of openings can be sized such that the non-clustered particles can pass through it and the clustered particles cannot pass through it.

[0021] In any of the embodiments disclosed herein, each opening in the plurality of openings can be square-shaped, and each square-shaped opening in the plurality of openings can have a side length of about 10 microns to about 17 microns.

[0022] In any of the embodiments disclosed herein, each opening in the plurality of openings may be circular.

[0023] In any of the embodiments disclosed herein, each opening in the plurality of openings may be oval in shape.

[0024] In any of the embodiments disclosed herein, each opening in the plurality of openings may be polygonal in shape.

[0025] In any of the embodiments disclosed herein, each opening in the plurality of openings may have the same shape.

[0026] In any of the embodiments disclosed herein, the clustered particles can be label-free.

[0027] In any of the embodiments disclosed herein, the clustered particles can be labeled.

[0028] In any of the embodiments disclosed herein, the device can have a diameter of about 5 millimeters to about 300 millimeters.

[0029] In any of the embodiments disclosed herein, the device can include a fluoropolymer.

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

[0031] In any of the embodiments disclosed herein, the device can include a thermosetting polymer.

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

[0033] In any of the embodiments disclosed herein, the device can include a metal.

[0034] In any of the embodiments disclosed herein, the device can include a semiconductor.

[0035] The disclosed technology can include a method of fabricating a separation device for separating clustered particles, the method including fabricating a silicon mold on a silicon wafer, fabricating a polymer mold, fabricating the separation device, and removing the separation device.

[0036] In any of the embodiments disclosed herein, fabricating the silicon mold on the silicon wafer can include depositing a first photoresist layer on the silicon wafer, patterning the first photoresist layer, etching the silicon wafer to form a plurality of pillars, 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 sloped sidewalls extending to each pillar in the plurality of pillars, depositing a third photoresist layer, patterning the third photoresist layer, and etching the silicon wafer to form the silicon mold.

[0037] In any of the embodiments disclosed herein, fabricating the polymer mold can include coating the silicon wafer with silane, depositing a first polymer layer on the silicon wafer, curing the first polymer layer to form the 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 the second polymer mold.

[0038] In any of the embodiments disclosed herein, the first polymer layer and the second polymer layer can include polydimethylsiloxane (PDMS).

[0039] In any of the embodiments disclosed herein, creating the polymer mold can further include removing the second polymer mold from the first polymer mold.

[0040] In any of the embodiments disclosed herein, fabricating the separation device can include attaching the second polymer mold to a substrate, filling the second polymer mold with a UV-curable polymer, exposing the UV-curable polymer to UV light, and curing the UV-curable polymer.

[0041] In any of the embodiments disclosed herein, a vacuum pump can be used to fill the second polymer mold with the UV curable polymer.

[0042] In any of the embodiments disclosed herein, the substrate can be a vinyl dicing tape.

[0043] In any of the embodiments disclosed herein, the substrate can be an acetate sheet.

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

[0045] In any of the embodiments disclosed herein, filling the second polymer mold with the thermosetting polymer can be performed on a thermoelectric cooler.

[0046] In any of the embodiments disclosed herein, the UV curable polymer can be a thermosetting polymer.

[0047] In any of the embodiments disclosed herein, removing the detached chip can include removing the second polymer mold and removing the detached chip from the substrate.

[0048] The disclosed technology can include a method for separating clustered particles, the method including providing a separation device including a plurality of microwells, each of which can have a plurality of sidewalls and a bottom surface with a meshed capture region; passing a fluid including a plurality of clustered particles and a plurality of non-clustered particles through the separation device; capturing the plurality of clustered particles within the meshed capture region; and outputting the fluid including the plurality of non-clustered particles.

[0049] In any of the embodiments disclosed herein, the fluid may be blood, the non-clustered particles may be cells, and the clustered particles may be cell clusters.

[0050] In any of the embodiments disclosed herein, the fluid may be urine, the non-clustered particles may be cells, and the clustered particles may be cell clusters.

[0051] In any of the embodiments disclosed herein, the method for separating clustered particles can further include positioning the separation device within a filter holder.

[0052] In any of the embodiments disclosed herein, passing the fluid through the separation device can be performed at a flow rate of about 20 mL / h to about 100 mL / h.

[0053] In any of the embodiments disclosed herein, the output fluid can be substantially free of clustered particles.

[0054] In any of the embodiments disclosed herein, the method for separating clustered particles can further include recovering the clustered particles from the meshed capture region.

[0055] In any of the embodiments disclosed herein, recovering the clustered particles from the meshed capture region can further include washing the clustered particles with PBS and transferring the cell clusters to a holding container.

[0056] In any of the embodiments disclosed herein, the cell clusters can be retrieved from the meshed trapping region directly by a micromanipulator.

[0057] In any of the embodiments disclosed herein, the method for separating clustered particles can further include analyzing the cell clusters.

[0058] In any of the embodiments disclosed herein, the clustered particles can comprise circulating tumor cell clusters.

[0059] In any of the embodiments disclosed herein, the clustered particles can include exfoliated cancer cells in urine.

[0060] In any of the embodiments disclosed herein, the method for separating clustered particles can further include coating the separation device with a growth medium.

[0061] In any of the embodiments disclosed herein, the captured clustered particles can be grown on the coated separation device.

[0062] In any of the embodiments disclosed herein, the grown clustered particles can be analyzed directly on the coated separation device.

[0063] In any of the embodiments disclosed herein, the method for separating clustered particles can further include coating the separation device with an inorganic material.

[0064] In any of the embodiments disclosed herein, the method for separating clustered particles can further include coating the separation device with an organic material.

[0065] The disclosed technology may further include a method of filtering a raw sample of blood using the device of claim 1.

[0066] The disclosed technology may further include a method for in-line filtering a sample of blood using the device of claim 1.

[0067] The disclosed technology may further include a method for detecting a thrombus using the device of claim 1.

[0068] The disclosed technique may further include a method of dissociating clustered particles using the apparatus of claim 1.

[0069] These and other aspects of the present invention are described in the following detailed description and accompanying drawings. Other aspects and features of embodiments of the present invention will become apparent to those skilled in the art upon review of the following description of specific, exemplary embodiments of the present invention in conjunction with the drawings. While features of the present invention may be described in connection with particular embodiments and figures, all embodiments of the present invention may include one or more of the features described herein. Furthermore, while one or more embodiments may be described as having certain advantageous features, one or more of such features may also be used with various embodiments of the present invention described herein. Similarly, while exemplary embodiments may be described below as device, system, or method embodiments, it will be understood that such exemplary embodiments may be implemented in various devices, systems, and methods of the present invention. [Brief explanation of the drawings]

[0070] Reference is now made to the accompanying drawings, which are not necessarily drawn to scale. In the drawings: [Figure 1A] FIG. 1A is a top view of a separation device according to some embodiments of the present disclosure. [Figure 1B] FIG. 1B is a bottom view of a separation device according to some embodiments of the present disclosure. [Figure 2] FIG. 2 is a diagram of a separation device within a filtration holder, according to some embodiments of the present disclosure. [Figure 3A] FIG. 3A illustrates a plurality of microwells of a separation device according to some embodiments of the present disclosure. [Figure 3B] FIG. 3B is a diagram illustrating forces acting on trapped clustered particles according to some embodiments of the present disclosure. [Figure 3C] FIG. 3C illustrates a microwell with trapped clustered particles, according to some embodiments of the present disclosure. [Figure 4A] FIG. 4A illustrates variations of meshed capture regions of microwells according to some embodiments of the present disclosure. [Figure 4B] FIG. 4B illustrates variations of meshed capture regions of microwells according to some embodiments of the present disclosure. [Figure 4C] FIG. 4C illustrates a variation of a meshed capture region of a microwell according to some embodiments of the present disclosure. [Figure 4D] FIG. 4D illustrates variations of meshed capture regions of microwells according to some embodiments of the present disclosure. [Figure 5A] FIG. 5A illustrates a cross-section of a plurality of microwells according to some embodiments of the present disclosure. [Figure 5B] FIG. 5B illustrates a top view of the plurality of microwells of FIG. 5A, according to some embodiments of the present disclosure. [Figure 6A] FIG. 6A illustrates a cross-section of a plurality of microwells according to some embodiments of the present disclosure. [Figure 6B] FIG. 6B illustrates a top view of the plurality of microwells of FIG. 6A, according to some embodiments of the present disclosure. [Figure 7A] FIG. 7A illustrates a cross-section of a plurality of microwells according to some embodiments of the present disclosure. [Figure 7B] FIG. 7B illustrates a top view of the plurality of microwells of FIG. 7A, according to some embodiments of the present disclosure. [Figure 8A] FIG. 8A illustrates a cross-section of a microwell according to some embodiments of the present disclosure. [Figure 8B] FIG. 8B illustrates a top view of the microwell of FIG. 8A, according to some embodiments of the present disclosure. [Figure 9] FIG. 9 is a flow diagram outlining a method for making a separation device according to some embodiments of the present disclosure. [Figure 10A] FIG. 10A illustrates a method of making a silicon mold according to some embodiments of the present disclosure. [Figure 10B]FIG. 10B illustrates a method of making a silicon mold according to some embodiments of the present disclosure. [Figure 10C] FIG. 10C illustrates a method of making a silicon mold according to some embodiments of the present disclosure. [Figure 10D] FIG. 10D illustrates a method of making a silicon mold according to some embodiments of the present disclosure. [Figure 10E] FIG. 10E illustrates a method of making a silicon mold according to some embodiments of the present disclosure. [Figure 10F] FIG. 10F illustrates a method of making a silicon mold according to some embodiments of the present disclosure. [Figure 10G] FIG. 10G illustrates a method of making a silicon mold according to some embodiments of the present disclosure. [Figure 10H] FIG. 10H illustrates a method of making a silicon mold according to some embodiments of the present disclosure. [Figure 10I] FIG. 10I illustrates a method of making a silicon mold according to some embodiments of the present disclosure. [Figure 11A] FIG. 11A illustrates a method of making a polymer mold according to some embodiments of the present disclosure. [Figure 11B] FIG. 11B illustrates a method of making a polymer mold according to some embodiments of the present disclosure. [Figure 11C] FIG. 11C illustrates a method of making a polymer mold according to some embodiments of the present disclosure. [Figure 12A] FIG. 12A illustrates a method for making and removing a separation device according to some embodiments of the present disclosure. [Figure 12B] FIG. 12B illustrates a method for making and removing a separation device according to some embodiments of the present disclosure. [Figure 12C] FIG. 12C illustrates a method for making and removing a separation device according to some embodiments of the present disclosure. [Figure 13] FIG. 13 illustrates a method for separating clustered particles according to some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0071] The present disclosure relates to a separation device for separating clustered particles from a fluid sample containing non-clustered and clustered particles. The separation device can include a plurality of microwells having a bottom surface with a meshed capture region. The meshed capture region can be divided into a plurality of openings using one or more dividing lines. The fluid sample can be poured into the microwells when passed through the separation device at a high volumetric flow rate. The openings can be sized so that the non-clustered particles pass through the openings and the clustered particles are loosely captured within the meshed capture region. Once captured, the clustered particles can be recovered from the meshed capture region for molecular and functional analysis. Separation and analysis of the captured clustered particles can provide valuable diagnostic information and insight into potential therapeutic strategies.

[0072] The disclosed technology is more fully described below with reference to the accompanying drawings. However, the disclosed technology can be embodied in many different forms and should not be construed as limited to the examples set forth herein. The components described below as constituting various elements of the disclosed technology are intended to be exemplary and not limiting. Many suitable components that would perform the same or similar functions as the components described herein are intended to be included within the scope of the disclosed electronic devices and methods. Such other components not described herein may include, for example, but are not limited to, components developed after the development of the disclosed technology.

[0073] In the following description, many specific details are set forth. However, it should be understood that examples of the disclosed technology may be practiced without these specific details. In other instances, well-known methods, structures, and techniques have not been shown in detail so as not to obscure an understanding of the specification. References to "one embodiment," "embodiment," "exemplary embodiment," "some embodiments," "particular embodiments," "various embodiments," etc., indicate that embodiments of the disclosed technology so described may include particular features, structures, or characteristics, but not all embodiments necessarily include the 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.

[0074] Throughout the specification and claims, the following terms have at least the meaning expressly associated therewith, unless the context clearly dictates otherwise: The term "or" is intended to mean an inclusive "or." Furthermore, the terms "a," "an," and "the" are intended to mean one or more unless otherwise specified or clear from the context to be singular.

[0075] Unless otherwise specified, the use of ordinal adjectives "first," "second," "third," etc. to describe general objects is intended only to indicate reference to different instances of similar objects and is not intended to suggest that the objects so described must be in any particular order, whether temporally, spatially, hierarchically, or otherwise.

[0076] Unless otherwise specified, the terms "clustered particle" and "clustered particles" refer to any cluster of two or more particles, including microparticles and nanoparticles.

[0077] Unless otherwise specified, the term "cell cluster" includes any cluster of two or more cells, where the cells may be of any type, including, but not limited to, circulating tumor cells, exfoliated tumor cells, red blood cells, and artificially synthesized nanoparticles and microparticles.

[0078] FIG. 1A illustrates a top view of a separation device 100. The separation device 100 can have an inlet 112 configured to receive a fluid. The separation device 100 can include a plurality of microwells 102 configured to capture clustered particles. The microwells 102 can be recesses in the separation device 100. The microwells 102 can include a plurality of sidewalls 104. The sidewalls 104 can extend from the top surface to the bottom surface of the microwells 102. The microwells 102 can have a depth of any size. The depth of the microwells 102 can facilitate the separation and capture of clustered particles. The depth of the microwells 102 can be based on the application for which the separation device 100 is being used and the size of the clustered particles being captured by the separation device 100. In some embodiments, the depth of the microwells 102 can be between about 10 microns and about 500 microns. In some embodiments, the separation device 100 can be used to capture nanoparticle-clustered particles or extracellular vesicle-clustered particles. In the present application, the microwells 102 can be submicron deep. The bottom surface of the separation device 100 can include a meshed capture region 106. A plurality of thin partition lines 110 can divide the meshed capture region 106 into a plurality of openings 108.

[0079] 1B is a bottom view of the separation device 100. The separation device 100 can include an outlet 114 configured to output fluid. The inlet 112 and outlet 114 can be any type of inlet or outlet configured to route fluid to and from the microwells 102. In some embodiments, the inlet 112 can be an open surface above the microwells 102. In some embodiments, the outlet 114 can be an open surface adjacent to the opening 108 of the meshed capture region 106.

[0080] 2 illustrates separation device 100 disposed within a filter holder 202. Filter holder 202 can be any commercially available filter holder. Filter holder 202 can be customized based on the desired size and shape of separation device 100 and the application for which separation device 100 will be used.

[0081] The separation device 100 can be any size and shape. In some embodiments, the separation device 100 can be generally rectangular, as illustrated in FIGS. 1A and 1B. In some embodiments, the separation device 100 can be generally circular, as illustrated in FIG. 2. The separation device 100 can have a diameter D, as illustrated in FIGS. 1A, 1B, and 2. The diameter D of the separation device 100 in FIGS. 1A and 1B can be the length of the separation device 100 relative to its longitudinal axis. The diameter D can be based on the diameter of a substrate, such as a silicon wafer, used in the method of fabricating the separation device 100. The separation device 100 can have a diameter D of about 5 millimeters to about 300 millimeters. The diameter D of the separation device 100 can be based on the application in which the separation device 100 will be used. For applications requiring a volumetric flow rate of more than 1000 mL / h, the separation device 100 can have a larger diameter D than applications requiring a volumetric flow rate of 20 mL / h to 100 mL / h.

[0082] The flow rate at which a fluid can pass through separation device 100 can depend on the diameter D of separation device 100 and the application for which separation device 100 is being used. In some embodiments, a fluid can pass through separation device 100 at a flow rate of about 20 mL / h to about 100 mL / h. At this volumetric flow rate, separation device 100 can have a diameter D of about 25 millimeters or greater and can effectively separate and capture clustered particles. In some embodiments, a fluid can pass through separation device 100 at a volumetric flow rate greater than 1000 mL / h. At this volumetric flow rate, separation device 100 can have a diameter D of about 150 millimeters to about 300 millimeters and can effectively separate and capture clustered particles.

[0083] The speed at which fluid can pass through separator 100 can likewise depend on the size of separator 100 and the application in which separator 100 is used. In some embodiments, fluid can pass through separator 100 at a speed between about 20 microns per second and about 260 microns per second.

[0084] Separation device 100 can include any number of microwells 102. The number of microwells 102 can depend on the surface area of ​​separation device 100. The number of microwells 102 can depend on the size of the clustered particles to be separated by separation device 100. In some embodiments, separation device 100 can have from about 40 to about 280 microwells per square millimeter. When separation device 100 is being used to separate nanoparticle-clustered particles, separation device 100 can have from about 40,000 to about 280,000 microwells 102 per square millimeter, with each microwell 102 having a size on the order of nanometers.

[0085] Separation device 100 can be made of any material that can be flowed and subsequently solidified as needed, and that can be micropatterned and / or nanopatterned. In some embodiments, separation device 100 can be made substantially of a polymer. The polymer can be a UV-curable polymer. Alternatively, or in addition, the polymer can be a thermosetting polymer. The polymer can be a fluoropolymer, such as a perfluoropolyether-based polymer. Fluoropolymers can facilitate easy removal of separation device 100 from various molds during fabrication of separation device 100. In some embodiments, separation device 100 can be made substantially of a metal. In some embodiments, separation device 100 can be made substantially of a semiconductor.

[0086] FIG. 3A illustrates a plurality of microwells 102 of a separation device 100. A fluid sample can be passed through the inlet 112 of the separation device 100. The fluid can include a plurality of non-clustered particles 302 and a plurality of clustered particles 304. The fluid can vary depending on the application for which the separation device 100 is being used. In some embodiments, the fluid can be blood. Alternatively, in some embodiments, the fluid can be urine. The non-clustered particles 302 can include non-clustered cells, such as a single red blood cell or white blood cell. In some embodiments, the non-clustered particles 302 can include a single cancer cell 306, such as a single circulating tumor cell. The clustered particles 304 can include cell clusters. The clustered particles 304 can be any number of clustered cells, such as, but not limited to, a two-cell cluster, a three-cell cluster, a ten-cell cluster, or the like. The clustered particles 304 can be unlabeled. Alternatively, the clustered particles 304 can be labeled. The labeling can include molecular labeling, such as fluorescent imaging, bead labeling, and the like. The cell clusters can be cancer cell clusters. By way of example, the cell clusters can include circulating tumor cell (CTC) clusters, ovarian cancer cell clusters, breast cancer cell clusters, prostate cancer cell clusters, and the like. In some embodiments, the cell clusters can include clusters of blood cells, which indicate the possible presence of a thrombus. In some embodiments, the clustered particles 304 can include nanoparticle-clustered particles. In some embodiments, the clustered particles 304 can include extracellular vesicle-clustered particles.

[0087] When a fluid sample passes through the inlet 112 of the separation device 100, the microwells 102 can funnel the non-clustered particles 302 and clustered particles 304 into the meshed capture region 106. As illustrated in FIG. 3A , the sidewalls 104 of the microwells 102 can have sloped portions 104a. The sloped sidewalls 104a can be sloped at any angle, including positive angles, negative angles, and 0 degrees. The sloped sidewalls 104a can facilitate the non-clustered particles 302 and clustered particles 304 being funneled into the meshed capture region 106. The sloped sidewalls 104a can also minimize the movement of the captured clustered particles 304 so that the captured clustered particles 304 can remain securely within the microwells 102.

[0088] The dividing lines 110 forming the openings 108 can divide the fluid flow into multiple flow paths. Depending on the application for which the separation device 100 is being used, the openings 108 can be sized to allow non-clustered particles 302 to pass through the openings 108 and exit through the outlet 114. However, the geometry of the clustered particles 304, relative to the size of the openings 108, may prevent the clustered particles 304 from passing through the openings 108. In some embodiments, the size of the openings 108 can be approximately 100 to 300 square microns. In some embodiments, if the separation device 100 is being used to capture nanoparticle-clustered particles, the openings 108 can be sized accordingly. The size of the openings 108 can be optimized to allow the microwells 102 to capture two-cell and three-cell clustered particles 304 while minimizing the unwanted capture of white blood cells. Because unclustered particles 302 can easily pass through opening 108 without interference, separation device 100 is capable of processing large volumes of fluid, including unprocessed whole blood, without risk of clogging separation device 100. By minimizing the risk of clogging, separation device 100 may be ideal for clinical settings.

[0089] 3B illustrates the forces that may act on clustered particles 304 within the meshed capture region 106 of the microwell 102. The flow of the fluid sample as it passes through the microwell 102 generates a Dean drag force F D When the clustered particles 304 engage with the barrier lines 110 of the meshed capture area 106, a reaction force F R Reaction force F R can form a dynamic force balance that can provide a stable equilibrium for the trapped clustered particles 304. Furthermore, when the clustered particles 304 engage with the sloped sidewall 104a, a friction force F F The combination of these forces allows the microwells 102 to gently immobilize the clustered particles 304 without dissociating the clustered particles 304.

[0090] 3C is an additional diagram illustrating clustered particles 304 trapped within microwells 102. This configuration of meshed trapping region 106 allows microwells 102 to gently trap clustered particles 304. This gentle trapping can minimize dissociation of clustered particles 304. Preventing dissociation of clustered particles 304 can be important because clustered particles 304 may occur relatively rarely within a fluid sample and may provide valuable information during analysis.

[0091] 4A-4D illustrate various configurations of the meshed capture region 106. Each meshed capture region 106 can include one or more partition lines 110 configured to divide the meshed capture region 106 into multiple openings 108 and support captured clustered particles 304. As shown in FIG. 4A, the partition lines 110 can divide the meshed capture region 106 into four openings 108 having a square shape. The openings 108 can be arranged in a 2x2 array. In some embodiments, each square-shaped opening 108 can have a side length of about 10 microns to about 17 microns. As illustrated in FIG. 4B, the meshed capture region 106 can be divided into four openings 108 having a generally circular shape. As illustrated in FIG. 4C, the meshed capture region 106 can be divided into four openings 108 having a generally elliptical shape. As illustrated in FIG. 4D, the meshed capture region 106 can be divided into five openings 108 having a generally polygonal shape. In some embodiments, each opening 108 may have a hexagonal shape.

[0092] 4A-4D show modified meshed capture region 106, it is contemplated that meshed capture region 106 can include any number of partition lines 110 to form any number of openings 108 having any geometric shape. The size and shape of openings 108 can be based on the size and shape of clustered particles 304 and the application for which separation device 100 is to be used. In some embodiments, openings 108 can have the same geometric shape and size. In some embodiments, openings 108 can have different geometric shapes and sizes. When separation device 100 is used to capture nanoparticle-clustered particles and / or extracellular vesicle-clustered particles, the size of openings 108 can be made accordingly.

[0093] 5A to 8B illustrate cross-sectional views and top views of exemplary configurations of multiple microwells 102.

[0094] 5A and 5B illustrate cross-sectional and top views, respectively, of a plurality of microwells 102. The microwells 102 can include sloping sidewalls 104a configured to funnel fluid into a meshed capture region 106. A partition line 110 can divide the meshed capture region 106 into a plurality of square apertures 108 arranged in a 2x2 aperture array. The microwells 102 can be separated from each other by flat portions of the top surface of the separation device 100.

[0095] 6A and 6B illustrate cross-sectional and top views, respectively, of a plurality of microwells 102 having a modified top portion from the plurality of microwells illustrated in FIGS. 5A and 5B. Adjacent microwells 102 can be connected to one another to create a substantially pointed tip. A partition line 110 can divide the meshed capture region 106 of each microwell 102 into four square apertures 108 arranged in a 2×2 aperture array. The microwells 102 include sloping sidewalls 104a, which can facilitate funneling and capture of clustered particles 304 within the meshed capture region 106.

[0096] 7A and 7B illustrate cross-sectional and top views, respectively, of a plurality of microwells 102 having a linear array of apertures 108. A partition line 110 can divide the meshed capture region 106 of each microwell 102 into 12 apertures 108. The apertures 108 can be arranged in a 2 x 6 aperture array such that the array is generally linear. The microwells 102 can include sloping sidewalls 104a to facilitate pouring and capture of clustered particles 304 within the meshed capture region 106. The microwells 102 can be separated from each other by a flat portion of the top surface of the separation device 100.

[0097] 8A and 8B illustrate cross-sectional and top views, respectively, of a plurality of microwells 102 having a generally mesh-like configuration. Dividing lines 110 can divide the meshed capture region 106 of the microwells 102 into 36 apertures. The apertures 108 can be arranged in a 9x4 aperture array. The microwells 102 include sloping sidewalls 104a, which can facilitate pouring and trapping of clustered particles 304 within the meshed capture region 106.

[0098] 5A-8B show variations of the microwells 102, it is contemplated that the microwells 102 can have any configuration. The meshed capture region 106 can include any array of apertures 108. The array of apertures 108 can be an array of any number of apertures, such as, but not limited to, a 2x2 aperture array, a 3x5 aperture array, a 4x6 aperture array, a 5x10 aperture array, and the like.

[0099] The disclosed technology can also include a method 900 for fabricating a separation device 100. As illustrated in Figure 9, the method 900 can include fabricating a silicon mold on a silicon wafer (902), fabricating a polymer mold (904), fabricating a separation device (906), and removing the separation device (908). The method 900 for fabricating a separation device 100 can be performed in a non-cleanroom environment, thereby reducing costs and labor hours.

[0100] 10A-10I illustrate a method of making a silicon mold 1012. As illustrated in Figure 10A, a silicon wafer 1002 can be provided. In some embodiments, the silicon wafer 1002 can have a thickness of about 300 microns to 600 microns.

[0101] 10B and 10C, 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 be the basis for a desired array of openings 108 in the meshed capture region 106.

[0102] 10D, silicon wafer 1002 can be etched to form pillars 1006. Silicon wafer 1002 can be etched to a depth of approximately 10 microns using deep reactive ion etching.

[0103] In FIG. 10E, a nitride layer 1006 can be deposited. The nitride layer 1006 can be approximately 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 illustrated in FIG. 10F, 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.

[0104] As shown in FIG. 10G, the nitride layer 1006 can be etched using reactive ion etching 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. Etching the silicon wafer 1002 can create sloped walls. The sloped walls can extend to multiple pillars. The formation of the sloped walls can serve as the basis for forming the sloped sidewalls 104a of the separation device 100.

[0105] As illustrated in Figure 10H, a third photoresist layer 1010 can be deposited and patterned on a silicon wafer 1002. The silicon wafer 1002 can be etched to a depth of about 50 microns using deep reactive ion etching. Etching of the silicon wafer 1002 can form a silicon mold 1012.

[0106] 11A-11C illustrate a method for fabricating a polymer mold. The method for fabricating a polymer mold can include double-molding a polymer. A silicon mold 1012 can be coated with silane under vacuum conditions for 8 hours before fabricating the polymer mold. Coating the silicon mold 1012 with silane facilitates easy removal of the first polymer mold 1102 from the silicon mold 1012. In some embodiments, metal layer sputtering, including gold layer sputtering, can be used to reduce and / or eliminate the 8-hour waiting time. FIG. 11A illustrates fabrication of the first polymer mold 1102. A first polymer layer can be poured 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. The cured first polymer mold 1102 can be peeled from the silicon mold 1012, as shown in FIG. 11B. The surface of the first polymer mold 1102 can be activated using oxygen plasma and coated with silane for approximately 8 hours. As illustrated in FIG. 11C, the first polymer mold 1102 can serve as a mold for fabricating the second polymer mold 1104. A second polymer layer can be poured onto the first polymer mold 1102 and cured to form the second polymer mold 1104. Once the second polymer mold 1104 is fabricated, the second polymer mold 1104 can be removed from the first polymer mold 1102.

[0107] In some embodiments, the first polymer layer and the second polymer layer can include polydimethylsiloxane (PDMS).

[0108] 12A-12C illustrate methods for fabricating and removing the separation device 100. As illustrated in FIG. 12A, the second polymer mold 1104 can be attached to a substrate 1202. In some embodiments, the second polymer mold 1104 can be attached to the non-adhesive side of vinyl dicing tape. Alternatively, the substrate 1202 can comprise an acetate sheet, a PET sheet, or other similar material. Once the second polymer mold 1104 is attached 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 an inlet port in the second polymer mold 1104. A vacuum can be applied to an outlet port to facilitate filling the UV-curable polymer into the second polymer mold 1104. 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 and forming the separation device 100. In some embodiments, the UV light can have a wavelength of approximately 365 nanometers. In some embodiments, the second polymer mold 1104 can be filled with a UV-curable polymer over a thermoelectric cooler. The thermoelectric cooler can reduce the temperature of the UV-curable polymer, thereby increasing the viscosity of the UV-curable polymer. Increasing the viscosity of the UV-curable polymer allows for the use of higher vacuum levels without creating bubbles, improving manufacturing yields.

[0109] Once the UV-curable polymer is cured, the second polymer mold 1104 can be peeled off the detachment device 100, as shown in Figure 12B. The detachment device 100 can then be removed from the substrate 1202, as illustrated in Figure 12C. In some embodiments, the detachment device 100 can be placed on a thermoelectric cooler to facilitate easy removal of the detachment device 100.

[0110] In some embodiments, the UV-curable polymer can be a fluorine-based polymer, including perfluoropolyether-based polymers. In some embodiments, the UV-curable polymer can be a thermosetting polymer. By way of example, if exposure to UV light is undesirable, a thermosetting polymer such as PDMS can be used to form the separation device 100.

[0111] While FIGS. 10A-12C illustrate one example method for fabricating separation device 100, other methods are contemplated. In some embodiments, hot embossing can be used to fabricate separation device 100 made substantially of a polymer. Hot embossing can be a low-cost, scalable technique for fabricating separation device 100, making it applicable to a wide range of applications. Polymers in this technique can include polymethyl methacrylate, cyclic olefin copolymers, polycarbonate, polyethylene, and the like. The technique generally involves heating, molding, and demolding. The polymer can be softened by heating above the polymer's glass transition temperature. Pressure can be applied to cause the softened polymer to assume the shape of the underlying mold. In a demolding process, the polymer can be cooled and removed from the mold. The polymer can then be punched to create through-holes and create openings 108 in separation device 100. The temperature, pressure, and choice of polymer can vary depending on the application and desired parameters, such as thickness, of separation device 100.

[0112] Additionally, in some embodiments, conventional electroless electroplating can be used to fabricate a separation device 100 made substantially of metal. This technique can include fabricating a second polymer mold 1104 as described herein. A metal seed layer can be deposited on the surface of the second polymer mold 1104 using an electron beam evaporator in high vacuum. Metal ions can adhere to the surface of the second polymer mold 1104 and then grow. The grown metal can have approximately the same shape as the second polymer mold 1104. Varying the thickness of the electroplated metal can change the strength and flexibility of the separation device 100.

[0113] In some embodiments, silicon micromachining can be used to fabricate separation device 100. Unlike the fabrication methods illustrated in Figures 10A through 12C, which can be performed without a clean room, silicon micromachining allows separation device 100 to be fabricated from a silicon wafer in a clean room. In this technique, a silicon nitride layer can be deposited on a silicon wafer. The silicon nitride layer can be patterned by reactive ion etching, and the silicon wafer can be etched with a KOH (or TMAH) solution. Backside photolithography can be performed, followed by plasma etching of the nitride layer, to fabricate separation device 100.

[0114] 13 shows a method 1300 for separating clustered particles. Method 1300 can include providing 1302 a separation device 100 including a plurality of microwells 102. Each microwell 102 can include a plurality of sidewalls 104 and a bottom surface having a meshed capture region 106. Separation device 100 can further include any of the features described herein.

[0115] The method 1300 can include passing 1304 a fluid through the separation device 100. The fluid can include a plurality of unclustered particles 302 and a plurality of clustered particles 304. As the fluid passes through the separation device 100, the unclustered particles 302 and the clustered particles 304 can be funneled into the microwells 102.

[0116] The method 1300 can include trapping 1306 a plurality of clustered particles 304 within a meshed trapping region 106 .

[0117] The method 1300 may include outputting 1308 a sample of the fluid. The output sample includes a plurality of non-clustered particles 302. Because the clustered particles 304 may remain trapped within the microwells 102, the output sample may be substantially free of the clustered particles 304.

[0118] The method 1300 can further include recovering the clustered particles 304 from the meshed capture region 106. To recover the clustered particles 304, the clustered particles 304 can be washed with PBS. Following washing with PBS, the captured clustered particles 304 can be output at a different relative counterflow rate relative to the volumetric flow rate at which the fluid flows through the separation device 100. The output clustered particles 304 can then be transferred to a holding vessel. Alternatively, the clustered particles 304 can be directly recovered from the meshed capture region 106. In some embodiments, the clustered particles 304 can be directly recovered from the meshed capture region 106 using a micromanipulator. Unlike conventional pore filters, in which the captured clustered particles 304 can adhere to the surface of the filter, the recesses of the meshed capture region 106 within the microwells 102 allow the separation device 100 to be moved to a system or device configured for analysis without risk of losing the captured clustered particles 304.

[0119] The recovered clustered particles can be imaged and subjected to any form of molecular or functional analysis. Analysis of the clustered particles 304 can provide valuable information about the clustered particles 304, such as the primary site of cancer or cellular mutations. Additionally, possible courses of treatment can be explored. In some embodiments, the clustered particles 304 can be treated with possible drugs and / or other forms of therapy. The results of these drugs and treatments can be used to improve personalized medicine.

[0120] In some embodiments, the method 1300 for separating clustered particles can include coating the separation device 100 with an organic or inorganic coating. In some embodiments, the inorganic coating can increase the surface adhesion properties of the separation device 100. The inorganic coating can include antibodies with specific affinities so that the separation device 100 can capture the clustered particles 304. In some embodiments, an organic coating, such as a PEG or BSA coating, can reduce non-specific adhesion so that the captured clustered particles 304 can be detached.

[0121] In some embodiments, the method 1300 for separating clustered particles can include coating the separation device 100 with a growth culture. When the separation device 100 is coated with a growth culture, the captured cluster particles 304 can be grown directly on the separation device 100. In this sense, the separation device 100 can function similarly to a human organ and / or tissue. The captured clustered particles 304 can easily survive because fluid flow (e.g., blood flow) can provide a continuous source of nutrients. The grown clustered particles 304 can then be further analyzed by various techniques. In some embodiments, the grown clustered particles 304 can be removed and cultured to develop new cell lines or new drug therapies.

[0122] The separation device 100 and / or method 1300 for separating clustered particles can be used in a variety of additional applications. For example, urine cytology is a technique in which abnormal cells in urine can be examined under a microscope to diagnose urinary tract cancers, including bladder cancer. This technique may require enrichment of rare exfoliated cancer cells from a large voided urine sample obtained from a patient. Instead of using current centrifugation and cytospin methods, the separation device 100 can be used to filter large urine samples without damaging or losing significant numbers of the rare exfoliated cancer cells. After capturing the exfoliated cancer cells, they can be characterized using fluorescent staining or Pap staining protocols.

[0123] In some embodiments, the separation device 100 can be used to filter raw blood samples. Furthermore, the separation device 100 can be used in an in-line blood purification system. Because circulating tumor cell clusters have a higher metastatic potential compared to individual circulating tumor cells, removing CTC clusters from the blood can be important. In this application, blood can be removed from a patient. A blood pump can be used to pump the blood, and an anticoagulant can be added. The blood can be allowed to flow through the separation device 100. CTC clusters can be gently captured within the microwells 102 of the separation device 100, while single red blood cells, white blood cells, and single CTCs can pass through the separation device 100. The washed blood, substantially free of CTC clusters, can be returned to the patient. This technique can be performed continuously in a portable system and / or at intervals depending on the severity of the patient's condition.

[0124] In some embodiments, the separation device 100 can be used to break up the clustered particles. In this technique, the volumetric flow rate at which a sample of blood is passed through the separation device 100 can be increased so that the shear forces on the captured clustered particles are also increased. The increased shear forces can cause the clustered particles 304 to dissociate into non-clustered particles 302. As an example, CTC clusters can be dissociated into single CTCs. This technique can facilitate therapeutic intervention and improve the course of treatment, as single CTCs have been shown to be less prone to metastasis.

[0125] It is to be understood that the embodiments and claims disclosed herein are not limited in their application to the details of the construction and arrangement of components described in the specification and illustrated in the drawings. Rather, the specification and drawings provide examples of contemplated embodiments. The embodiments and claims disclosed herein are further capable of other embodiments and of being practiced and carried out in various ways. It is also to be understood that the phrases and terms employed herein are for the purpose of description and should not be regarded as limiting the scope of the claims.

[0126] As such, those skilled in the art will appreciate that the conception underlying the present application and claims may be readily utilized as a basis for the designing of other structures, methods, and systems for carrying out some of the purposes of the embodiments and claims presented herein, and it is important, therefore, that the claims be regarded as including such equivalent structures.

[0127] Moreover, the purpose of the Abstract is to enable the U.S. Patent and Trademark Office and the public, particularly those skilled in the art who are not familiar with patent and legal terminology, to quickly assess the nature and substance of the technical disclosure of the present application at a glance. The Abstract is not intended to define the claims of the present application, nor is it intended to limit the scope of the claims in any way. Instead, the present invention is intended to be defined by the claims appended hereto.

Claims

1. 1. A separation apparatus for separating clustered particles, comprising: an inlet configured to receive a flow in a flow direction of an inlet fluid comprising a plurality of non-clustered particles and a plurality of clustered particles; a plurality of microwells downstream of the inlet, each microwell including a bottom surface having at least one sidewall and a plurality of sidewalls and a meshed capture region configured to capture the plurality of clustered particles and allow the plurality of non-clustered particles to pass through, the meshed capture region including a plurality of partition lines defining a plurality of openings configured to divide the fluid flow into a plurality of flow paths; an outlet downstream of the plurality of microwells configured to output fluid that has passed through the microwells containing the plurality of unclustered particles; a sidewall of at least one of the plurality of microwells includes a first portion and a second portion; the meshed capture area is downstream of the second portion; the first portion is parallel to the flow direction; the second portion is downstream of the first portion, inclined relative to the first portion at an angle of between 1 and 89 degrees relative to the flow direction, and configured to capture the clustered particles in the microwells in combination with the mesh capture region by providing a reaction force on the plurality of clustered particles in a direction at least partially opposite to the flow direction.

2. At least one opening of the plurality of openings is square; The separation device according to claim 1 , wherein a distance between adjacent ones of the plurality of openings is smaller than a length of a side of the at least one opening of the plurality of openings.

3. 2. The separation device of claim 1, wherein the total permeable area defined by the plurality of openings is greater than 50% of the total area defined by the meshed capture region.

4. 10. The separation device of claim 1, wherein the inlet fluid is blood, the non-clustered particles comprise non-clustered cells, and the clustered particles comprise cell clusters.

5. 10. The separation device of claim 1, wherein the inlet fluid is urine, the non-clustered particles comprise non-clustered cells, and the clustered particles comprise cell clusters.

6. 5. A separation device according to any one of claims 1 to 4, wherein the device is configured to provide a volumetric flow rate through the inlet and outlet of between 20 mL / h and 100 mL / h.

7. 7. The separation device according to claim 1, wherein the depth of each microwell is between 10 microns and 500 microns.

8. 8. A separation device according to any one of claims 1 to 7, wherein at least a portion of each side wall is sloped.

9. 9. The separation device of claim 1, wherein the device comprises between 40 and 280 microwells per square millimeter.

10. The separation device according to claim 1 , wherein the plurality of openings are arranged in an array.

11. 11. The separation device of claim 1, wherein each opening in the plurality of openings is sized such that the non-clustered particles pass through it and the clustered particles do not pass through it.

12. 12. The separation device of claim 1, wherein each opening in the plurality of openings has a shape selected from the group consisting of a square, a circle, an oval, and a polygon.

13. 13. The separation device of claim 12, wherein each opening in the plurality of openings is square-shaped with a side length between 10 microns and 17 microns.

14. The separation device of claim 1 , wherein each opening in the plurality of openings has the same shape.

15. 15. The separation device of claim 1, wherein the clustered particles are label-free.

16. 15. A separation device according to any one of claims 1 to 14, wherein the clustered particles are labeled.

17. A separation device according to any one of claims 1 to 16, wherein the device has a diameter of between 5 millimetres and 300 millimetres.

18. 18. The separation device of any one of claims 1 to 17, wherein the device is fabricated from a material selected from the group consisting of perfluoropolyether-based polymers, thermosetting polymers, UV-curable polymers, metals, semiconductors, and combinations thereof.

19. A separation device comprising: The entrance and microwells downstream of the inlet, each microwell including a sidewall and a bottom surface having a meshed capture region; an outlet downstream of the microwell; receiving a flow of fluid comprising non-clustered particles and clustered particles from the inlet in a flow direction; configured to output a separated fluid containing the non-clustered particles and not containing the clustered particles from the outlet; at least one sidewall of the microwell includes a first portion and a second portion; the meshed capture area is downstream of the second portion; the first portion is parallel to the flow direction; the second portion is downstream of the first portion, is inclined relative to the first portion at an angle of between 1 and 89 degrees relative to the flow direction, and is configured to capture the clustered particles in the microwells in combination with the mesh capture region by providing a reaction force on the plurality of clustered particles in a direction at least partially opposite to the flow direction; the meshed capture region is configured to capture the clustered particles of the fluid and to allow the non-clustered particles of the fluid to pass through; A separation device, wherein the meshed capture region comprises openings configured to divide the fluid flow into channels.

20. At least one opening is square; 20. The separating device of claim 19, wherein the spacing between adjacent openings is less than a length of a side of the at least one opening.

21. 20. The separation device of claim 19, wherein the total permeable area defined by the openings is greater than 50% of the total area defined by the meshed capture region.

22. 20. A method for manufacturing a separation device according to claim 19, comprising the steps of: preparing a silicon mold on a silicon wafer; creating a polymer mold; making the separation device; and removing the separation device.

23. preparing the silicon mold on the silicon wafer, depositing a first photoresist layer on the silicon wafer; patterning the first photoresist layer; Etching the silicon wafer to form a plurality of pillars; 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 sloped sidewalls extending to each pillar in the plurality of pillars; depositing a third photoresist layer; patterning the third photoresist layer; and etching the silicon wafer to form the silicon mold.

24. preparing the polymer mold includes: coating the silicon mold with silane; depositing a first polymer layer on the silicon mold; curing the first polymer layer to form a first polymer mold; removing the first polymer mold from the silicon mold; coating the first polymer mold with a silane; depositing a second polymer layer on the first polymer mold; and curing the second polymer layer to form a second polymer mold.

25. 25. The method of claim 24, wherein the first polymer layer and the second polymer layer comprise polydimethylsiloxane (PDMS).

26. The method of claim 24 , wherein creating the polymer mold further comprises removing the second polymer mold from the first polymer mold.

27. preparing the silicon mold on the silicon wafer, depositing a first photoresist layer on the silicon wafer; patterning the first photoresist layer; Etching the silicon wafer to form a plurality of pillars; 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 sloped sidewalls extending to each pillar in the plurality of pillars; depositing a third photoresist layer; patterning the third photoresist layer; Etching the silicon wafer to form the silicon mold; preparing the polymer mold includes: coating the silicon mold with silane; depositing a first polymer layer on the silicon mold; curing the first polymer layer to form a first polymer mold; removing the first polymer mold from the silicon mold; coating the first polymer mold with a silane; depositing a second polymer layer on the first polymer mold; and curing the second polymer layer to form a second polymer mold. Producing the separation device comprises: attaching the second polymer mold to a substrate; filling the second polymer mold with a UV curable polymer; exposing the UV curable polymer to UV light; and curing the UV curable polymer.

28. further comprising removing the second polymer mold from the first polymer mold; 28. The method of claim 27, wherein the first polymer layer and the second polymer layer are made of polydimethylsiloxane (PDMS).

29. 30. The method of claim 28, wherein the second polymer mold is filled with the UV curable polymer using a vacuum pump.

30. 30. The manufacturing method of any one of claims 28 to 29, wherein the substrate is a vinyl dicing tape.

31. 30. The method of any one of claims 28 to 29, wherein the substrate is an acetate sheet.

32. 30. The method of any one of claims 28 to 29, wherein the substrate is a PET sheet.

33. 33. The method of any one of claims 28 to 32, wherein filling the second polymer mold with the UV curable polymer occurs on a thermoelectric cooler.

34. 34. The method of any one of claims 28 to 33, wherein the UV curable polymer is a thermosetting polymer.

35. Removing the separation device includes: removing the second polymer mold; and removing the separation device from the substrate.

36. 20. A method for separating clustered particles using the separation device of claim 19, comprising the steps of: passing a fluid comprising a plurality of clustered particles and a plurality of non-clustered particles through the separation device; capturing the plurality of clustered particles within the mesh-like capture region; and outputting a separated fluid comprising the plurality of unclustered particles.

37. 37. The method of claim 36, wherein the fluid is blood, the non-clustered particles are cells, and the clustered particles are cell clusters.

38. 37. The method of claim 36, wherein the fluid is urine, the non-clustered particles comprise non-clustered cells, and the clustered particles comprise cell clusters.

39. 39. The method of any one of claims 36 to 38, further comprising positioning the separation device in a filter holder.

40. 40. The method of any one of claims 36 to 39, wherein passing the fluid through the separation device is carried out at a flow rate of between 20 mL / h and 100 mL / h.

41. 41. The method of any one of claims 36 to 40, further comprising recovering the clustered particles from the meshed capture area.

42. Recovering the clustered particles from the meshed capture region washing the clustered particles with PBS; and transferring the clustered particles to a holding vessel.

43. 42. The method of claim 41, wherein a micromanipulator directly retrieves the clustered particles from the meshed trapping region.

44. 44. The method of any one of claims 36 to 43, further comprising analyzing the clustered particles.

45. 45. The method of any one of claims 36 to 44, wherein the clustered particles comprise circulating tumor cell clusters.

46. 46. ​​The method of any one of claims 36 to 45, wherein the clustered particles comprise exfoliated cancer cells in urine.

47. 47. The method of any one of claims 36 to 46, further comprising coating the separation device with a growth medium.

48. 48. The method of claim 47, wherein the captured clustered particles grow on the coated separation device.

49. 49. The method of claim 48, further comprising directly analyzing the grown clustered particles on the coated separation device.

50. 20. A method of filtering a raw sample of blood using the separation device of claim 19.

51. 20. A method for in-line filtering a sample of blood using the separation device of claim 19.

52. 20. A method for detecting thrombi using the separation device of claim 19.

53. 20. A method for dissociating clustered particles using the separation device of claim 19.

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